Gene-Based Therapies for Cancer (Current Cancer Research)

Current Cancer Research

For other titles published in this series, go to www.springer.com/series/7892

Jack A. Roth Editor

Gene-Based Therapies for Cancer

Editor Jack A. Roth, M.D., F.A.C.S. Professor and Bud Johnson Clinical Distinguished Chair, Department of Thoracic & Cardiovascular Surgery; Professor of Molecular and Cellular Oncology; Director, W.M. Keck Center for Innovative Cancer Therapies; Chief, Section of Thoracic Molecular Oncology; The University of Texas MD Anderson Cancer Center Houston, TX USA [email protected]

ISBN 978-1-4419-6101-3 e-ISBN 978-1-4419-6102-0 DOI 10.1007/978-1-4419-6102-0 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2010931279 © Springer Science+Business Media, LLC 2010 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in ­connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. While the advice and information in this book are believed to be true and accurate at the date of going to press, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

Foreword

Cancer is a disease of dysfunctional genes. Normal cellular genes that regulate cell proliferation develop carcinogen-induced, or rarely, germline mutations that alter the gene product so that it is permanently in an active configuration. Examples include the Kras oncogene and the epidermal growth factor receptor. These oncogenes confer the property of unlimited replication to cancer cells. Genes that normally suppress cell growth or induce programmed cell death, after sensing DNA damage, develop inactivating mutations in the cancer cell. Examples include the p53 tumor suppressor gene and the retinoblastoma gene. Inactivation of these tumor suppressor genes removes essential growth control mechanisms from the cell. Strategies for replacing inactivated tumor suppressor genes or inactivating oncogenes are logical extensions of the gene therapy concept. Investigators have been pursuing these concepts for almost 20 years, but slow progress in developing systemic delivery vehicles for genes and the ability to target tumors, and fragmentary knowledge of the critical genes to target, have limited progress. Recently, significant progress in sequencing the cancer genome, combined with advances in personalized cancer treatment has converged to accelerate the development of cancer gene therapy. Personalized or targeted cancer treatments rely on high throughput technologies, including DNA sequencing, expression arrays, and proteomics to identify critical pathways for cancer cell survival. Small molecule drug libraries are used to identify drugs that may specifically inhibit a pathway. Dramatic regression of metastatic cancers have occurred in a small percentage of cases with lower toxicity than seen with conventional chemotherapy agents, with these drugs indicating proof-ofprinciple for this approach. However, it is clear that additional critical pathways must be targeted as tumor recurrence occurs rapidly. Importantly, research in this field has identified critical targets for gene therapy approaches and raises the possibility of combining targeted small molecules with gene-based therapies. Gene therapy offers the potential of highly selective targeting of multiple critical pathways with minimal toxicity. Additional progress in the development of targeted and less toxic viral and nonviral vectors has also improved the outlook for cancer gene therapy. In this book, translational gene therapy approaches are emphasized. Chapters include discussions of specific gene delivery technologies as well as their application in the treatment of various cancers with extensive discussions of ongoing clinical trials. One approach that is not discussed at length in this book is the v

vi

Foreword

use of genes to modify the immune response to cancer as this is more appropriate for reviews of the immunotherapy of cancer. As is evident from numerous positive clinical trials, gene therapy for cancer has established proof-of-principle. The next step will be to enable this technology to be widely applied for the systemic treatment of cancer. Jack A. Roth, M.D.

Contents

1 RNAi: A New Paradigm in Cancer Gene Therapy................................. Edna M. Mora, Selanere L. Mangala, Gabriel Lopez-Berestein, and Anil K. Sood

1

2 Gene-Based Therapy for Cancer: Brain Tumors.................................... Hong Jiang and Juan Fueyo

17

3 Gene Therapy of Prostate Cancer............................................................ Svend O. Freytag, Hans Stricker, Benjamin Movsas, Mohamed Elshaikh, Ibrahim Aref, Kenneth Barton, Stephen Brown, Farzan Siddiqui, Mei Lu, and Jae Ho Kim

33

4 siRNA Versus shRNA for Personalized Cancer Therapy: Mechanisms and Applications................................................. John S. Vorhies, Donald D. Rao, Neil Senzer, and John Nemunaitis 5 Tumor Suppressor Gene Therapy............................................................ Jack A. Roth, John Nemunaitis, Lin Ji, and Rajagopal Ramesh 6 Targeted Oncolytic Adenovirus for Human Cancer Therapy: Gene-Based Therapies for Cancer........................................................... Toshiyoshi Fujiwara 7 Gene Therapy for Malignant Pleural Mesothelioma.............................. Edmund K. Moon, Sunil Singhal, Andrew R. Haas, Daniel H. Sterman, and Steven M. Albelda

51 63

79 95

8 Mesenchymal Stem/Stromal Cells as Cellular Vehicles for Tumor Targeting.................................................................................. 113 Frank Marini, Matus Studeny, Jennifer Dembinski, Keri L. Watson, Shannon Kidd, Erika Spaeth, Zhizong Zeng, Xiaoyang Ling, Ann Klopp, Fredrick Lang, Brett Hall, and Michael Andreeff

vii

viii

Contents

  9 Retargeting Adenovirus for Cancer Gene Therapy.............................. 141 Erin E. Thacker and David T. Curiel 10 Lentiviruses: Vectors for Cancer Gene Therapy.................................. 155 Yuan Lin, Amar Desai, and Stanton L. Gerson 11 Interleukin-24 Gene Therapy for Melanoma........................................ 181 Nancy Poindexter, Rajagopal Ramesh, Suhendan Ekmekcioglu, Julie Ellerhorst, Kevin Kim, and Elizabeth A. Grimm 12 Herpes Simplex Virus 1 for Cancer Therapy........................................ 203 Richard L. Price, Balveen Kaur, and E. Antonio Chiocca 13 Telomerase as a Target for Cancer Therapeutics.................................. 231 Jerry W. Shay 14 Gene Therapy for Sarcoma..................................................................... 251 Keila E. Torres and Raphael E. Pollock Index.................................................................................................................. 269

Contributors

Steven M. Albelda, M.D. William Maul Massey Professor of Medicine; Vice Chief, Pulmonary, Allergy, and Critical Care Division, University of Pennsylvania Medical Center; Abramson Research Center, Philadelphia PA, USA Michael Andreeff, M.D., Ph.D. Professor of Medicine and Haas Chair in Genetics; Chief, Molecular Hematology and Therapy, The University of Texas MD Anderson Cancer Center, Departments of Leukemia and Stem Cell Transplantation and Cellular Therapy, Houston TX, USA Ibrahim Aref, M.D. Medical Director, Department of Radiation Oncology, Henry Ford Macomb Hospital, Detroit MI, USA Kenneth Barton, Ph.D. Staff Scientist, Department of Radiation Oncology, Henry Ford Health System, Detroit MI, USA Stephen Brown, Ph.D. Staff Scientist, Department of Radiation Oncology, Henry Ford Health System, Detroit MI, USA E. Antonio Chiocca, M.D., Ph.D. Chairman, Department of Neurological Surgery, Dardinger Family Professor of Oncologic Neurosurgey; Physician Director, OSUMC Neuroscience Signature Program; Co-Director, Dardinger Center for Neuro-oncology and Neurosciences; Co-Director, Viral Oncology Program of the Comprehensive Cancer Center; James Cancer Hospital/Solove Research Institute; The Ohio State University Medical Center, Columbus OH, USA David T. Curiel, M.D., Ph.D. Distinguished Professor of Radiation Oncology (Endowed Chair); Director, Cancer Biology Division and Therapeutics Center, Department of Radiation Oncology, Washington University School of Medicine, St. Louis, MO, USA ix

x

Contributors

Jennifer Dembinski, Ph.D. Department of Stem Cell Transplantation and Cellular Therapy, Section of Molecular Hematology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Amar Desai, B.S. Ph.D. Student, Department of Pharmacology, Case Western Reserve University, Cleveland OH, USA Suhendan Ekmekcioglu Ph.D. Associate Professor, Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Julie Ellerhorst M.D., Ph.D. Associate Professor, Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Mohamed Elshaikh, M.D. Residency Program Director, Department of Radiation Oncology, Henry Ford Health System, Detroit MI, USA Svend O. Freytag, Ph.D. Division Head of Research in Department of Radiation Oncology, Henry Ford Health System, Detroit MI, USA Juan Fueyo, M.D. Associate Professor, Department of Neuro-Oncology, The University of Texas MD Anderson Cancer Center, Houston TX, USA Toshiyoshi Fujiwara, M.D. Professor and Chairman, Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Phamaceutical Sciences and Vice Director of Center for Gene and Cell Therapy of Okayama University Hospital, Okayama, Japan Director of the Board, Oncolys BioPharma, Minato-ku Tokyo, Japan Stanton Gerson, M.D. Director, University Hospitals Ireland Cancer Center and Director, Case Comprehensive Cancer Center; Professor of Medicine, Oncology and environmental Health Sciences; Case Western Reserve University, Cleveland OH, USA Elizabeth A. Grimm, Ph.D. Professor, Department of Experimental Therapeutics; Francis King Black Memorial Professor of Cancer Research; Deputy Head for Research Affairs, Division of Cancer Medicine; and Co-Director, Melanoma Research Program;

Contributors

xi

The University of Texas MD Anderson Cancer Center, Houston, TX, USA Andrew R. Haas, M.D., Ph.D. Assistant Professor, Pulmonary, Allergy, and Critical Care Division, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia PA, USA Brett Hall, Ph.D. Team Lead Biomarkers, Division of Janssen Pharmaceutica, Ortho Biotech Oncology R&D, Oncology Biomarkers Beerse, Johnson & Johnson, Beerse, Belgium Lin Ji, Ph.D. Associate Professor, Department of Thoracic and Cardiovascular Surgery Research, The University of Texas MD Anderson Cancer Center, Houston TX, USA Hong Jiang, Ph.D. Assistant Professor, Department of Neuro-Oncology, The University of Texas MD Anderson Cancer Center, Houston TX, USA Balveen Kaur, Ph.D. Assistant Professor, Dardinger Laboratory for Neuro-oncology and Neurosciences, Department of Neurological Surgery, James Comprehensive Cancer Center and The Ohio State University Medical Center, Columbus OH, USA Shannon Kidd, Ph.D. Graduate Research Assistant, Department of Stem Cell Transplantation and Cellular Therapy, Section of Molecular Hematology, The University of Texas MD Anderson Cancer Center, Houston TX, USA Jae Ho Kim, M.D. Chairman Emeritus, Department of Radiation Oncology, Henry Ford Health System, Detroit MI, USA Kevin Kim, M.D. Associate Professor of Medicine, Department of Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Ann Klopp, M.D. Assistant Professor, Department of Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston TX, USA Fredrick Lang, M.D. Professor, Department of Neurosurgery, The University of Texas MD Anderson Cancer Center, Houston TX, USA

xii

Contributors

Yuan Lin, Ph.D. Student, Division of Hematology/Oncology, Case Western Reserve University, Cleveland OH, USA Xiaoyang Ling, Ph.D. Instructor, Department of Stem Cell Transplantation and Cellular Therapy, Section of Molecular Hematology, Houston TX, USA Gabriel Lopez-Berestein, M.D. Professor, Department of Experimental Therapeutics, Center for RNA Interference and Non-Coding RNA, Department of Cancer Biology, The University of Texas MD Anderson Cancer Center, Houston TX, USA Mei Lu, Ph.D. Senior Research Biostatician, Department of Biostatistics and Research Epidemiology, Henry Ford Health System, Detroit MI, USA Lingegowdda S. Mangala, Ph.D. Research Scientist, USRA, Division of Life Sciences, NASA Johnson Space Center, Houston TX, USA Frank Marini, Ph.D. Associate Professor, Department of Stem Cell Transplantation and Cellular Therapy, Section of Molecular Hematology, The University of Texas MD Anderson Cancer Center, Houston TX, USA Edmund K. Moon, M.D. Post-doctoral Fellow Pulmonary, Allergy, and Critical Care Division, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia PA, USA Edna M. Mora, M.D. Department of Surgery, School of Medicine, University of Puerto Rico, San Juan Puerto Rico; University of Puerto Rico Comprehensive Cancer Center, San Juan Puerto Rico; Adjunct Associate Professor, Department of Surgical Oncology, The University of Texas MD Anderson Cancer Center, Houston TX, USA Benjamin Movsas, M.D. Chairman, Department of Radiation Oncology, Henry Ford Health System, Detroit MI, USA John Nemunaitis, M.D. Co-founder Gradalis, Inc, Executive Medical Director and Oncologist, Mary Crowley Cancer Research Centers, Texas Oncology PA, Baylor Sammons Cancer Center, Dallas TX, USA

Contributors

xiii

Nancy Poindexter, Ph.D. Associate Professor, Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Raphael E. Pollock, M.D., Ph.D. Professor and Head, Division of Surgery; Senator A.M. Aiken, Jr. Distinguished Chair; Director, Sarcoma Research Center; Chair, Department of Surgical Oncology; The University of Texas MD Anderson Cancer Center, Houston TX, USA Richard L. Price, B.S. M.D., Ph.D. Student, Dardinger Laboratory for Neuro-oncology and Neurosciences, Department of Neurological Surgery, James Comprehensive Cancer Center and The Ohio State University Medical Center, Columbus OH, USA Rajagopal Ramesh, Ph.D. Associate Professor, Department of Thoracic and Cardiovascular Surgery Research, The University of Texas MD Anderson Cancer Center, Houston TX, USA Donald D. Rao, Ph.D. Director of Interference Technology, Gradalis, Inc, Dallas TX, USA Jack A. Roth, M.D., F.A.C.S. Professor and Bud Johnson Clinical Distinguished Chair, Department of Thoracic and Cardiovascular Surgery; Professor of Molecular and Cellular Oncology; Director, W.M. Keck Center for Innovative Cancer Therapies; Chief, Section of Thoracic Molecular Oncology; The University of Texas MD Anderson Cancer Center, Houston TX, USA Neil Senzer, M.D. Gradalis, Inc, Scientific Director, Mary Crowley Cancer Research Centers, Texas Oncology PA; Adjunct Associate Professor, Baylor University Institute of Biomedical Studies, Dallas TX, USA Jerry W. Shay, Ph.D. Professor and Vice Chairman, Department of Cell Biology, Associate Director, Simmons Comprehensive Cancer Center at The University of Texas Southwestern Medical Center, Dallas TX, USA Farzan Siddiqui, M.D., Ph.D. Resident, Department of Radiation Oncology, Henry Ford Health System, Detroit MI, USA

xiv

Contributors

Sunil Singhal, M.D. Assistant Professor, Pulmonary, Allergy, and Critical Care Division, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia PA, USA Anil K. Sood, M.D. Professor, Departments of Gynecologic Oncology and Cancer Biology; Co-Director, Center for RNA Interference and Non-Coding RNA; Co-Director, Blanton-Davis Ovarian Cancer Research Program, Department of Cancer Biology, The University of Texas MD Anderson Cancer Center, Houston TX, USA Erika Spaeth, B.S. Graduate Research Assistant, Department of Stem Cell Transplantation and Cellular Therapy, Section of Molecular Hematology, The University of Texas MD Anderson Cancer Center, Houston TX, USA Daniel H. Sterman, M.D. Associate Professor, Pulmonary, Allergy, and Critical Care Division, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia PA, USA Hans Stricker, M.D. Henry Ford Health System, Vattikuti Urology Institute, Detroit MI, USA Matus Studeny, M.D. Clinical Research Director, Boehringer Ingelheim Pharmaceuticals RCV GmbH and Co KG; Division of Medicine and Clinical Development Department; Vienna, Austria Erin E. Thacker, Ph.D. Adjunct Professor at Birmingham-Southern College; Division of Human Gene Therapy, Departments of Medicine, Obstetrics and Gynecology, Pathology, Surgery, University of Alabama at Birmingham, Birmingham AL, USA Keila E. Torres, Ph.D. Consultant, Department of Surgical Oncology, The University of Texas MD Anderson Cancer Center, Houston TX, USA John S. Vorhies, B.A. Consultant, Gradalis, Inc, Dallas TX, USA Zhizong Zeng, M.D. Research Scientist, Department of Stem Cell Transplantation and Cellular Therapy, Section of Molecular Hematology, The University of Texas MD Anderson Cancer Center, Houston TX, USA

Chapter 1

RNAi: A New Paradigm in Cancer Gene Therapy Edna M. Mora, Selanere L. Mangala, Gabriel Lopez-Berestein, and Anil K. Sood

Abstract  RNA interference (RNAi) has revolutionized the field of gene therapy and opened up new opportunities for personalized treatments. However, several challenges remain for gene therapy. Therefore, new approaches for gene regulatory therapies are needed to overcome these challenges. In this chapter, we discuss the clinical significance of the RNAi machinery, clinical applications, delivery systems, off-target effects, imaging, and clinical trials. The remarkable advances in the design, delivery, and understanding of RNAi-based therapeutics predict a bright future for their development as therapeutic agents. It is well established that once a gene is identified as an important player in tumor progression or metastasis, siRNA is a feasible alternative to modulate its expression. Moreover, the development of new delivery systems will further advance the efficiency and localization of siRNA delivery to specific tissues and organs. Concurrently, the development of “intelligent probes” to identify siRNA function in addition to localization will further advance the evaluation of new formulations using imaging techniques. Keywords  RNAi • Delivery systems • Clinical applications • RNAi formulations

A.K. Sood (*) Department of Gynecologic Oncology, U.T.M.D. Anderson Cancer Center, 1155 Herman Pressler, Unit 1362, Houston, TX 77030, USA and Center for RNA Interference and Non-Coding RNA, U. T. M. D. Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030, USA and Department of Cancer Biology, U.T.M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Unit 173, Houston, TX 77030 USA e-mail: [email protected] J.A. Roth (ed.), Gene-Based Therapies for Cancer, Current Cancer Research, DOI 10.1007/978-1-4419-6102-0_1, © Springer Science+Business Media, LLC 2010

1

2

E.M. Mora et al.

1 Introduction Genetic dysregulation is a hallmark of cancer development and progression (Hanahan and Weinberg 2000). It is well known that accumulation of genetic changes in normal cells including mutations, changes in gene copy number, transcription of abnormal genes, and changes in protein translation results in phenotypic changes that support the development of malignant changes followed eventually by the growth of metastatic foci. As a result, these genetic changes provide new opportunities as targets for cancer therapy. Gene regulatory therapy is an attractive alternative to other systemic cancer therapies for several reasons. Systemic therapies such as chemotherapy and hormonal therapy not only affect the growth of primary and metastatic tumors, but also affect normal tissues and organs. As a result, secondary effects (e.g., alopecia, nausea, vomiting, etc.) occur at the expense of the patient’s quality of life. In contrast, therapy designed to target cells with specific genetic abnormalities may translate into a “personalized” approach. For example, FUSI is a tumor suppressor gene that is lost in a large number of lung cancer patients. Treatment of lung cancer patients with a FUSI-gene nanoparticle-based therapy assumes that FUSI expression in the tumor is known prior to therapy (Guinn and Mulherkar 2008). However, several challenges remain for gene therapy, including the limited intratumoral spread of viral particles delivered in the blood stream, development of neutralizing antibodies in the plasma, complement activation, development of non-neutralizing antibodies, unwanted infection of irrelevant cells, and phagocytosis by Kupffer cells (Guinn and Mulherkar 2008). Therefore, new approaches for gene regulatory therapies are needed. Traditionally, gene therapy was conceived as the replacement for “damaged” genes. However, RNA interference (RNAi) has broadened this concept to include the regulation of gene expression. In fact, depending on the specific targets (e.g., IL-8, miRNAs), the RNAi pathway can modulate (increase or decrease) gene function. RNAi has revolutionized the field of gene therapy and opened up new opportunities for personalized treatments. For example, Colombo and colleagues (2008) recently reviewed the role of RNAi in the development of Aurora kinases as a group of antineoplastic drugs. SiRNA was instrumental for documenting the role of Aurora kinases in cell proliferation, and identified these kinases as important targets for cancer therapy. At present, there are several groups pursuing the development of Aurora kinase inhibitors as antineoplastic drugs. While many new therapeutic targets have been identified, it may not be possible to target all of these with conventional approaches such as small molecule inhibitors due to several reasons, including: (1) complex protein structure (e.g., p130Cas) that would be difficult to target with a small molecule inhibitor; (2) nonenzymatic functions, (3) multiple structural domains with independent functions, (4) multiple phosphorylation sites that are critical for function, and (5) incompletely known three-dimensional structure. Moreover, most small molecule inhibitors lack specificity

1  RNAi: A New Paradigm in Cancer Gene Therapy

3

and can be associated with undesirable side effects (Rix and Superti-Furga 2009). Similarly, while monoclonal antibodies have shown promise against specific targets such as VEGF, their use is limited to either ligands or cell surface receptors. The use of siRNA is preferable compared to other approaches such as antisense oligomers due to evidence suggesting longer duration of target inhibition as well as reduced toxicity. The development of RNAi-based gene therapies is possible due to a better understanding of the biological basis of this complex system. The discovery of RNAi by Fire and colleagues (1998) has been seminal to our understanding of cellular transcriptional and translational regulation. Furthermore, the description of specific gene silencing by intracellular delivery of exogenous siRNA by Tushl and colleagues (Elbashir et al. 2001) opened a new era in targeted therapeutics. This process is highly conserved and prevalent in a wide variety of organisms including plants, worms, and vertebrates. Subsequent studies in mammalian cells have opened new possibilities for the use of RNAi-mediated gene silencing against various diseases, including cancer.

2 Clinical Significance of the RNAi Processing Machinery The RNAi machinery is a complex system requiring a series of steps including processing by the key enzymes, Dicer and Drosha (Fig. 1). Evaluation of Dicer in lung cancer patients showed a correlation between low Dicer levels and poor prognosis (Karube et  al. 2005). Similarly, Muralidhar and coworkers (2007) found a correlation between the levels of Drosha in cervical squamous cell carcinoma. Our group has evaluated the expression of Dicer and Drosha in clinical samples from ovarian cancer patients using quantitative RT-PCR analysis (Merritt et al. 2008a). The levels of Dicer and Drosha mRNA correlated with protein levels, and were decreased in 60 and 51% of ovarian cancer specimens, respectively. Low Dicer expression correlated with advanced tumor stage ( p = 0.007), and low Drosha expression with suboptimal surgical cytoreduction ( p = 0.02). High expression of both proteins correlated with increased median survival ( p < 0.001). There were three predictors of reduced disease-free survival: low Dicer expression ( p = 0.02), high grade histologic features ( p = 0.03), and poor response to chemotherapy ( p < 0.001). These data support an active role for Dicer and Drosha in the clinical outcome of ovarian cancer patients. Similar findings were observed in separate cohorts of ovarian, lung, and breast cancer patients. However, in some tumor types, such as, prostate and esophageal cancers, varying results with regard to the effect of Dicer expression on patient outcome have been observed, suggesting that RNAi regulatory processes may be tumor-specific (Chiosea et  al. 2006; Sugito et  al, 2006). Characterization of the specific RNAi components in different tumor types will further guide the development of effective cancer gene therapies.

4

E.M. Mora et al.

Fig.  1  The RNA-interference Cascade in humans. Long precursor microRNA (miRNA) segments, called pri-miRNA, are first cleaved in the nucleus by Drosha, a RNase III endonuclease, into segments of approximately 70 nucleotides each (called pre-miRNA). Transportation into the cytoplasm by means of exportin five leads to cleavage by Dicer, another RNase III endonuclease, which produces mature miRNA segments. Host degradation of messenger RNA (mRNA) and translational repression occurs after miRNA binds to the RNA-induced silencing complex (RISC). Cytoplasmic long doubled-stranded RNA (dsRNA) is cleaved by Dicer into small interfering RNA (siRNA), which is incorporated into RISC, resulting in the cleavage and degradation of specific target mRNA

3 Clinical Application of RNAi The use of siRNA for therapy in clinical settings is gradually being evaluated. Local delivery of siRNA can be achieved in anatomical areas accessible to direct injection. This strategy delivers high local concentrations of siRNA with minimal systemic degradation. For example, the use of naked siRNA to the eye for the treatment of macular degeneration (Whitehead et al. 2009) has been achieved successfully. Clinically, this strategy of local delivery may be useful for the treatment of tumors with low likelihood of distant spread. Local delivery of siRNA has been tested in several organs including lung (Durcan et al. 2008), urinary bladder, brain, and cervix. Garbuzenko and colleagues (2009) compared the delivery of free siRNA when given by the intravenous or intratracheal routes in non-tumor-bearing mice. In vitro and in vivo data suggested that intratracheal injection resulted in higher (4 to 7.5 times) delivery of siRNA to the lungs compared to intravenous delivery. Similarly, Oliveira Reis and coworkers

1  RNAi: A New Paradigm in Cancer Gene Therapy

5

(2009) proposed the use of siRNA for the treatment of bladder cancer. In addition, the local delivery of siRNA has been tested in an in vivo model of glioblastoma multiforme (Crzelinski et al. 2006). Finally, the topical application of siRNA in the genital tract was shown to be effective in the treatment of herpes simplex virus lesions in preclinical studies (Paliser et al. 2006). Given that cancer is mostly a systemic disease, effective and safe systemic delivery of siRNA is essential to provide a significant impact on cancer therapy. However, systemic injection of naked siRNA results in rapid degradation. In addition, the doses of naked siRNA needed to achieve target modulation systemically are too high and can elicit toxicities and off-target effects. SiRNA is a high molecular weight polyanionic molecule that requires a delivery system to be able to penetrate into the cell. Nuclease digestion in the circulation and at intracellular sites is a potential barrier for the clinical applications of siRNA (Fernandez et  al. 2007). Chemical modifications of one or more components of the siRNA molecule have been attempted in order to enhance cell uptake, avoid nuclease degradation, decrease immunogenicity and improve the therapeutic index of the drug (Table 1). In addition to modifications of the sugars, phosphates and bases, modifications to the duplex architecture have been evaluated. These include blunt-end duplexes, 25/27 mer dicer-substrates, sisiRNA, single antisense strand, hairpin (shRNA), and dumbbell (shRNA). Of these, sisiRNA showed reduced off-target effects and increased potency (Watts et al. 2008). However, RNAi chemical modifications may result in decreased effectiveness and the development of toxic metabolites as a result of degradation of a non-natural molecule (Li and Shen 2009). The first generation of effective systemic delivery methods for siRNA was based on lipid formulations. These include liposomes, micelles, emulsions, and solid lipid nanoparticles (Table  2). Liposomes are lipid structures, characterized in most instances by an assembly of a phospholipid bilayer membrane and an aqueous internal compartment (Sanguino et al. 2008). Liposomes constitute an effective carrier for in vivo siRNA delivery. It has been shown that the liposomes protect siRNA from degradation, increase its half-life, and prolong target modulation following systemic delivery. However, some liposomal preparations, especially cationic lipids, may induce the release of oxygen radicals, complement activation, and generate an interferon response (Dass 2002). In addition, they can alter the gene expression of treated cells when analyzed by microarray-based gene expression profiling. Conversely, neutral liposomes such as DOPC have shown low toxicity with no changes in hematological parameters (Gutierrez-Puente et al. 2003). We have extensively used the 1,2-dioleoyl-sn-glycero-phosphatidylcholine (DOPC) nanoliposomes to deliver siRNA in vivo for therapeutic applications against key targets in ovarian and other cancers (Landen et  al. 2005; Halder et  al. 2006; Gray et  al 2008; Merritt et  al. 2008b). Pegylated (PEG) liposomes may increase the half-life of siRNA and decrease complement activation. However, administration of pegylated liposomes can contribute to toxicity, which depends on the exact composition, charge density, and primary amine groups in the formulation (Van Mil et al. 2009). Biodegradable and nontoxic polymer-based delivery systems have been used recently for siRNA delivery. Positively charged polymers are potentially viable carriers

6

E.M. Mora et al.

Table 1  SiRNA chemical modifications General category Chemical modification Sugars 2¢-OH-methylation 2¢-methoxyethyl;2¢-O-allyl 2,4, dinitrophenyl ethers (2¢-O-DNP) 2¢-F-RNA

Phosphates

Bases

DNA-RNA with no electronegative substitutes at 2¢ positions 4¢-S-RNA 4¢S-2¢-FANA Insertion of LNP nucleotide Phosphorothioate (PS) linkages Boranophosphates 5-Br-Ura and 5¢-I-Ura Diaminopurine Thiouracil Pseudouracil

Termini

5-methylation of pyrimidines Difluorotoluyl base Dihydrouracil Antisense strand: 5¢-phosphorylation Sense strand: inverted abasic end cap Sense strand: fluorescent dyes or biotin Sense strand: membrane-penetrating peptides Sense strand: lipophilic groups Sense strand: 5-8 dA and dT units

Effects Increases binding affinity and nuclease stability Reduced activity Higher binding affinity, nuclease resistance, high potency Increased serum stability; increased binding affinity Reduce off-target effects Increase nuclease stability Low binding affinity Increased binding affinity Same or low potency; cytotoxicity Increased potency; increased nuclease stability Reduced potency Reduced potency Increase binding affinity, increased potency and specificity Increased binding affinity, increased potency and specificity Used with sugar modifications Same potency Lower binding affinity High potency Helps exonuclease stability Useful for imaging studies Helps delivery Helps delivery Improves delivery

for the negatively charged DNA and RNA. The most commonly used carriers are cationic polymers like polyethyleneimine (PEI) and cyclodextrin. The conjugation of PEI with RNA/DNA molecules results from electrostatic attraction. These particles show high transfection efficiency coupled with low toxicity. Natural cationic polymers include chitosan and atellocollagen (Oh and Park 2009). Chitosan is a biodegradable polysaccharide that has low toxicity and high transfection efficiency (Mangala et al. 2008). Its dual role as a base and salt makes it ideal for interaction with negatively and positively charged molecules. Given the multiple examples of effective in vivo siRNA delivery with polymeric delivery systems, further development appears to be warranted. An alternative strategy for in vivo delivery of siRNA is the direct conjugation of siRNA with specific molecules to enhance intracellular delivery. Several formulations

1  RNAi: A New Paradigm in Cancer Gene Therapy Table 2  Systemic delivery systems General category Description Lipid complex Cationic liposomes Neutral liposomes (DOPC) Lipoplexes Stable nucleic acid-lipid particles Conjugated Polymer-functional polymers peptides Polymer-lipophilic molecules (e.g., cholesterol) Polymer-PEG Aptamers and antibodies Cationic Chitosan polymers Atellocollagen

7

Natural vs. synthetic siRNA/shRNA Synthetic siRNA Synthetic siRNA Synthetic Synthetic

siRNA siRNA

Synthetic

siRNA

Synthetic

siRNA

Synthetic Synthetic Natural

siRNA siRNA/shRNA

Natural

siRNA/shRNA

PegylatedCyclodextrin Poly-l-lysine Transkingdom RNA plasmid

Synthetic Synthetic Synthetic Synthetic

siRNA/shRNA siRNA/shRNA siRNA/shRNA shRNA

Viral vectors

Adenovirus Bachulavirus Herpes Lentivirus

Bacterialderived carriers Peptide transduction domains (PTD)

Minicells

Natural Natural Natural Natural Natural Synthetic

shRNA shRNA shRNA shRNA shRNA siRNA/shRNA

Penetratin

Synthetic

siRNA

Transportan

Synthetic

siRNA

Amphipathic peptide

Synthetic

siRNA

Poly-arginine

Synthetic

siRNA

Plasmid vectors

Comments

Biodegradablenontoxic Biodegradablenontoxic Cytotoxicity

Used in nondividing and difficult to transfect cells

High efficiency delivery to primary cells; low cytotoxicity High efficiency delivery to primary cells: low cytotoxicity High efficiency delivery to primary cells High efficiency delivery to primary cells

8

E.M. Mora et al.

have been developed with the goal of delivering siRNA into specific cell types (e.g., tumor cells, endothelial cells, etc.). Folate-conjugated nanoparticles may allow specific delivery since many tumor cells overexpress the folate receptor (Gosselin and Lee 2002). Other conjugated formulations have used transferrin as a target molecule because tumor cells overexpress the transferrin receptor (Hogrefe et al. 2006). This type of system is very effective and has shown low toxicity. In addition, a lipidoid-siRNA delivery system that preferentially targets liver cells has also been developed (Akinc et  al. 2009a). Langer and colleagues showed that 90% of this formulation is delivered to the liver and can induce reversible but prolonged gene silencing without the loss of activity following repeated administrations (Akinc et al. 2009b). Finally, other targeted approaches have been developed using RGDbased formulations, which aim to exploit the selective expression of the avb3 integrin in the tumor vasculature. A recent novel development in siRNA delivery involves the use of minicells as carriers of siRNA in tumor cells (Karagiannis and Anderson 2009; MacDiarmid et al. 2009). Minicells are produced from Salmonella typhimurium by derepressing cryptic polar sites of cell fusion through the inactivation of genes controlling normal bacterial cell division. The minicells can be loaded with siRNA or chemotherapeutic drugs, and may be very useful for overcoming drug resistance. Dowdy and associates have recently reported the delivery of siRNA to primary cells using peptide transduction domains (PTD) (Eguchi et al. 2009; Eguchi and Dowdy, 2009). SiRNA was coupled to PTD by covalent and noncovalent bonds. Uptake was mainly through the endosomal pathway. Developing PTDs with increased endosomalytic properties may be useful for achieving biological effects at low concentrations. An alternative to the delivery of naked siRNA using carrier-dependent delivery methods or chemically modified siRNA is the delivery of plasmids encoding siRNA or shRNA under the control of different promoters (e.g., RNA polymerase II- or RNA polymerase III-dependent promoters). Likewise, inducible promoters, like tetracycline and metallothionein have also been used. Finally, the viral delivery of siRNA and shRNA permits the delivery of siRNA and shRNA in cells that are difficult to transfect or in nondividing cells. The most widely used viral vectors for shRNA delivery include adenovirus, adeno-associated virus, lentivirus, retrovirus, herpes and baculovirus vectors. They vary in their transfection efficiency, capacity to integrate into the genome, and the duration of expression.

4 Off-Target Effects A potential barrier for the development of siRNA as cancer therapy is the stimulation of off-target effects upon siRNA administration. Unintended effects on gene expression mediated by RNAi are termed “off-target effects”. Off-target effects can be classified as specific or nonspecific. Specific off-target effects are mediated by partial sequence complementarity of the RNAi constructs to mRNAs other than the

1  RNAi: A New Paradigm in Cancer Gene Therapy

9

intended target. Nonspecific off-target effects include a variety of immune and toxicity-related effects that are intrinsic to the siRNA construct itself or its delivery vehicle (Robbins et al. 2009). Judge and colleagues (2005) showed that both siRNA and shRNA constructs with complementarity in the “seed region” can produce the same off-target expression profiles, even across cell lines, independent of the delivery method. Even limited sequence (seven nucleotides) siRNA:mRNA complementarity with the intended target can produce off-target suppression (Akinc et al. 2009b). In addition, some sequences, such as those containing certain motifs including 5¢-UGUGU-3¢, and 5¢-GUCCUUCAA-3¢ appear to be particularly associated with immune activation (Sioud 2008). To avoid these effects, several programs have been designed to predict the efficiency and specificity of siRNA molecules (Li et al. 2008). Some off-target effects can be minimized or eliminated by careful and strategic design of the siRNA sequence. There are several search engines with different criteria that allow the design and validation of siRNA sequences (Pushparaj et al. 2008). For example, AsiDesigner is a freely accessible web tool (http://sysbio.kribb. re.kr/AsiDesigner/) that provides stepwise off-target searching with BLAST and FASTA algorithms (Kim and Rossi 2009). In addition, validated sequences have been characterized and collated by the scientific community in (http://www.ncbi.nlm. nih.gov/projects/genome/RNAi). However, due to the long running time needed to predict secondary structures of target mRNA, some of these programs are mostly based on sequence characteristics, which limits their capacity to predict some off-target effects that result from homology between the RNA’s secondary structure. Nonspecific effects are more difficult to predict than specific effects. By far, the immunological effects of siRNAs are the most studied off-target effects. Systemic delivery of siRNA duplexes can activate the innate immune response, resulting in increased levels of inflammatory cytokines such as tumor necrosis factor alpha, interleukin-6, and interferon (Kleinman et  al. 2008). This response can be either Toll-like receptor (TLR)-mediated or non-TLR mediated (Vankoningsloo et  al. 2008). The TLR-mediated response induced by siRNA is mainly mediated through TLR-7, TLR-8, and TLR-3. The TLR-7 and TLR-8 ligands include siRNA and RNA oligonucleotides, while TLR-3 recognizes only duplex siRNA. TLR-3 is expressed in primary human endothelial, epithelial, and fibroblast cells. Activation of TLR-3 induces the production of interferon gamma and IL-12. Recently, Kleinman et al. (2008) showed that the inhibition of angiogenesis in a choroidal neovascularization model may be a nonspecific siRNA effect. Even siRNAs targeting nonmammalian genes or noncoding genes (e.g., GFP) were able to induce an antiangiogenesis effect through the activation of TLR3 (Oh and Park 2009). Other cytoplasmic proteins such as ds-RNA-binding protein kinase and RIG1 can also mediate nonspecific immune responses in the cytoplasm upon exposure to siRNA. Structural features such as asymmetrical siRNA can activate RIG1 mediated IFN activation. The purity and quality of siRNA batches can also influence the induction of IFN response, even in in vitro models. Nonhematological cell lines do not express TLR-7/8, but the cytoplasmic proteins are widely expressed in mammalian cells. Cell culture conditions, cell type, duration of transfection, and posttransfection time before analysis could impact the results of siRNA experiments (Robbins et al. 2009).

10

E.M. Mora et al.

Immune stimulation by siRNA can induce not only the production of inflammatory cytokines but also the production of antibodies against components of the delivery method. This effect decreases the efficiency of transfection. Other examples of nonspecific off-target effects include changes in telomeres and nontarget genes. Ho-Sze and colleagues (2008) showed that siRNA can induce significant reversible changes in telomeres. They found distinct heterochromatin features after siRNA treatment such as the increased binding of Argonaute-1, telomeric-repeatbinding factor-1, heterochromatin protein-1b, histoneH3 hypermethylation, association of chromosome ends with an unidentified RNA, downregulation of the expression of a transgene inserted adjacent to the telomere, increased expression of heterogeneous high molecular weight RNA containing telomeric repeat sequences, and formation of transient nuclear foci. Interestingly, other studies identified siRNA-mediated activation of nontarget genes by noncoding siRNA. Tschuch and colleagues (2008) found that GFP-siRNA inhibits the GFP gene and also deregulates a set of endogenous target genes. This effect was dependent on the amount of siRNA used, as well as the cell type. Using genome-wide expression profiling, they identified several genes that were consistently deregulated. Based on the above analysis, tools that can predict and identify significant offtarget effects elicited by siRNA are highly desirable. Specifically, it is important to assess nontargeted gene and immune stimulation upon exposure to siRNA. Among the various alternatives, microarrays have been used successfully to identify siRNAinduced off-targets effects (Anderson et al. 2008). Another alternative (vanDongen et al. 2008) is the use of Sylamer technology to detect miRNA and siRNA off target signals from expression data. It is freely available, and simple to use, making it a good alternative for genome-scale studies. There is considerable controversy as to how to assess immunostimulation by siRNAs. ELISA kits for the quantification of IFN and inflammatory cytokines are widely available. For in  vitro studies, the detection of IFN-b, IL-6 and IL-8 in the supernatant of culture cells can be used as a measurement of siRNA-mediated activation through the RIG1.MDA5/PKR pathways. In vivo, the measurement of IFN and cytokines needs to be assessed in a continuous fashion since the response can be transient and varies widely between different cell lines. Robbins and coworkers (2009) suggested the use of IFN-inducible IFITI (p56) mRNA as a more sensitive method for assessing immune stimulation than plasma cytokines.

5 RNAi Imaging: Biodistribution and Target Modulation One of the most critical issues for the biological evaluation of protein downregulation/ biodistribution and clinical development of RNAi therapies is the need for noninvasive assessment of siRNA delivery to the tissues of interest. Initially, bioluminescent techniques were used to evaluate the kinetics of siRNA-mediated gene silencing in live cells and live animals. For example, Barlett and colleagues (2006) demonstrated that the combination of bioluminescent and mathematical models provide useful insights into imidazole-modified cyclodextrin-containing polycations mixed with

1  RNAi: A New Paradigm in Cancer Gene Therapy

11

amantane-PEG conjugate and adamantane-PEG-Transferrin. However, the use of nanoparticles for siRNA delivery imposes another level of complexity to the assessment of biodistribution because macromolecules alone tend to accumulate within tumors after systemic delivery, also called the enhanced permeability and retention effect (Matsumura et al. 1987). As a result, several researchers proposed the use of multimodality imaging approaches to evaluate biodistribution and the effectiveness of nanoparticle delivery of siRNA. For example, Barlett and colleagues (2007) used a combination of bioluminescence with positron electron tomography (PET)/computer tomography (CT) to assess the biodistribution of cyclodextrin-containing polycations-siRNA-transferrin (CD-siRNA-Tf ) nanoparticles. They found that the biodistribution of cyclodextrin-containing nanoparticles (CD-siRNA) and CD-siRNATf was the same based on PET data. However, bioluminescence indicated that CD-siRNA-Tf reduced the luciferase activity to 50% of CD-siRNA controls. Other studies have used magnetic resonance (MR) imaging and bioluminescence to assess cationic liposome-mediated delivery of siRNA (Mikhaylova et  al. 2009). In these studies, fluorescently labeled COX-2-specific siRNA cationic liposomes containing MR contrast agents were used to assess the delivery of siRNA in vitro and in vivo. A new approach to assess the efficiency and biodistribution of siRNA has been the development of probes linked to the siRNA molecule. For example, Medarova and colleagues (2007) developed a probe in which magnetic nanoparticles labeled with a near-infrared dye are covalently linked to siRNA. This probe also contains a membrane trasnslocation peptide for enhancing intracellular delivery. They showed tumor uptake of the probe by MRI and optical imaging in glioma and colon cancer models. Similarly, So and associates (2008) demonstrated siRNA-mediated gene silencing in vivo with a new application of a ribozyme-based reporter. In this application, the reporter mRNA is attached to the 3¢ exon and the internal guiding sequence and antisense sequence are attached to the 5¢ end of the molecule. After attachment of the antisense sequence to the target RNA, the ribozyme splices the reporter into the target RNA in-frame, resulting in a fusion RNA consisting of the reporter and part of the target. After translation of the fusion RNA, the reporter is activated. This system is compatible with MRI and PET technology. However, this method does not allow subcellular resolution or the evaluation of fast RNA dynamics.

6 Development of RNAi-Based Gene Therapy: Clinical Trials In 2008, the first cancer patient completed the first successful dosing cycle with siRNA for the treatment of cancer in a clinical trial. This patient was administered four doses of Calando’s CALAA-01, which is an siRNA that targets ribonucleotide reductase. This Phase I trial (NCT-00689-65) was designed to deliver siRNA systemically using transferrin-targeted CD nanoparticles in patients with nonresectable or metastatic solid tumors (NIH clinical trial 597982). The complex formulation contains four components: a duplex synthetic nonchemically modified siRNA, a cyclodextrin-containing polymer (AD), a stabilizing agent (PEG) and a cell-targeting

12

E.M. Mora et al.

agent (transferrin). The cationic polymer interacts electrostatically with anionic siRNA to assemble into nanocomplexes below approximately 100 nm in diameter that protect the siRNA from nuclease degradation in serum. The siRNA-containing nanocomplexes are targeted to cells that overexpress the transferrin receptor. Upon reaching a target cell, transferrin binds to the transferrin receptor on the cell surface. The siRNA-containing nanocomplex enters the cell by endocytosis. Inside the cell, there is unpacking of the siRNA, permitting the function of the RNAi. In 2010, Davis and colleagues reported the first evidence for effective regulation of the M2 subunit ribonucleotide reductase (RRM2) in melanoma patients refractory to standard therapy. They showed that after intravenous administration of MMR2-siRNA, the nanoparticles were localized in tissue biopsies. Furthermore, there was downregulation of MMR2 at the RNA and protein levels. These results show specific gene regulation by a RNAi mechanism. Other details of the trial are not available at this time. A second Phase I clinical trial (NCT 00672542) lead by the Duke Comprehensive Cancer Center is actively recruiting metastatic melanoma patients to assess the safety of vaccination with melanoma tumor-associated antigen-encoding RNAtransfected in mature dendritic cells (NIH clinical trial 595941). The dendritic cells are derived from monocytes that have been either untreated, transfected with control siRNA, or transfected with siRNA, targeting the inducible immunoproteosome beta subunits LMP2, LMP7, and MECL1. A combination of siRNAs encoding melanoma tumor-associated antigens MART-1, tyrosinase, gp100, and MAGE-3 will be utilized for dendritic cells transfection. The vaccine will be administered by intradermal injection in the extremities. Clinical and laboratory toxicities will be characterized for each arm of the study. As a secondary objective, the study will assess antimelanoma immune responses as well as clinical responses induced by vaccination with this dendritic cell-based product. It is expected that in the next 2 years, the number of clinical trials evaluating RNAi strategies for clinical applications in different types of cancers is expected to increase substantially.

7 Future Development The remarkable advances in the design, delivery, and understanding of RNAi-based therapeutics predict a bright future for their development as therapeutic agents. It is well established that once a gene is identified as an important player in tumor progression or metastasis, siRNA is a feasible alternative to modulate its expression. Moreover, the development of new delivery systems will further advance the efficiency and localization of siRNA delivery to specific tissues and organs. Concurrently, the development of “intelligent probes” to identify siRNA function and localization will further advance the evaluation of new formulations using imaging techniques. Acknowledgements  Portions of this work were supported by NIH grants (CA 110793, 109298, CA128797, and RC2GM092599), the Ovarian Cancer Research Fund, Inc. (Program Project Development Grant), U. T. M. D. Anderson Cancer Center SPORE (P50CA083639), the Zarrow

1  RNAi: A New Paradigm in Cancer Gene Therapy

13

Foundation, the Marcus Foundation, the Betty Anne Asche Murray Distinguished Professorship, and the EIF Foundation to A.K.S. E.M. was supported by the U. T. M. D. Anderson Cancer Center Ovarian Cancer Spore (P50CA083639), Ovarian Cancer Program of the Department Of Defense (OC-073399), the National Cancer Institute Partnership Program (U54 96297, U54 96300) and a grant from the Puerto Rico Comprehensive Cancer Center.

References Akinc A, Goldberg M, Qin J, et al (2009a) Development of lipidoid-siRNA formulations for systemic delivery to the liver. Mol Ther 17(5): 872–879. Akinc A, Zumbuelh A, Goldberg M, et al. (2009b) A combinatorial library of lipid-like materials for delivery of RNAi therapeutics. Nat Biotechnol 26(5): 561–569. Anderson E, Boese Q, Khvorova A, et  al. (2008) Identifying siRNA-induced off targets by microarray analysis. Methods Mol Biol 442: 45–63. Bartlett DW, Davis ME. (2006) Insights into the kinetics of siRNA-mediated gene silencing from live-cell and live-animal bioluminescent imaging. Nucleic Acids Res 34(1): 322–333. Bartlett DW, Su H, Hildebrandt IJ, et al. (2007) Impact of tumor-specific targeting on the biodistribution and efficiency of siRNA nanoparticles measured by multimodality in vivo imaging. Proc Natl Acad Sci USA 104(39): 15549–15554. Chiosea S, Jelezcova E, Chandran U, et  al. (2006) Up-regulation of dicer: a component of the MircoRNA machinery in prostate adenocarcinoma. Am J Pathol 169(5):1812–1820. Colombo R, Moll J (2008) Target validation and biomarker identification in oncology. Mol Diagn Ther 12(2): 71–76. Crzelinski M, et  al. (2006) RNA interference mediates RNA silencing of pleiotrophin through polyethylenimine complexed small interfering RNAs in  vivo exerts antitumoral effects in glioblastoma xenografts. Hum Gen Ther 17: 751–766. Dass, CR. (2002) Vehicles for oligonucleotide delivery to tumors. J Pharm Pharmacol 54 (1): 593–627. Davis ME, Zukerman JE, et al. (2010) Evidence of RNAi in humans from systemically administred siRNA via targeted nanoparticles, Nature 464(7291):1067–1070. Durcan N, Murphy C, Cryan S-A. (2008) Inhalable siRNA: potential as a therapeutic agent in the lungs. Mol Pharm 5(4): 559–566. Eguchi A, Dowdy SF. (2009) siRNA delivery using peptide transduction domains. Trends Pharmacol Sci 30(7): 341–345. Eguchi A, Maede BR, Chang Y-C, Fredrickson CT, et al. (2009) Efficient siRNA delivery into primary cells by a peptide transduction domain-dsRNA binding domain fusion protein. Nat Biotechnol 27(6): 567–571. Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tushl T. (2001) Duplexes of 21-nucleotide RNA’s mediate RNA interference in cultured mammalian cells. Nature 411: 494–498. Fernandez A, Sanguino A, Peng Z, et al. (2007) An anticancer C-kit kinase inhibitor is reengineered to make it more active and less cardiotoxic. J Clin Invest 117(12): 4044–4054. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. (1998) Potent and specific genetic interference by double stranded RNA in Caenorhabditis elegans. Nature 391: 806–811. Garbuzenko OB, Saad M, Betigeri S, et  al. (2009) Intratracheal versus intravenous liposomal delivery of siRNA, antisense oligonucleotides and anticancer drug. Pharm Res 26(2): 382–394. Gosselin MA, Lee RJ. (2002) Folate receptor-targeted liposomes as vectors for therapeutic agents. Biotechnol Annu Rev 8: 103–133. Gray MJ, VanBuren G, Dallas NA, et al. (2008) Therapeutic targeting of neuropilin-2 by siRNA reduces in vivo tumor progression in colorectal carcinoma cells in a murine orthotopic model. J Natl Cancer Inst 100: 109–120.

14

E.M. Mora et al.

Guinn BA, Mulherkar R. (2008) International progress in cancer gene therapy. Cancer Gene Ther 15: 765–775. Gutierrez-Puente Y, Tari AM, Ford RJ, et al. (2003) Cellular pharmacology of P-ethoxy antisense oligonucleotides targeted to Bcl-2 in a follicular lymphoma cell line. Leuk Lymphoma 44: 1979–1985 Halder J, Kamat AA, Landen CN, et  al. (2006) Focal adhesion kinase silencing augments docetaxel-mediated apoptosis in ovarian cancer cells. Clin Cancer Res 12(16): 4916–4924. Hanahan D, Weinberg RA. (2000) The hallmarks of cancer. Cell 100: 57–70. Hogrefe RI, Lebedev AV, Zon G, et  al. (2006) Chemically modified short interfering hybrids (siHYBRIDS): nanoimmunoliposomes delivery in  vitro and in  vivo for RNAi of Her-2. Nucleosides Nucleotides Nucleic Acids 25(8): 889–907. Ho-Sze CY, Murnane JP, Ying Yeung AK, et al. (2008) Telomeres Acquire distinct heterochromatin characteristics during siRNA-Induced RNA interference in mouse cells. Curr Biol 18(3): 183–187. Judge AD, Sood V, Shaw JR, et  al. (2005) Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA. Nat Biotechnol 23: 457–462. Karagiannis ED, Anderson DG. (2009) Minicells overcome tumor drug-resistance. Nat Biotechnol 27(7): 620–621. Karube Y, Tanaka H, Osada H, et  al. (2005) Reduced expression of dicer associated with poor prognosis in lung cancer patients. Cancer Sci 96(2): 111–115. Kim DH, Rossi JJ. (2009) Overview of gene silencing by RNA interference. Curr Protoc Nucleic Acid Chem 16.1.1–16.1.10. Kleinman ME, Yamada K, Takeda A, et al. (2008) Sequence- and target-independent angiogenesis suppression by siRNA via TLR3. Nature 452(3): 591–598. Landen CN, Chavez-Reyes A, Bucana C, et  al. (2005) Therapeutic AphA2 gene targeting by in vivo liposomal siRNA delivery. Cancer Res 65(15): 6910–6918. Li L, Shen Y. (2009) Overcoming obstacles to develop effective and safe siRNA therapeutics . Expert Opin Biol Ther 9(5): 609–619. Li Z, Fortin Y, Shen S-H. (2008) Forward and robust selection of the most potent and noncellular toxic siRNA from RNAi libraries. Nucleic Acids Res 37(1) doi:1-.1093/nar/gkn953. MacDiarmid JA, Amaro-Mugridge NB, Madrid-Weiss J, et  al. (2009) Sequential treatment of drug-resistant tumors with targeted minicells containing siRNA or a cytotoxic drug. Nat Biotechnol 27(7): 643–654. Mangala LS, Han HD, Lu C, et al. (2008) In vivo vascular and tumor cell gene silencing with chitosan nanoparticles in ovarian carcinoma. The 99th Annual Meeting of the American Association for Cancer Research, San Diego, CA. Matsumura Y, Oda T, Maeda H. (1987) General mechanism of intratumor accumulation of macromolecules: advantages of macromolecular therapeutics. Gan To Kagaku Ryoho 14: 821–829. Medarova Z, Pham W, Farrar C, et al. (2007) In vivo imaging of siRNA delivery and silencing in tumors. Nat Med 13(3): 372–377. Merritt WM, Lin YG, Han LY, et al. (2008a) Dicer, drosha, and outcomes in patients with ovarian cancer. N Engl J Med 359(25): 2641–2650. Merritt WM, Lin YG, Spannuth WA, et al. (2008b) Effects of IL-8 targeted therapy with liposome incorporated siRNA on ovarian cancer growth. J Natl Cancer Inst 100: 359–372. Mikhaylova M, Stasinopoulos I, Kato Y, et al. (2009) Imaging of cationic multifunctional liposome-mediated delivery of COX-2 siRNA. Cancer Gene Ther 16: 217–226. Muralidhar B, Goldstein LD, Ng G, et al. (2007) Global microRNA profiles in cervical squamous cell carcinoma depend on Drosha expression levels. J Pathol 212: 368–377. NIH http://www.cancer.org/search/viewclinicaltrials.aspx?cdrid=595941&version=healthprofe sionals NIH http://www.cancer.org/search/viewclinicaltrials.aspx?cdrid=597982&version=health profesionals Oh Y-K, Park TG. (2009) siRNA delivery systems for cancer treatment. Adv Drug Deliv Rev 61: 850–862.

1  RNAi: A New Paradigm in Cancer Gene Therapy

15

Oliveira Reis L, Campos Pereira T, Favaro WJ, et al (2009) Experimental animal model and RNA interference: a promising association for bladder cancer research. World J Urol 27: 353–361. Paliser D, et  al. (2006) An siRNA-based microbicide protects mice from lethal herpes simplex virus-2 infection. Nature 439: 89–94. Pushparaj PN, Aarthi JJ, Manikandan J, et al.(2008) siRNA, miRNA, and shRNA: in vivo applications. J Dent Res 11: 992–1003. Rix U, Superti-Furga G. (2009) Target profiling of small molecules by chemical proteomics. Nat Chem Biol 5(9): 616–624. Robbins M, Judge A, MacLachlan I. (2009) siRNA and innate immunity. Oligonucleotides 19(2): 89–101. Sanguino A, Lopez-Berestein G, Sood AK (2008) Strategies for in vivo siRNA delivery in cancer. Mini Rev Med Chem 8: 248–255. Sioud M. (2008) Does the understanding of immune activation by RNA predict the design of safe siRNA’s? Front Biosci 13: 4379–4392. So M-K, Gowrishankar G, Hasegawa S, et al. (2008) Imaging target mRNA and siRNA-mediated gene silencing in vivo with ribozyme-based reporters. Chembiochem 9: 2682–2691. Sugito N, Ishiguro H, Kuwabara Y, et al. (2006) RNASEN regulates cell proliferation and affects survival in esophageal cancer patients. Clin Cancer Res 19(6): 454–458. Tschuch C, Schulz A, Pscherer A, et al. (2008) Off-target effects of siRNA specific for GFP. BMC Mol Biol 9: 60, doi: 10.1186/1471-2199-9-60. Van Mil A, Doevendans PA, Sluijter JPG. 2009 The potential of modulating small rna activity in vivo. Mini Rev Med Chem 9: 235–248. vanDongen S, Abreu-Goodger C, Enright AJ. (2008) Detecting microRNA binding and siRNA off-target effects from expression data. Nat Methods 5(12): 1023–1025. Vankoningsloo S, de Longueville F, Evrard S, et al. (2008) Gene expression silencing with “specific” small interfering RNA goes beyond specificity- a study of key parameters to take into account in the onset of small interfering RNA off-target effects. FEBS J 275: 2736–2753. Watts JK, Deleavey GF, Damha MJ. (2008) Chemically modified siRNA: tools and applications. Drug Discov Today 13(19/20): 842–854. Whitehead KA, Langer R, Anderson DG (2009) Knocking down barriers: advances in siRNA delivery. Nat Rev Drug Discov 8: 129–136.

Chapter 2

Gene-Based Therapy for Cancer: Brain Tumors Hong Jiang and Juan Fueyo

Abstract  Malignant gliomas are a devastating disease with dismal prognosis. Gene-based therapies hold the promise for more specific and efficacious intervention. Clinical experiences have revealed the limits of gene transfer by replicationdeficient adenoviral or retroviral vectors. The problem has been partially improved with the bystander effect of suicide gene therapy. Replication-competent oncolytic viruses are expected to bridge the vector gap since the viruses replicate and spread the progeny to the adjacent cancer cells. The oncolytic viruses discussed in this review represent either genetically engineered (adenovirus, herpes simplex virus-1 and measles virus) or naturally occurring (reovirus and newcastle disease virus) strains of viruses that exhibit relatively selective replication in tumor cells. Clinical trials demonstrate that these viruses are well tolerated in glioma patients. Thereafter, certain challenges need to be addressed in future clinical studies to achieve desirable efficacy. Keywords  Glioma • Oncolytic virus

1 Introduction Malignant gliomas account for approximately 70% of the 22,500 new cases of malignant primary brain tumors that are diagnosed in adults in the US each year (Wen and Kesari 2008). Although relatively uncommon, malignant gliomas are associated with disproportionately high morbidity and mortality. Despite optimal treatment, the median survival is only 12–15 months for patients with glioblastomas

J. Fueyo (*) Department of Neuro-Oncology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Unit 1002, Houston, TX 77030, USA e-mail: [email protected] J.A. Roth (ed.), Gene-Based Therapies for Cancer, Current Cancer Research, DOI 10.1007/978-1-4419-6102-0_2, © Springer Science+Business Media, LLC 2010

17

18

H. Jiang and J. Fueyo

and 2–5 years for patients with anaplastic gliomas (Wen and Kesari 2008). The clinical outcome of patients with glioblastomas almost remained the same for the past decade (Furnari et  al. 2007). According to the CRS Brain & CNS Cancers Workshop, June 2007, high rates of mortality convert malignant glioma into the first leading cause of cancer death in children, the third leading cause of cancerrelated death among men 15–54 years of age and the fourth leading cause of death for women 15–34 years of age. Malignant gliomas are highly resistant to conventional therapies (i.e., surgery, radiation, and chemotherapy). Tumor recidivism followed by unstoppable progression is the rule. Thus, more efficacious and specific therapies are urgently needed. As the genetic basis of brain tumors has been delineated, many molecular defects have been tagged as promising targets for therapy. Viral vectors have been used in preclinical and clinical studies to transfer therapeutic genes into cancer cells. However, experience has shown that targeting only one gene or one pathway at a time may not be an effective approach to tackle the heterogeneous glioblastomas, in which many of the genes involved in regulating cell proliferation, differentiation, and death are abnormal. Moreover, replication-deficient adenoviruses lack the ability to reach to enough number of cancer cells to induce significant anti-cancer effect. A goal is now to design strategies to target universal defects in cancer cells and develop agents with expandable cytotoxic power only in cancer cell population. Oncolytic viruses hold the promise for implementing such strategies. Oncolytic viruses represent either genetically engineered or naturally occurring strains of viruses that exhibit relatively selective replication in tumor cells. Naturally, viruses induce changes in host cells to create an environment favorable for viral replication that are similar to the processes involved in cellular transformation (Thorne et al. 2005). These include uncontrolled cellular proliferation, prevention of apoptosis, and resistance to host organism immune effector mechanisms (Thorne et al. 2005). Viral strains or mutants that are defective in these processes are exploited as oncolytic viruses. In this review, we summarize the principle and the application of replicationdeficient and replication-competent (oncolytic) viral vectors in the therapy for malignant brain tumors (Table  1). Our focus is on those therapies that have already arrived to clinical trials in glioma patients.

2 Replication-Deficient Viral Vectors 2.1 Ad-p53 One of the most frequent genetic alterations in cancer is p53 mutation which occurs in more than 50% of human cancers (Levine 1997). p53 is frequently inactivated in brain tumors either through mutation of the p53 gene or posttranslational regulations (Lang et al. 2003). The inactivation of p53 is a critical event in the formation

HSV-1

Reovirus Measles virus

Newcastle disease virus

HSV1716

Reolysin MV-CEA

HuJ

Ras pathway CD46 Ras pathway Ras pathway

RNA export RB pathway Ras pathway Cell proliferation Ras pathway

Cell proliferation

Cell proliferation

Cell proliferation

p53 pathway

I.T. I.T. I.V.

Phase I/II

I.B. I.T. I.T. I.B. I.T. I.B.

I.T. I.B. Virus: I.B. GCV: I.V. RVPC: I.T GCV: I.V. Virus: I.B. GCV: I.V.

Route of delivery

Phase I/II Phase I

Phase I

Phase I Phase I Phase I/Ib

Phase III

Phase I/II

Phase III

Phase I

Clinical trial status

I.T. intratumoral, I.B. injected into adjacent brain posttumor resection, I.V. intravenous, RVPC retroviral vector-producing cell

Adenovirus Adenovirus HSV-1

Replication-deficient adenovirus Replication-deficient retrovirus Replication-deficient retrovirus Replication-deficient adenovirus

Onyx-015 Delta-24-RGD G207

Oncolytic viruses

Ad-HSVtk/GCV (Cerepro)

HSVtk/IL-2

RV-HSVtk/GCV

Ad-p53

Table 1  Clinical trials testing gene-based therapies in glioma patients Targeted defects in Therapy Virus type glioma Replication-deficient viral vectors

Freeman et al. (2006)

Rampling et al. (2000), Papanastassiou et al. (2002), Harrow et al. (2004) Forsyth et al. (2008) N/A

Chiocca et al. (2004) N/A Markert et al. (2000, 2009)

Immonen et al. (2004)

Colombo et al. (2005)

Rainov (2000)

Lang et al. (2003)

References

2  Gene-Based Therapy for Cancer: Brain Tumors 19

20

H. Jiang and J. Fueyo

and progression of gliomas (Lang et al. 2003). Since wild-type p53 is a primary mediator of cell cycle arrest and apoptosis, it is rational to transfer p53 into cancerous cells to control tumor growth and achieve therapeutic benefits. Ad-p53 is a type 5 replication-deficient human adenovirus in which the E1 region has been replaced with the cDNA of the wild-type p53 gene driven by the cytomegalovirus promoter (Zhang et al. 1993; Zhang et al. 1995). Ad-p53 has been shown to be effective against a variety of tumor types (Lang et  al. 2003). Only minimal toxicity has been observed in patients with lung or head and neck cancer after direct intratumoral injection during Phase I clinical trials (Lang et al. 2003). Because of the promising preclinical results in brain tumors and the encouraging clinical results in other tumor types, the clinical potential of Ad-p53 in the treatment of human gliomas has been tested in a Phase I clinical trial. In all 15 patients enrolled, exogenous p53 protein was detected within the nuclei of astrocytic tumor cells (Lang et al. 2003). Exogenous p53 transactivated p21CIP/WAF and induced apoptosis were observed (Lang et al. 2003). However, transfected cells resided on average within 5  mm of the injection site (Lang et  al. 2003). Although toxicity was minimal, the widespread distribution of this agent remains a significant goal (Lang et al. 2003).

2.2 HSVtk/GCV Gene Therapy Because tumor cells divide more rapidly than normal cells, cytotoxic nucleotide analogs are widely used in chemotherapy to reach a therapeutic index in which damage to the cancer cells is maximized while keeping the toxicity to the normal host cells acceptable. To further achieve an optimal therapeutic effect while limiting systemic toxicity, gene-directed enzyme prodrug therapy (GDEPT, suicide gene therapy) has been developed (Fillat et  al. 2003). This is a two-step therapeutic approach for cancer gene therapy. In the first step, the transgene is delivered into the tumor and expressed. In the second step, a prodrug is administered and is ­selectively activated by the expressed enzyme. The first GDEPT system described was the thymidine kinase gene of the herpes simplex virus (HSVtk) in combination with the prodrug Ganciclovir (GCV). The thymidine kinase enzyme (tk), produced by the herpes simplex virus (HSV), is harmless to humans since it lacks a substrate in the human body. However, in cells that express tk, the tk can metabolize intravenously administered GCV to produce a cytotoxic GCV triphosphate, which is selective for dividing cells (Fillat et al. 2003). This is of particular significance in the brain where the normal neurons surrounding the tumor are nonproliferative and therefore not susceptible to toxic metabolites. The treatment effect is further strengthened by a “bystander effect” (Fillat et al. 2003). Using the retrovirus (RV)-mediated transduction of glioblastoma cells with HSVtk gene and subsequent systemic treatment with GCV, a Phase III, multicenter, randomized, open-label, parallel-group, controlled trial has been performed in the treatment of 248 patients with newly diagnosed, previously untreated

2  Gene-Based Therapy for Cancer: Brain Tumors

21

glioblastoma (Rainov 2000). The results revealed that the adjuvant treatment improved neither time to tumor progression nor overall survival time, although the feasibility and good biosafety profile of this gene therapy strategy were further supported. The failure of this specific protocol may be due mainly to the presumably poor rate of delivery of the HSVtk gene to tumor cells. Since antitumor immune response was observed during HSVtk/GCV therapy (Barba et  al. 1994), to amplify this antitumor immune response, a strategy was developed based on the combined delivery of a cytokine gene (human interleukin-2, IL-2) together with HSV-TK (Pizzato et al. 1998; Barzon et al. 2002; Barzon et al. 2003). In preclinical experimental models, it demonstrated not only the efficient killing of transduced cancer cells but also the growth inhibition of distant nontransduced tumor masses (Barzon et al. 2003). In a Phase I/II clinical study, a total of 12 patients with recurrent glioblastoma multiforme received intratumor injection of retroviral vector-producing cells (RVPCs), followed by intravenous GCV (Colombo et al. 2005). The results demonstrated that the intratumor injection of RVPCs was safe, provided effective transduction of the therapeutic genes to target tumor cells, and activated a systemic cytokine cascade, with tumor responses in 50% of cases. In another randomized, controlled study, HSVtk gene was transduced by a replication-deficient adenoviral vector (type 5) (Immonen et  al. 2004). AdvHSV-tk treatment produced a clinically and statistically significant increase in median survival time of 36 patients with operable primary or recurrent malignant glioma. Six patients had increased anti-adenovirus antibody titers, without adverse effects. The treatment was well tolerated. It is concluded that AdvHSV-tk gene therapy and GCV is a potential new treatment for operable primary or recurrent high-grade glioma. Recently, a Phase III multicentre, standard care-controlled, pivotal trial in 236 patients with operable high grade glioma revealed results that are consistent with those previously reported. This therapeutic regimen (Cerepro), developed by Ark Therapeutics Group plc, works by harnessing healthy cells to produce GCV triphosphate that is either incorporated into DNA or inhibits the polymerase to destroy newly growing cancer cells (bystander effect) (Fillat et al. 2003). It has been granted Orphan Drug Status by the European Committee for Orphan Medicinal Products and by the Office of Orphan Products Development, FDA. Approvals for named patient supply of Cerepro have been given by the French Medicines Control Agency (AFSSAPS) in February 2009 and also by the Finnish Medicines Authorities (NAM) in May 2009.

3 Oncolytic Viruses 3.1 Adenovirus Adenoviruses are a nonenveloped virus with a single, linear, double-stranded DNA genome of approximately 36–38  kpb in size (Fields et  al. 2007). Wild-type adenoviruses induce cell death through replicating in and lysing infected cells.

22

H. Jiang and J. Fueyo

This cytotoxic capacity, together with the efficiency with which viruses can spread from one cell to another, inspired the notion that replication-competent viruses could be a solution for the problems encountered in delivering gene therapy for cancer. Because adenoviruses require host factors for replication, they express a series of genes immediately after infection to reprogram the host cell to facilitate replication. These so-called early genes encode proteins that bind and inactivate cellular regulators of cell cycle and apoptosis. The first adenoviral gene expressed after infection is called E1A. The E1A products bind to and inactivate critical cellular proteins, such as the RB family of pocket proteins (Jiang et al. 2006). Inactivation of these proteins propels the cell into unscheduled DNA synthesis, which creates a favorable environment for adenoviral replication. However, forced entry into S phase may trigger p53-mediated apoptosis during the early stage of viral infection, aborting the process of replication. To prevent the p53-induced apoptotic response, adenovirus encodes other early proteins, such as E1B-55K, E4orf6 and E1B-19K, to bind and inactivate p53 and Bax, resulting in a prolonged cell life that ensures the propagation of virions (Jiang et al. 2006). Such biological logic allows the design of replicationcompetent adenoviruses unable to express proteins that interact with cell cycle regulators or apoptosis inducers. Thus, these mutant viruses can propagate selectively in cancer cells, but are unable to acquire a replication phenotype in normal cells. 3.1.1 ONYX-015 Based on the knowledge of virus–host cell interactions, a mutant adenovirus dl1520/ONYX-015, which does not express the E1B-55K protein to inactivate p53, was constructed and tested for anticancer effect (Bischoff et al. 1996). ONYX-015 was demonstrated to have preferential replication as well as anti-tumor efficacy in some p53-deficient human tumor cells (Bischoff et al. 1996; Heise et al. 1997). The work on ONYX-015 pioneered the field of oncolytic adenoviral research and application. Further studies showed that loss of E1B-55K-mediated late viral RNA export, rather than p53 degradation, restricts ONYX-015 replication in primary cells (O’Shea et al. 2004). In contrast, tumor cells, which have altered mechanisms for RNA export to complement the RNA export function of E1B-55K, support ONYX-015 replication (O’Shea et al. 2004). ONYX-015 has undergone extensive testing in the clinic and has proved safe at the doses up to 2 × 1012 viral particles (McCormick 2003). A dose-escalation trial of intracerebral injections of ONYX-015 for patients with recurrent malignant glioma, conducted by the National Cancer Institute’s New Approaches to Brain Tumor Therapy (NABTT) CNS Consortium, showed that injection of ONYX-015 into the tumor cavity after glioma resection is well tolerated at doses up to 1010 plaque-forming units of the virus (Chiocca et  al. 2004). ONYX-015 has been administered to over 250 cancer patients in roughly 15 clinical trials in a variety of tumor types involving intratumoral, intravenous, intraperitoneal, and hepatic arterial administration (Wildner 2005). Controlled clinical trials using the mutant as an oncolytic agent have led to important insights into the use of this virus as an

2  Gene-Based Therapy for Cancer: Brain Tumors

23

a­ nticancer strategy (Wildner 2005). ONYX-015 as a single-agent treatment has disappointing efficacy, but in combination with chemotherapy shows encouraging antineoplastic activity (Wildner 2005). 3.1.2 Delta-24-RGD The RB pathway is disrupted in virtually all human cancers. After adenovirus infection, in normal cells, E1A protein binds and inactivates RB protein. This function of E1A is not required in Rb-deficient cancer cells (Whyte et  al. 1989; Pelicano et al. 2006). At M. D. Anderson Cancer Center, we have tested an oncolytic adenovirus Delta-24 with a mutant E1A protein that is unable to bind RB (Fueyo et al. 2000). The Delta-24 adenovirus replicates in and lyses cancer cells with great efficiency. In vivo, one dose of the virus induced a dramatic inhibition of tumor growth in nude mice. However, normal fibroblasts or cancer cells with restored RB activity were resistant to Delta-24. These findings suggest that Delta-24 may be therapeutically useful against gliomas, and also possibly against other cancers with a disrupted RB pathway (Fueyo et al. 2000). After the Delta-24 strategy was reported, this virus was tested in other laboratories, and it is currently being used at our center and others as a platform for the development of combined tumor-targeting approaches. Because glioma cells consist of low levels of CAR (a native adenovirus receptor) and high levels of RGD-related integrins (Fueyo et al. 2003), adenoviruses retargeted to bind integrins should be able to circumvent the lack of CAR expression on the glioma cell surface, thus improving the ability of adenovirus to enter into cancer cells to achieve higher efficacy. In the first report of an oncolytic adenovirus (Delta24) modified by the genetic introduction of an RGD sequence in the fiber HI loop, Suzuki and colleagues (2001) showed that the fiber-knob protein modification fostered CAR-independent transduction, enhancing viral propagation and an oncolytic effect in vitro and in vivo in prostate and lung cancers. When this RGD-modified vector Delta-24-RGD was later tested in gliomas, it was more cytopathic to both low- and high-CAR-expressing glioma lines than was Delta-24, and it replicated more efficiently in both types of cell lines (Fueyo et  al. 2003). Comparing the results of intratumoral injection of Delta-24 and Delta-24-RGD in mice-bearing glioma xenografts, Delta-24-RGD was associated with an improved regression of the glioma xenografts and with longer survival (Fueyo et  al. 2003). In addition, when combined with agents currently used in glioma therapy, such as RAD001 and temozolomide, Delta-24-RGD synergistically enhanced the therapeutic effect (Alonso et al. 2008; Alonso et al. 2007). In a recent study, Delta-24-RGD showed robust efficacy against brain tumor stem cells (BTSCs) that are responsible for cancer initiation and resistance to conventional chemo- and radiotherapy (Jiang et al. 2007). Delta-24-RGD significantly improved the survival of the mice-bearing gliomas derived from BTSCs (Jiang et al. 2007). The virus-induced autophagic cell death in BTSCs and the drastic upregulation of autphagic protein ATG5 can be used as surrogate markers to monitor the therapeutic effect of Delta-24-RGD in the future clinical trials (Jiang et  al. 2007). In December, 2008, a Phase I clinical trial of

24

H. Jiang and J. Fueyo

Delta-24-RGD started in patients with recurrent malignant gliomas at the Brain Tumor Center, UT M. D. Anderson Cancer Center.

3.2 Herpes Simplex Virus-1 HSV-1 is a well-characterized enveloped, double-stranded DNA virus whose genome is ~152 kb (Roizman 1996). This virus is especially attractive for the development of oncolytic vectors because it is highly infectious, replicates rapidly, and can be readily modified to achieve vector attenuation in normal brain tissue (Grandi et al. 2009). Tumor specificity can be achieved by deleting viral genes that are only required for virus replication in normal cells and permit mutant virus replication selectively in tumor cells (Grandi et  al. 2009). G207 is an attenuated replicationcompetent HSV-1 with deletions of both copies of neurovirulence gene g34.5 (encoding ICP34.5) and an inactivating mutation of UL39, which encodes ICP6, the large subunit of HSV ribonucleotide reductase (RR) (Mineta et al. 1995). Loss of ICP6 expression restricts the replication of the virus to cells with elevated RR activity, presumably because of RB pathway abnormality that is common in glioma cells (Furnari et al. 2007). During viral infection, a stress response occurs in the host cell. Protein kinase R (PKR) activation shuts down translation in the infected cell as an anti-viral protective mechanism by phosphorylating and inactivating eukaryotic initiation factor-2a (eIF-2a) (He et  al. 1997). The g134.5 protein product ICP34.5 recruits protein phosphatase-1a in order to dephosphorylate eIF-2a and allow protein synthesis to proceed (He et al. 1997). Thus, defects in the ICP34.5 function restrict replication to cancer cells that possess decreased PKR activity, possibly due to the expression of a constitutively active form of RAS through mutations in upstream receptor tyrosine kinases (RTKs) or mutations of Ras itself (Farassati et al. 2001). In malignant gliomas, Ras mutations can be rare (Guha et al. 1997). However, the RTKs epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFR) are commonly overexpressed in malignant glioma (Furnari et  al. 2007), leading to overexpression of Ras and upregulation of the Ras signaling pathway. G207 has been tested in Phase I/Ib trial in patients with malignant glioma (Markert et al. 2000; Markert et al. 2009). Administration was carried out by direct stereotactic injection into the tumor or the brain surrounding the resection cavity. Doses of up to 3 × 109 infectious units were well tolerated and a maximum tolerated dose was not achieved. The trials showed that the inoculation of an attenuated HSV, the wild-type counterpart’s main pathogenic effect of which is encephalitis, remained relatively safe in a human brain. Recently, it was reported that temozolomide exhibited strong synergy with G207 through the induction of GADD34 and RR expression in malignant glioma cells and mice with intracranial gliomas (Aghi et al. 2006). These findings unveil the potential of HSV to target cells that survive temozolomide treatment. Another HSV-1 mutant HSV1716 bears a deletion of 759 bp in each copy of the gI34.5 gene, resulting in the null expression of the PKR inhibitor ICP34.5

2  Gene-Based Therapy for Cancer: Brain Tumors

25

(Kesari et  al. 1995). Due to this mutant’s preferential proliferation in malignant cells, several studies have employed HSV1716 to treat glioma or brain tumors. Three Phase I clinical trials have been carried out to evaluate the safety of HSV1716. In one of these trials, patients with high-grade glioma were treated with the virus. HSV1716 DNA was detected by PCR at the sites of inoculation. In 5 patients (of 12 total), an immune response to the virus was detected. Although it remains unclear whether the immune response to the virus contributes to the eradication of cancer cells infected by the virus, a significant increase in long-term survival following surgery was also observed. These trials demonstrated that HSV1716 can replicate selectively in high-grade glioma without severe adverse effects in patients (Rampling et al. 2000; Papanastassiou et al. 2002; Harrow et al. 2004). To enhance tumor cytotoxicity induced by HSV1716, noradrenaline transporter gene (NAT) was inserted into its backbone (Quigg et al. 2005). The resultant new construct HSV1716/NAT enabled active uptake of the radiopharmaceutical [131I] MIBG and resulted in significantly enhanced cytotoxicity compared to either agent alone. These studies show that the combination of oncolytic HSV therapy with targeted radiotherapy has the potential for effective tumor cell kill and warrants further investigation as a treatment for malignant glioma.

3.3 Reovirus Reovirus is a nonenveloped virus with a segmented, double-stranded RNA genome and can be associated with mild respiratory or gastrointestinal tract symptoms although infections tend to be asymptomatic (Fields et al. 2007). RNA viruses are attractive for cancer therapy because during their life cycle, double-stranded RNA activates type I interferon system that is crippled in tumor cells, providing a more permissive environment for the propagation of the viruses than in normal cells (Stojdl et al. 2000). When reovirus infects cells, double-stranded RNA can activate the host interferon-induced PKR which shuts down protein synthesis to protect the cell from viral infection (Strong et al. 1998). Activated Ras (or an activated element of the Ras pathway) inhibits (or reverses) PKR activation and allows viral protein synthesis and a lytic infection to occur (Strong et al. 1998). Since Ras-activated pathways are present in the majority of malignant gliomas (Guha et  al. 1997; Libermann et  al. 1985; Shamah et  al. 1993; Helseth et  al. 1988), the virus was tested in glioma cell cultures and in human gliomas growing in mice (Wilcox et al. 2001). When tested against cell cultures, live reovirus killed 20 of 24 established glioma cell lines. It also killed all nine primary cell cultures from gliomas removed from patients, but none of the cultured meningiomas. The reovirus was also effective against human gliomas established in the hind flank of mice. A single injection of live reovirus into these well-established tumors led to a statistically significant decrease in tumor size. In a Phase I clinical trial, reovirus was administered intratumorally stereotactically at up to 1 × 109 pfu in 12 patients with recurrent malignant gliomas (Forsyth et al. 2008). Maximum tolerated dose was not reached and the

26

H. Jiang and J. Fueyo

treatment was well tolerated. In July 2006, Oncolytics Biotech Inc. started a Phase I/II trial of reovirus (Reolycin) in patients with recurrent malignant gliomas. The primary objective of the study is to determine the maximum tolerated dose, dose limiting toxicity, and safety profile of Reolycin. Secondary objectives include the evaluation of viral replication, immune response to the virus, and any evidence of antitumor activity. Patients will be treated with Reolycin through infusion delivery. An additional group of patients will be treated at the maximum tolerated dose.

3.4 Measles Virus Measles virus is a negative strand RNA virus with a nonsegmented genome of 15,894 nucleotides in size (Fields et al. 2007; Blechacz and Russell 2008). The enveloped virions are pleomorphic and range in size from 100 to 300  nm (Fields et  al. 2007). Measles virus causes cell–cell fusion and apoptotic cell death (Fields et al. 2007). Wild-type measles virus is a virulent pathogen, causing deadly infection in children (Fields et al. 2007). Attenuated strains have been developed as vaccines. One of them is Edmonston B strain (MV-Edm) which demonstrates tumor selectivity (Blechacz and Russell 2008). One factor contributes to the tumor selectivity is that this strain enters cell preferentially using CD46 which is overexpressed in a variety of human cancers (Blechacz and Russell 2008; Fishelson et al. 2003). Another factor is that, in normal cells, MV-Edm is unable to inhibit innate immune response pathways that are defective in tumor cells (Blechacz and Russell 2008). These two factors determine that MV-Edm propagates efficiently in tumor cells but not in normal cells. In a preclinical study, MV-CEA, a genetically engineered MV-Edm that produces carcinoembryonic antigen (CEA), has been tested in glioblastomas (Phuong et al. 2003). The results reveal that MV-CEA has potent antitumor activity against gliomas in vitro, as well as in both subcutaneous and orthotopic U87 animal models. Monitoring CEA levels in the serum can serve as a low-risk method of detecting viral gene expression during treatment, and could allow dose optimization and individualization of treatment. Recently, the same group reported that a combination of MV-CEA and radiotherapy showed synergistic effect against glioblastoma in  vitro and in  vivo (Liu et  al. 2007). The synergistic effect of the combination seems to be due to increase in viral burst size and apoptotic cell death. These studies have just been translated into a Phase I clinical trial in patients with recurrent glioblastoma multiforme, starting from July 2009 at Mayo Clinic.

3.5 Newcastle Disease Virus Newcastle disease virus (NDV) is an enveloped avian RNA virus which is potentially fatal to birds, but only causes minor illness in humans (Alexander and Allan 1974). The use of NDV as an anti-tumor agent dates back to a 1964 study by Wheelock

2  Gene-Based Therapy for Cancer: Brain Tumors

27

and Dingle (Wheelock and Dingle 1964), who published their observations of a patient who was provided repeated injections of NDV in an attempt to treat his acute leukemia. NDV exploits cancer selective replication through defected interferon system in tumor cells as mentioned previously (Stojdl et al. 2000). Certain NDV strains can replicate up to 10,000 times better in tumor cells than in normal cells (Schirrmacher et al. 1999). In addition, NDV-infected cancer cells exhibit an enhanced recruitment and activation of natural killer cells and cytotoxic T cells compared to their uninfected counterparts (Haas et al. 1998). Thus, NDV facilitates the recognition of tumors cells as “foreign” (Zorn et al. 1994), which should enhance the efficacy of the virus in patients. The distinct strains of NDV can be either lytic or nonlytic (Shah et al. 2003). Several lytic strains of NDV have undergone preclinical animal studies for safety and efficacy against human cancers (Shah et al. 2003). MTH-68/H, a live attenuated strain of NDV, had been used in the treatment of different malignancies. It was tested in a small number of patients with glioblastoma multiforme (Csatary and Bakacs 1999; Csatary et  al. 2004; Wagner et  al. 2006). Anecdotal responses to MTH68/H in the patients have been reported. In a Phase I/II trial, HuJ, a lentogenic strain of NDV that has a selective cytopathogenicity for human and animal cancer cell lines, has been tested in patients with apparent recurrent glioblastoma multiforme (Freeman et al. 2006). The study was based on imaging studies to determine the safety and tumor response of repetitive intravenous administration of HuJ. Maximum tolerated dose was not achieved. Anti-NDV hemagglutinin antibodies appeared within 5–29 days. HuJ was recovered from blood, saliva, and urine samples and one tumor biopsy. One patient achieved a complete response. Intravenous HuJ is well tolerated.

4 Future Perspectives Gene-based cancer therapies reflect the achievements of cancer research. Defects in cancers are targeted specifically at molecular levels. Therefore, compared to conventional surgery, chemo- and radio-therapy, this type of therapies is expected to optimize the therapeutic index in cancer patients. However, despite the success in cultured cancer cells and laboratory animal models, gene-based cancer therapies in human patients encountered difficulties such as vector safety, transfer efficiency, vector targeting, host immune response, efficacy, and specificity of the therapeutic genes, etc. During the last decade, tremendous efforts have been made to address these issues. Since brain tumors are localized diseases that are suitable for gene-based therapies (Table  2), stereotactic intratumoral injection and injection into tumor cavity wall postresection are desirable delivery modes. Due to the infiltrative nature of gliomas, complete remission of the tumor largely depends on the efficient delivery of the therapeutics to the cancerous cells infiltrated into normal brain tissue. Clinical experience with replication-deficient viral vectors revealed minimal delivery

28 Table  2  Specific conditions of malignant gliomas

H. Jiang and J. Fueyo • • • •

Normally only one single tumor mass No metastasis Easy assessed through stereotactic or localized surgery Immune privileged (absence of a lymphatic drainage pathway; paucity of professional antigen-presenting cells) • Lack of optima therapy • Blood-brain barrier diminishes the efficacy of systemic interventions (e.g., chemotherapy) • Clinical trials have shown lack of toxicity for approaches using intratumoral delivery of vectors and viruses

with these agents (Lang et al. 2003; Rainov 2000). The problem has been partially improved with the bystander effect of suicide gene therapy (Immonen et al. 2004). Replication-competent oncolytic viruses are promising to bridge the vector gap in cancer gene therapies since the viruses replicate and spread the progeny to the adjacent cancer cells. To improve efficacy in glioma therapy, oncolytic virus was combined with suicide gene therapy by using the virus to carry the suicide gene (Conrad et  al. 2005). The preclinical study is promising in glioma cell lines and intracranial mouse model. Further challenges will be the anatomic barriers of the tumor and the clearance of the viruses by the host initial innate immune responses (Jiang et  al. 2006; Chiocca 2008). The anatomic barriers can be address by stereotactic techniques and protease expression by the viruses (Jiang et al. 2006). As to the host immunity, it might comprise the viral replication by the innate immune responses at the beginning (Chiocca 2008). Using cyclophosphamide to modulate host immune response, prior oncolytic virus inoculation has been shown to significantly improve the efficacy of the virus in glioma-bearing mice (Kambara et  al. 2005). However, the development of antitumor immunity later during gene therapy should help to eliminate remnant malignant cells (Barba et al. 1994). For example, oncolytic viruses expressing immune enhancing gene IL-12 demonstrates increased efficacy in part because of antitumor actions of immune-related infiltrating cells (Hellums et  al. 2005; Parker et al. 2000). Thus, a better understanding of the interaction between the oncolytic viruses and the host immune system will help to modulate the immune response to minimize antiviral immunity, while at the same time maximizing antitumor immunity.

References Wen PY, Kesari S. Malignant gliomas in adults. N Engl J Med 2008;359(5):492–507. Furnari FB, Fenton T, Bachoo RM, Mukasa A, Stommel JM, Stegh A, et al. Malignant astrocytic glioma: genetics, biology, and paths to treatment. Genes Dev 2007;21(21):2683–710. Thorne SH, Hermiston T, Kirn D. Oncolytic virotherapy: approaches to tumor targeting and enhancing antitumor effects. Semin Oncol 2005;32(6):537–48. Levine AJ. p53, the cellular gatekeeper for growth and division. Cell 1997;88(3):323–31.

2  Gene-Based Therapy for Cancer: Brain Tumors

29

Lang FF, Bruner JM, Fuller GN, Aldape K, Prados MD, Chang S, et al. Phase I trial of adenovirusmediated p53 gene therapy for recurrent glioma: biological and clinical results. J Clin Oncol 2003;21(13):2508–18. Zhang WW, Fang X, Branch CD, Mazur W, French BA, Roth JA. Generation and identification of recombinant adenovirus by liposome-mediated transfection and PCR analysis. Biotechniques 1993;15(5):868–72. Zhang WW, Alemany R, Wang J, Koch PE, Ordonez NG, Roth JA. Safety evaluation of Ad5CMV-p53 in vitro and in vivo. Hum Gene Ther 1995;6(2):155–64. Fillat C, Carrio M, Cascante A, Sangro B. Suicide gene therapy mediated by the Herpes Simplex virus thymidine kinase gene/Ganciclovir system: fifteen years of application. Curr Gene Ther 2003;3(1):13–26. Rainov NG. A phase III clinical evaluation of herpes simplex virus type 1 thymidine kinase and ganciclovir gene therapy as an adjuvant to surgical resection and radiation in adults with previously untreated glioblastoma multiforme. Hum Gene Ther 2000;11(17):2389–401. Barba D, Hardin J, Sadelain M, Gage FH. Development of anti-tumor immunity following thymidine kinase-mediated killing of experimental brain tumors. Proc Natl Acad Sci U S A 1994; 91(10):4348–52. Pizzato M, Franchin E, Calvi P, Boschetto R, Colombo M, Ferrini S, et al. Production and characterization of a bicistronic Moloney-based retroviral vector expressing human interleukin 2 and herpes simplex virus thymidine kinase for gene therapy of cancer. Gene Ther 1998; 5(7):1003–7. Barzon L, Bonaguro R, Castagliuolo I, Chilosi M, Gnatta E, Parolin C, et  al. Transcriptionally targeted retroviral vector for combined suicide and immunomodulating gene therapy of thyroid cancer. J Clin Endocrinol Metab 2002;87(11):5304–11. Barzon L, Bonaguro R, Castagliuolo I, Chilosi M, Franchin E, Del Vecchio C, et al. Gene therapy of thyroid cancer via retrovirally-driven combined expression of human interleukin-2 and herpes simplex virus thymidine kinase. Eur J Endocrinol 2003;148(1):73–80. Colombo F, Barzon L, Franchin E, Pacenti M, Pinna V, Danieli D, et al. Combined HSV-TK/IL-2 gene therapy in patients with recurrent glioblastoma multiforme: biological and clinical results. Cancer Gene Ther 2005;12(10):835–48. Immonen A, Vapalahti M, Tyynela K, Hurskainen H, Sandmair A, Vanninen R, et al. AdvHSV-tk gene therapy with intravenous ganciclovir improves survival in human malignant glioma: a randomised, controlled study. Mol Ther 2004;10(5):967–72. Fields BN, Knipe DM, Howley PM. 5th ed. Philadelphia: Wolters Kluwer Health/Lippincott Williams & Wilkins; 2007. Jiang H, McCormick F, Lang FF, Gomez-Manzano C, Fueyo J. Oncolytic adenoviruses as antiglioma agents. Expert Rev Anticancer Ther 2006;6(5):697–708. Bischoff JR, Kirn DH, Williams A, Heise C, Horn S, Muna M, et al. An adenovirus mutant that replicates selectively in p53-deficient human tumor cells. Science 1996;274(5286):373–6. Heise C, Sampson-Johannes A, Williams A, McCormick F, Von Hoff DD, Kirn DH. ONYX-015, an E1B gene-attenuated adenovirus, causes tumor-specific cytolysis and antitumoral efficacy that can be augmented by standard chemotherapeutic agents [see comment]. Nat Med 1997; 3(6):639–45. O’Shea CC, Johnson L, Bagus B, Choi S, Nicholas C, Shen A, et al. Late viral RNA export, rather than p53 inactivation, determines ONYX-015 tumor selectivity. Cancer Cell 2004;6(6): 611–23. McCormick F. Cancer-specific viruses and the development of ONYX-015. Cancer Biol Ther 2003;2(4 Suppl 1):S157–60. Chiocca EA, Abbed KM, Tatter S, Louis DN, Hochberg FH, Barker F, et al. A phase I open-label, dose-escalation, multi-institutional trial of injection with an E1B-Attenuated adenovirus, ONYX-015, into the peritumoral region of recurrent malignant gliomas, in the adjuvant setting. Mol Ther 2004;10(5):958–66. Wildner O. Clinical trials: the sensitizing side of ONYX-015. Gene Ther 2005;12(5):386–7.

30

H. Jiang and J. Fueyo

Whyte P, Williamson NM, Harlow E. Cellular targets for transformation by the adenovirus E1A proteins. Cell 1989;56(1):67–75. Pelicano H, Martin DS, Xu RH, Huang P. Glycolysis inhibition for anticancer treatment. Oncogene 2006;25(34):4633–46. Fueyo J, Gomez-Manzano C, Alemany R, Lee PS, McDonnell TJ, Mitlianga P, et al. A mutant oncolytic adenovirus targeting the Rb pathway produces anti-glioma effect in vivo. Oncogene 2000;19(1):2–12. Fueyo J, Alemany R, Gomez-Manzano C, Fuller GN, Khan A, Conrad CA, et al. Preclinical characterization of the antiglioma activity of a tropism-enhanced adenovirus targeted to the retinoblastoma pathway. J Natl Cancer Inst 2003;95(9):652–60. Suzuki K, Fueyo J, Krasnykh V, Reynolds PN, Curiel DT, Alemany R. A conditionally replicative adenovirus with enhanced infectivity shows improved oncolytic potency. Clin Cancer Res 2001;7(1):120–6. Alonso MM, Jiang H, Yokoyama T, Xu J, Bekele NB, Lang FF, et al. Delta-24-RGD in combination with RAD001 induces enhanced anti-glioma effect via autophagic cell death. Mol Ther 2008;16(3):487–93. Alonso MM, Gomez-Manzano C, Bekele BN, Yung WK, Fueyo J. Adenovirus-based strategies overcome temozolomide resistance by silencing the O6-methylguanine-DNA methyltransferase promoter. Cancer Res 2007;67(24):11499–504. Jiang H, Gomez-Manzano C, Aoki H, Alonso MM, Kondo S, McCormick F, et al. Examination of the therapeutic potential of Delta-24-RGD in brain tumor stem cells: role of autophagic cell death. J Natl Cancer Inst 2007;99(18):1410–4. Roizman B. The function of herpes simplex virus genes: a primer for genetic engineering of novel vectors. Proc Natl Acad Sci U S A 1996;93(21):11307–12. Grandi P, Peruzzi P, Reinhart B, Cohen JB, Chiocca EA, Glorioso JC. Design and application of oncolytic HSV vectors for glioblastoma therapy. Expert Rev Neurother 2009;9(4):505–17. Mineta T, Rabkin SD, Yazaki T, Hunter WD, Martuza RL. Attenuated multi-mutated herpes simplex virus-1 for the treatment of malignant gliomas. Nat Med 1995;1(9):938–43. He B, Gross M, Roizman B. The gamma(1)34.5 protein of herpes simplex virus 1 complexes with protein phosphatase 1alpha to dephosphorylate the alpha subunit of the eukaryotic translation initiation factor 2 and preclude the shutoff of protein synthesis by double-stranded RNAactivated protein kinase. Proc Natl Acad Sci U S A 1997;94(3):843–8. Farassati F, Yang AD, Lee PW. Oncogenes in Ras signalling pathway dictate host-cell permissiveness to herpes simplex virus 1. Nat Cell Biol 2001;3(8):745–50. Guha A, Feldkamp MM, Lau N, Boss G, Pawson A. Proliferation of human malignant astrocytomas is dependent on Ras activation. Oncogene 1997;15(23):2755–65. Markert JM, Medlock MD, Rabkin SD, Gillespie GY, Todo T, Hunter WD, et al. Conditionally replicating herpes simplex virus mutant, G207 for the treatment of malignant glioma: results of a phase I trial. Gene Ther 2000;7(10):867–74. Markert JM, Liechty PG, Wang W, Gaston S, Braz E, Karrasch M, et al. Phase Ib trial of mutant herpes simplex virus G207 inoculated pre-and post-tumor resection for recurrent GBM. Mol Ther 2009;17(1):199–207. Aghi M, Rabkin S, Martuza RL. Effect of chemotherapy-induced DNA repair on oncolytic herpes simplex viral replication. J Natl Cancer Inst 2006;98(1):38–50. Kesari S, Randazzo BP, Valyi-Nagy T, Huang QS, Brown SM, MacLean AR, et al. Therapy of experimental human brain tumors using a neuroattenuated herpes simplex virus mutant. Lab Invest 1995;73(5):636–48. Rampling R, Cruickshank G, Papanastassiou V, Nicoll J, Hadley D, Brennan D, et al. Toxicity evaluation of replication-competent herpes simplex virus (ICP 34.5 null mutant 1716) in patients with recurrent malignant glioma. Gene Ther 2000;7(10):859–66. Papanastassiou V, Rampling R, Fraser M, Petty R, Hadley D, Nicoll J, et  al. The potential for efficacy of the modified (ICP 34.5(-)) herpes simplex virus HSV1716 following intratumoural injection into human malignant glioma: a proof of principle study. Gene Ther 2002;9(6): 398–406.

2  Gene-Based Therapy for Cancer: Brain Tumors

31

Harrow S, Papanastassiou V, Harland J, Mabbs R, Petty R, Fraser M, et al. HSV1716 injection into the brain adjacent to tumour following surgical resection of high-grade glioma: safety data and long-term survival. Gene Ther 2004;11(22):1648–58. Quigg M, Mairs RJ, Brown SM, Harland J, Dunn P, Rampling R, et al. Assessment in vitro of a novel therapeutic strategy for glioma, combining herpes simplex virus HSV1716-mediated oncolysis with gene transfer and targeted radiotherapy. Med Chem 2005;1(5):423–9. Stojdl DF, Lichty B, Knowles S, Marius R, Atkins H, Sonenberg N, et  al. Exploiting tumorspecific defects in the interferon pathway with a previously unknown oncolytic virus. Nat Med 2000;6(7):821–5. Strong JE, Coffey MC, Tang D, Sabinin P, Lee PW. The molecular basis of viral oncolysis: usurpation of the Ras signaling pathway by reovirus. EMBO J 1998;17(12):3351–62. Libermann TA, Nusbaum HR, Razon N, Kris R, Lax I, Soreq H, et al. Amplification, enhanced expression and possible rearrangement of EGF receptor gene in primary human brain tumours of glial origin. Nature 1985;313(5998):144–7. Shamah SM, Stiles CD, Guha A. Dominant-negative mutants of platelet-derived growth factor revert the transformed phenotype of human astrocytoma cells. Mol Cell Biol 1993;13(12): 7203–12. Helseth E, Unsgaard G, Dalen A, Fure H, Skandsen T, Odegaard A, et al. Amplification of the epidermal growth factor receptor gene in biopsy specimens from human intracranial tumours. Br J Neurosurg 1988;2(2):217–25. Wilcox ME, Yang W, Senger D, Rewcastle NB, Morris DG, Brasher PM, et al. Reovirus as an oncolytic agent against experimental human malignant gliomas. J Natl Cancer Inst 2001; 93(12):903–12. Forsyth P, Roldan G, George D, Wallace C, Palmer CA, Morris D, et al. A phase I trial of intratumoral administration of reovirus in patients with histologically confirmed recurrent malignant gliomas. Mol Ther 2008;16(3):627–32. Blechacz B, Russell SJ. Measles virus as an oncolytic vector platform. Curr Gene Ther 2008; 8(3):162–75. Fishelson Z, Donin N, Zell S, Schultz S, Kirschfink M. Obstacles to cancer immunotherapy: expression of membrane complement regulatory proteins (mCRPs) in tumors. Mol Immunol 2003;40(2–4):109–23. Phuong LK, Allen C, Peng KW, Giannini C, Greiner S, TenEyck CJ, et al. Use of a vaccine strain of measles virus genetically engineered to produce carcinoembryonic antigen as a novel therapeutic agent against glioblastoma multiforme. Cancer Res 2003;63(10):2462–9. Liu C, Sarkaria JN, Petell CA, Paraskevakou G, Zollman PJ, Schroeder M, et al. Combination of measles virus virotherapy and radiation therapy has synergistic activity in the treatment of glioblastoma multiforme. Clin Cancer Res 2007;13(23):7155–65. Alexander DJ, Allan WH. Newcastle disease virus pathotypes. Avian Pathol 1974;3(4):269–78. Wheelock EF, Dingle JH. Observations on the repeated administration of viruses to a patient with acute leukemia. A preliminary report. N Engl J Med 1964;271:645–51. Schirrmacher V, Haas C, Bonifer R, Ahlert T, Gerhards R, Ertel C. Human tumor cell modification by virus infection: an efficient and safe way to produce cancer vaccine with pleiotropic immune stimulatory properties when using Newcastle disease virus. Gene Ther 1999;6(1):63–73. Haas C, Ertel C, Gerhards R, Schirrmacher V. Introduction of adhesive and costimulatory immune functions into tumor cells by infection with Newcastle Disease Virus. Int J Oncol 1998; 13(6):1105–15. Zorn U, Dallmann I, Grosse J, Kirchner H, Poliwoda H, Atzpodien J. Induction of cytokines and cytotoxicity against tumor cells by Newcastle disease virus. Cancer Biother 1994;9(3):225–35. Shah AC, Benos D, Gillespie GY, Markert JM. Oncolytic viruses: clinical applications as vectors for the treatment of malignant gliomas. J Neurooncol 2003;65(3):203–26. Csatary LK, Bakacs T. Use of Newcastle disease virus vaccine (MTH-68/H) in a patient with high-grade glioblastoma. JAMA 1999;281(17):1588–9. Csatary LK, Gosztonyi G, Szeberenyi J, Fabian Z, Liszka V, Bodey B, et al. MTH-68/H oncolytic viral treatment in human high-grade gliomas. J Neurooncol 2004;67(1–2):83–93.

32

H. Jiang and J. Fueyo

Wagner S, Csatary CM, Gosztonyi G, Koch HC, Hartmann C, Peters O, et al. Combined treatment of pediatric high-grade glioma with the oncolytic viral strain MTH-68/H and oral valproic acid. APMIS 2006;114(10):731–43. Freeman AI, Zakay-Rones Z, Gomori JM, Linetsky E, Rasooly L, Greenbaum E, et al. Phase I/II trial of intravenous NDV-HUJ oncolytic virus in recurrent glioblastoma multiforme. Mol Ther 2006;13(1):221–8. Conrad C, Miller CR, Ji Y, Gomez-Manzano C, Bharara S, McMurray JS, et al. Delta24-hyCD adenovirus suppresses glioma growth in vivo by combining oncolysis and chemosensitization. Cancer Gene Ther 2005;12(3):284–94. Chiocca EA. The host response to cancer virotherapy. Curr Opin Mol Ther 2008;10(1):38–45. Kambara H, Saeki Y, Chiocca EA. Cyclophosphamide allows for in  vivo dose reduction of a potent oncolytic virus. Cancer Res 2005;65(24):11255–8. Hellums EK, Markert JM, Parker JN, He B, Perbal B, Roizman B, et al. Increased efficacy of an interleukin-12-secreting herpes simplex virus in a syngeneic intracranial murine glioma model. Neuro Oncol 2005;7(3):213–24. Parker JN, Gillespie GY, Love CE, Randall S, Whitley RJ, Markert JM. Engineered herpes simplex virus expressing IL-12 in the treatment of experimental murine brain tumors. Proc Natl Acad Sci U S A 2000;97(5):2208–13.

Chapter 3

Gene Therapy of Prostate Cancer Svend O. Freytag, Hans Stricker, Benjamin Movsas, Mohamed Elshaikh, Ibrahim Aref, Kenneth Barton, Stephen Brown, Farzan Siddiqui, Mei Lu, and Jae Ho Kim

Abstract  Gene therapy has been administered to over 1,000 men with prostate cancer. Overall, it has been associated with little toxicity when administered as a single agent or in combination with standard prostate cancer treatments. Some strategies have generated very provocative results in early stage clinical trials. When administered intraprostatically to men with newly-diagnosed disease, adenovirusbased approaches have resulted in demonstrable tumor destruction, better-thanexpected 2-year biopsy results when combined with prostate radiotherapy, and a slowing of disease progression following biochemical failure. In more advanced settings, poxvirus-based vaccines have been shown to bolster the effectiveness of subsequent chemotherapy thereby delaying the onset and progression of metastatic disease. Thus, gene therapy has demonstrated antitumor activity across nearly all disease settings particularly when combined with standard cancer therapies. These preliminary results are very encouraging and lead us to believe that gene therapy may someday earn a place in the management of prostate cancer. Keywords  Adenovirus • Suicide gene therapy • Radiation • Prostate cancer vaccines

1 Introduction Despite recent advancements in early detection and treatment, prostate cancer is still the second leading cause of cancer death in men in the United States and approximately 27,000 men would have died because of it in 2009. Treatment options for newly-diagnosed, clinically localized prostate cancer include surgery, radiation therapy, and expectant management. In cancer with a low-risk of recurrence

S.O. Freytag (*) Department of Radiation Oncology, Henry Ford Health System, One Ford Place, 5D, Detroit, MI 48202, USA e-mail: [email protected] J.A. Roth (ed.), Gene-Based Therapies for Cancer, Current Cancer Research, DOI 10.1007/978-1-4419-6102-0_3, © Springer Science+Business Media, LLC 2010

33

34

S.O. Freytag et al.

(Stage £ T2a and Gleason score £6 and prostate-specific antigen (PSA) £10  ng/ mL), surgery and radiation therapy result in excellent long-term disease-free survival (Zelefsky et al. 2008a). However, these treatments are less effective against more advanced forms of the disease and a significant fraction (³30%) of such patients will develop local and/or distant recurrence. Unfortunately, once the disease recurs, there are few therapeutic options that have a high likelihood of eradicating the cancer with a reasonable degree of safety. Hormone-naïve, recurrent prostate cancer is typically managed with androgen suppression therapy (AST). Although AST is effective at slowing disease progression, it is not curative, is associated with significant morbidity, and many men will ultimately develop hormonerefractory, metastatic cancer. Despite recent gains with taxane-based chemotherapy, metastatic prostate cancer remains incurable and most men will succumb to their disease. Thus, there are three stages in prostate cancer progression where better treatments are needed and novel investigational therapies should be evaluated – aggressive forms of newly-diagnosed disease, locally recurrent disease, and hormone-refractory metastatic disease. Gene therapy has been applied in each of these disease settings. This chapter summarizes the gene therapy strategies that have been evaluated clinically to date. All have been associated with low toxicity when applied as single agents or in combination with standard prostate cancer treatments. Based on the provocative results obtained in early stage trials, at least two approaches have progressed to phase 3. Although it remains to be seen whether gene therapy can actually improve survival, there is a growing body of evidence indicating that it can result in significant tumor destruction, slow disease progression, and augment the effectiveness of radiotherapy and chemotherapy. These encouraging findings raise the possibility that gene therapy may earn a place in the management of prostate cancer if applied in the right setting.

2 Enzyme/Prodrug Gene Therapy At least ten enzyme/prodrug gene therapy trials targeting prostate cancer have been reported. All have used adenovirus-based platforms armed with one or more therapeutic genes, and have been evaluated in the settings of newly-diagnosed, locally recurrent, and metastatic disease. In the newly-diagnosed setting, most have been combined with prostate radiotherapy and two were applied prior to surgery. All strategies have demonstrated low toxicity and some have generated very provocative results in early stage trials.

2.1 Enzyme/Prodrug Gene Therapy Using Replication-Defective Adenoviruses The first such trial was conducted at Baylor College of Medicine using a replication-defective adenovirus (ADV/HSV-tk) expressing the wild-type herpes simplex

3  Gene Therapy of Prostate Cancer

35

virus thymidine kinase (HSV-1 TK) gene under the transcriptional control of a viral promoter (Herman et al. 1999). Unlike the mammalian TK enzyme, HSV-1 TK can convert nucleoside prodrugs such as ganciclovir (GCV, and its analogs) into their corresponding monophosphates that are potent DNA chain terminators ultimately resulting in cell death. The HSV-1 TK/GCV system exhibits a modest bystander effect in which neighboring tumor cells not expressing HSV-1 TK are killed owing to the local diffusion of toxic metabolites (Culver et al. 1992; Takamiya et al. 1992). Eighteen men with locally recurrent prostate cancer received an intraprostatic injection of adenovirus up to a dose of 2 × 1012 vp followed by 2 weeks of GCV prodrug therapy. The investigational therapy was well tolerated. Only one patient exhibited significant toxicity (grade 3 transaminitis, grade 4 thrombocytopenia) that was probably attributable to the leakage of the adenovirus into the circulation during the adenovirus injection. Three (17%) patients demonstrated an objective PSA response (>50% decline), one of which lasted over 1 year. Based on these encouraging results, the trial was expanded to examine the safety and efficacy of repeated gene therapy cycles (Miles et al. 2001). Three cycles of the gene therapy were found to be safe. Analysis of pre and posttreatment PSA kinetics demonstrated that the mean PSA doubling time (PSADT) increased from 16 to 43 months (median 12.2–12.7  months), suggesting that the gene therapy may have slowed disease progression. Many patients exhibited an elevation in circulating CD4+/ CD8+ T lymphocytes following the gene therapy. Whether these immune responses were directed against the injected adenovirus (which is likely) or patient’s prostate tumor was not examined. The same gene therapy approach was evaluated in the setting of newly-diagnosed disease in combination with prostate radiotherapy (Teh et al. 2001, 2004). The scientific rationale for combining suicide gene therapy with prostate radiotherapy is based on the original findings of Kim and Freytag who demonstrated, in preclinical models, that the HSV-1 TK suicide gene system had significant tumor radiosensitizing activity (Kim et al. 1994, 1995; Rogulski et al. 1997). Fifty-nine patients with low-, intermediate-, and high-risk prostate cancer received up to three ADV/HSV-tk injections at 5 × 1011 vp per injection each followed by 2 weeks of valganciclovir (vGCV) and standard prostate radiotherapy. Overall, the combined treatment was well tolerated. Short-term PSA responses were uninformative in the intermediate- and high-risk groups for these patients also received AST (as well as radiation therapy). Therefore, to assess efficacy, prostate biopsies were taken at several time points up to 24 months after the study therapy. Rather dramatic posttreatment biopsy results were reported across all prognostic risk groups (100% cancer-free). Unfortunately, only 2 biopsy cores, rather than the standard 6–12, were taken at the posttreatment biopsies thereby weakening the significance of the biopsy results. As was observed in the locally recurrent setting, patients exhibited an elevation in circulating CD4+/CD8+ T lymphocytes after administration of the gene therapy that persisted for up to 1 year in some (Satoh et al. 2004; Fujita et al. 2006). This same group examined the safety and efficacy of applying HSV-1 TK gene therapy prior to surgery (Ayala et  al. 2006). An advantage of this trial design is that detailed pathology can be obtained on resected prostate specimens.

36

S.O. Freytag et al.

Twenty-three men with localized, high-risk prostate cancer were administered ADV/HSV-tk followed by 2 weeks of vGCV. Histopathological analysis of postsurgical specimens showed marked tumor destruction at the injection sites that correlated with tumor size. Tumor immune infiltrates largely comprised CD8+ T lymphocytes, B lymphocytes, and macrophages, all of which increased following the gene therapy. Adenovirus-mediated HSV-1 TK gene therapy has also been evaluated in the setting of metastatic disease (Kubo et al. 2003). The approach taken is somewhat novel in that it utilized a replication-defective adenovirus (Ad-OC-hsv-TK) containing the HSV-1 TK gene under the transcriptional control of the osteocalcin promoter. The rationale for using the osteocalcin promoter to drive HSV-1 TK expression is based on the fact that metastatic prostate cancer, which typically localizes to bone, assimilates its new environment by taking on a bone stroma-like gene expression profile and expresses high levels of osteocalcin. Thus, HSV-1 TK gene expression is directed to metastatic lesions. Eleven men were administered two intralesional injections of Ad-OC-hsv-TK up to a dose of 5 × 1011 vp/injection followed by 3 weeks of vGCV. The investigational therapy was well tolerated. Most of the adverse events were mild to moderate and could be attributed to the dissemination of the adenoviral vector to collateral tissues (flu-like symptoms, transaminitis) or vGCV (lymphopenia, neutropenia). Biopsy of the injected lesions demonstrated that tumor cell apoptosis correlated with HSV-1 TK expression and coxsackie/ adenovirus receptor (CAR) levels. One patient exhibited an objective PSA response; however, this event lasted less than 2 weeks. There were no objective radiological responses. Recently, the safety and efficacy of the nitroreductase/CB1954 enzyme/prodrug system was evaluated in men with newly-diagnosed and locally recurrent prostate cancer (Patel et al. 2009). Nitroreductase (NTR) is a bacterial enzyme that converts the weakly monofunctional alkylating agent, CB1954, into a potent bifunctional alkylating agent resulting in DNA crosslinking and ultimately cell death. Thirty-nine men received an intraprostatic injection of a replication-defective adenovirus expressing NTR (CTL102) up to a dose of 1 × 1012 vp, 19 of whom also subsequently received CB1954 prodrug therapy. Fourteen patients received two cycles of the gene therapy. The investigational therapy was well tolerated. Gene therapy-related side effects included transient flu-like symptoms, transaminitis, and lymphopenia. NTR expression was detected in surgical specimens by immunohistochemistry, but did not appear to correlate with adenovirus dose. Of the 19 patients who received both the adenovirus and CB1954 prodrug, two (11%) showed an objective PSA response.

2.2 Enzyme/Prodrug Gene Therapy Using Replication-Competent Adenoviruses Four gene therapy trials using three different (but related) adenoviruses have been conducted at the Henry Ford Health System targeting locally recurrent and newly- diagnosed

3  Gene Therapy of Prostate Cancer

37

disease (Freytag et al. 2002a, 2003, 2007a, b; Barton et al. 2008). The viral platform utilized by this group consists of a replication-competent, oncolytic adenovirus armed with a cytosine deaminase (CD)/HSV-1 TK fusion gene in the E1 region (Freytag et al. 1998). CD converts the prodrug 5-fluorocytiosine (5-FC) into 5-fluorouracil (5-FU), which ultimately leads to the inhibition of the de novo DNA synthesis pathway. Preclinical studies showed that combining the CD/5-FC and HSV-1 TK/GCV enzyme/prodrug systems generated chemotherapeutic and radiosensitizing effects much greater than what could be achieved with either system alone (Rogulski et al. 1997, 2000; Freytag et al. 1998, 2002b; Khil et al. 1995). Moreover, the CD/5-FC system exhibits a bystander effect that is about tenfold greater than that of HSV-1 TK/GCV (Huber et al. 1994). This important attribute has the potential to extend the killing radius of the oncolytic adenovirus itself. There are several potential advantages of the replication-competent adenovirus platform. First, replication-competent adenoviruses are cytolytic and can result in significant tumor destruction, an effect that is not generally observed with replicationdefective adenoviruses. Not only would this effect reduce the local tumor burden, but it might also facilitate tumor antigen uptake and presentation by professional antigen-presenting cells (APC) following tumor antigen release. Second, owing to their DNA replicative ability, replication-competent adenoviruses result in much higher (103–104 times) therapeutic gene expression per cell relative to replicationdefective adenoviruses. Third, because of their ability to kill the host cell and reinfect neighboring cells, replication-competent adenoviruses have greater potential to spread throughout the tumor than replication-defective adenoviruses. And fourth, owing to the trans-activation activity of E1A, replication-competent adenoviruses express viral antigens de novo and, therefore, are likely to provide a much greater “danger signal” to the immune system relative to replication-defective adenoviruses. Although the latter effect is likely to blunt the short-term effects of adenovirus-based approaches by limiting viral persistence, it might also facilitate the development of long-lasting antitumor immunity by recruiting key immune cells to the tumor site. In the locally recurrent setting, 16 men received an intraprostatic injection of the first-generation Ad5-CD/TKrep adenovirus up to a dose of 1 × 1012 vp followed by 1–2 weeks of 5-FC + GCV prodrug therapy (Freytag et al. 2002a). There were no dose-limiting toxicities (DLTs) and 94% of the adverse events were mild to moderate. Roughly half of patients exhibited significant (>25%) declines in PSA and there were three (19%) objective PSA responses. However, all PSA responses were short-lived (<6 months). A follow-up analysis of this trial was conducted at 5 years (Freytag et al. 2007a). It was found that the gene therapy had a significant effect on PSADT, a nonvalidated, surrogate endpoint that is highly predictive for the development of distant metastases and prostate cancer-specific mortality. For illustration, the PSA profile of the best responding patient spanning 9 years is shown in Fig. 1. Prior to the gene therapy, this patient had a rising PSA with a PSADT of 10  months. Following the gene therapy, his PSA declined rapidly to a nadir of 1.9 ng/mL representing a 79% decline. Over the next 6 years, his PSA rose gradually with a PSADT of 33 months and he developed no symptoms of his disease.

38

S.O. Freytag et al.

Fig. 1  PSA profile of patient spanning 9 years. This patient with a rising PSA after definitive radiotherapy received a single intraprostatic injection of the Ad5-CD/TKrep adenovirus at 1 × 1012 vp followed by 2 weeks of 5-FC + GCV prodrug therapy. The point where the gene therapy (GT) was administered is indicated. PSA values are plotted (black circles). PSADT was calculated by log regression using all available PSA values. The dashed black lines represent the log regression curves. Assuming that salvage AST would be administered at a PSA of 15 ng/mL (black dashed line), the gene therapy would have delayed the implementation of salvage therapy by 6.4 years

He expired of natural causes 6.5 years after the gene therapy at the age of 87 years. His last PSA was 10.1 ng/mL and he never required salvage AST. Thus, the gene therapy moved this patient from a category of having a finite probability of dying from prostate cancer (PSADT <15 months) to one where the likelihood of dying from prostate cancer is remote (PSADT >15 months). Moreover, it freed him from having to experience the significant morbidities associated with AST. When considering all evaluable patients (n  = 14), the PSADT increased following the gene therapy from a mean of 17 to 31 months (median 16–22 months) (p  =  0.014). Three of eight (38%) patients treated with the lower adenovirus doses (1010 and 1011 vp), and five of six (83%) treated with the highest dose (1012 vp), exhibited a significant lengthening of PSADT suggesting a possible dose effect. Because the gene therapy slowed the rate of cancer growth in many patients, it delayed the point when salvage AST was indicated by a mean of 2.1 years in all patients and 2.6 years in patients that received the highest adenovirus dose. The results indicated that the gene therapy may have provided a potential long-term benefit to patients, as demonstrated by a lengthening of the PSADT and delay in when salvage therapy was indicated. At a median follow-up of 9 years, overall and cause-specific survival of this gene therapy (GT) cohort was compared to well-matched historical controls who did not receive the gene therapy (non-GT) (Sandler et al. 2000). The latter study involved 154 men who developed PSA recurrence following definitive radiotherapy and received “standard” salvage therapies. The baseline characteristics of the two groups

3  Gene Therapy of Prostate Cancer

39

Fig. 2  Kaplan–Meier survival analysis of gene therapy (GT) versus nongene therapy (non-GT) cohort. Survival data for non-GT cohort were taken from Sandler et al. (2000). Tick marks indicate the time at which patients were censored due to lack of follow-up. Patients who expired with evidence/symptoms of metastatic disease were declared a death due to prostate cancer

(GT vs. non-GT) were well-matched with respect to: (1) age at PSA recurrence (75 vs. 75 years), (2) preradiation prognostic risk factors (clinical stage – 94 vs. 83% T1/T2; Gleason score – 50 vs. 49% ³7; mean PSA – 12 vs. 16 ng/mL), (3) radiation dose received during primary treatment (67 vs. 72 Gy), and (4) time period in which they received definitive treatment (1984–1998 vs. 1986–1998). Overall (OS) and disease-specific (DSS) survival are depicted in Fig. 2. Fiveyear OS of the GT group was 81 versus 58% for the non-GT group, suggesting a trend effect (p = 0.12, Fisher Exact Test). Five-year DSS was 100 versus 73% (GT vs. non-GT; p = 0.03, Fisher Exact Test). The estimated median OS of the GT group is 8.6 versus 5.9 years for the non-GT group. Although no conclusions can be made from such retrospective analyses, the results raise the possibility that replicationcompetent adenovirus-mediated suicide gene therapy may have the potential to improve the survival of men with locally recurrent prostate cancer. Three phase 1 trials using the same replication-competent adenovirus platform were conducted in the newly-diagnosed setting (Freytag et al. 2003, 2007b; Barton et al. 2008). In all three trials, the gene therapy was combined with standard prostate radiotherapy and targeted men with intermediate- to high-risk disease (Gleason score ³7 or PSA >10 ng/mL). The major difference among these three trials is the investigational agent used. The first trial utilized the same first-generation Ad5-CD/ TKrep adenovirus that was used in the locally recurrent setting. The second and third trials used two different (but related) second-generation adenoviruses both of which contained an improved yeast CD/mutant HSV-1 TKSR39 fusion gene in the E1 region and either the adenovirus death protein (ADP) or human sodium iodide symporter (hNIS) gene in the E3 region. ADP increases the cytolytic effects of oncolytic adenoviruses. hNIS was used as a reporter gene to monitor adenovirus spread and persistence in patients using routine nuclear imaging (Barton et al. 2008). To date, a total of 42 patients have been treated up to an adenovirus dose of 5 × 1012 vp. Overall, the gene therapy/radiotherapy combination has been well tolerated. Gene therapy-related adverse events include transient flu-like symptoms

40

S.O. Freytag et al.

(25%) and transaminitis (33%), which are likely attributable to the oncolytic adenovirus, and hematologic events [lymphopenia (89%), anemia (44%), neutropenia (22%), thrombocytopenia (19%)], which are likely attributable to the 5-FC + vGCV prodrug therapy. Importantly, the gene therapy did not exacerbate the most common side effects of prostate radiotherapy (genitourinary and gastrointestinal events). Efficacy was assessed using two endpoints (1) prostate biopsy (³6 cores) positivity at 2 years, which is prognostic for the development of distant metastases and disease-specific survival, and (2) freedom from biochemical/clinical failure (FFF). Better-than-expected biopsy results were obtained in men treated with the gene therapy/radiotherapy combination (Table  1). When considering all patients biopsied thus far, 7 of 33 (21%) had a positive 2-year biopsy, which is better-thanexpected for men with intermediate- to high-risk disease. When the results were broken down by prognostic risk group, most of the treatment effect was observed in the intermediate-risk group with 2 of 21 (10%) having a positive 2-year biopsy. By contrast, 5 of 12 (42%) high-risk patients were positive for cancer at 2 years, which was not significantly different than what was expected for this risk group. Although longer follow-up is needed, the estimated 5-year FFF for the intermediaterisk group is 95%, which is slightly better than expected (60–85%). By contrast, the estimated 5-year FFF for the high-risk group is 63%, which is within the range expected (40–65%). Together, the biopsy and PSA results raise the possibility that replication-competent adenovirus-mediated suicide gene therapy may have the potential to improve the outcome of prostate radiotherapy in select (i.e., intermediaterisk) patient groups. It is possible that saturating the prostate gland with the adenovirus through the use of higher adenovirus doses will generate better results in the high-risk group. Table 1  Posttreatment prostate biopsy results Two-year prostate biopsy results All patients Expected for EBRT only Gene therapy + EBRT (n = 33) Intermediate-risk patients Expected for EBRT only Gene therapy + EBRT (n = 21) High-risk patients Expected for EBRT only Gene therapy + EBRT (n = 12)

Prostate biopsy status % positive p Value 46 21

0.03

42 10

0.02

51 42

0.55

Expected biopsy results are from Zelefsky et al. (2008b). Results for 70 Gy are shown. In the gene therapy cohort, biopsies were based on a mean of 9 cores taken at 2 years after external beam radiotherapy (EBRT) and those with adenocarcinoma and severe treatment effects were scored positive. Intermediate-risk: Stage T1/T2 and Gleason score 7 or PSA 10–20  ng/mL. High-risk: Stage ³ T3 or Gleason score ³8 or PSA >20 ng/mL

3  Gene Therapy of Prostate Cancer

41

3 Vaccine-Based Gene Therapy Strategies Several different vaccine-based strategies have been evaluated clinically. Such strategies have utilized poxvirus (vaccinia and fowlpox) expressing PSA either alone or in combination with costimulatory molecules, and cell-based vaccines expressing granulocyte-macrophage colony stimulating factor (GM-CSF). They have evaluated in the settings of newly-diagnosed, locally recurrent and metastatic disease. Several have generated provocative results in early stage trials justifying progression to phase 3.

3.1 Poxvirus-Based Vaccines Poxvirus-based vaccines expressing PSA were the first to be evaluated clinically. Poxviruses have several advantages, including proven safety track record, potent adjuvant activity, large genome size facilitating the insertion of therapeutic genes, and ease of manipulation. In the first study conducted at the University of Michigan (Sanda et al. 1999), six patients with locally recurrent prostate cancer after surgery were administered intradermally two doses of vaccinia virus expressing PSA (PROSTVAC). At the time of enrollment, patients were on salvage AST for 2–14 months and had undetectable PSA. Adverse events were limited to transient lowgrade flu-like symptoms, fatigue, and vaccination site erythema. To assess efficacy, AST was interrupted and the rise in PSA was monitored during recovery from the castrate state. Typically, patients will show a rise in PSA within 1–2 months after discontinuation of AST as their testosterone levels recover (although this varies with age). The PSA of one patient remained undetectable for over 8 months after the restoration of testosterone levels, indicative of a possible antitumor response. Several patients developed increased anti-PSA antibody titers following treatment. A number of poxvirus-based approaches have been conducted subsequently and some have generated very provocative results. In a study (E7897) conducted by the Eastern Cooperative Oncology Group (ECOG) (Kaufman et al. 2004), 62 patients with locally recurrent prostate cancer were randomly assigned to receive four vaccinations with (1) fowlpox-PSA (rF-PSA), (2) three rF-PSA followed by one vacciniaPSA (rV-PSA), or (3) one rV-PSA followed by three rF-PSA. All prime/boost vaccination regimens were well tolerated. The most frequent treatment-related adverse event was injection site reaction. When considering all patients, 45% remained free of biochemical progression (defined as £50% increase in PSA), and 78% remained free of clinical progression, for 19 months. Of the three vaccination regimens, the third (rV-PSA followed by three rF-PSA) resulted in the best PSA progression-free survival. Although there were no significant increases in anti-PSA antibody titers, about half of the patients showed PSA-specific T cell responses. In an approach being developed at the National Cancer Institute, recombinant poxvirus-expressing PSA and the T cell costimulatory B7.1 molecule has been

42

S.O. Freytag et al.

evaluated in the settings of newly-diagnosed disease concomitant with prostate radiotherapy, nonmetastatic hormone-refractory disease in combination with antiandrogen therapy, and metastatic disease in combination with docetaxel chemotherapy (Gulley et al. 2002, 2005, 2008; Arlen et al. 2005, 2006, 2007; Lattouf et al. 2006; Madan et al. 2008; Lechleider et al. 2008; Paola et al. 2006). In the newlydiagnosed setting, 30 patients were randomized 2:1 to receive vaccine plus radiotherapy or radiotherapy alone (Gulley et al. 2005). The vaccination course consisted of a single priming injection of two recombinant vaccinia viruses (rV) expressing either PSA or B7.1 as an admixture, followed by monthly booster vaccinations with recombinant fowlpox virus (rF)-expressing PSA. Vaccinations were accompanied by local injections of GM-CSF and low-dose systemic IL-2. Prostate radiotherapy was given between the fourth and sixth vaccinations. The investigational therapy was well tolerated. There were no treatment-related ³ grade 3 events. Thirteen of 17 (76%) patients who completed the full vaccination course developed ³threefold increase in PSA-specific T cells whereas patients in the control arm did not. Interestingly, some patients developed T cell reactivity to prostate antigens not contained in the vaccine indicating an “antigen cascade” effect. In the setting of nonmetastatic hormone-refractory disease, 42 patients who exhibited a rising PSA following salvage AST were randomized to receive either poxvirus-based PSA + B7.1 vaccination or nilutamide (Arlen et al. 2005). Patients in the vaccination arm also received GM-CSF and IL-2. Patients who subsequently exhibited biochemical, but not clinical, progression were allowed to crossover and receive treatment on the other arm. A number of patients on the vaccination arm exhibited prolonged declines and stabilization of PSA. Median time to treatment failure was 9.9 months on the vaccination arm versus 7.6 months in the nilutamide arm. Interestingly, in patients who crossed over, median time to treatment failure was 25.9 months (from initiation of therapy) in the vaccination-to-nilutamide group versus 15.5 months in the nilutamide-to-vaccination group. Follow-up analysis demonstrated that crossover patients who received the vaccination followed by nilutamide demonstrated improved overall survival versus the nilutamide-to-vaccination group (6.2 vs. 3.7 years, p = 0.045) (Madan et al. 2008). This vaccination approach was also evaluated in the setting of hormone-refractory metastatic disease (Gulley et al. 2002; Arlen et al. 2006). In a phase 2 study, 28 patients were randomized to receive either the vaccine and weekly docetaxel or vaccine alone. Patients on the vaccine only arm were allowed to cross over to the combined therapy arm at the time of disease progression. Patients on the combined therapy arm exhibited a greater decrease in PSA velocity relative to those on the vaccine arm (79% of whom crossed over) or historical controls who received docetaxel only. Median biochemical/clinical progression-free survival was 6.1 months for patients on the vaccine arm who crossed over, 3.2 months for patients who received the combined therapy up front, 1.8 months for patients who received the vaccine only, and 3.7 months for historical controls. As was observed in previous trials, a number of patients developed T cell reactivity to PSA as well as prostate antigens not contained in the vaccine. There appeared to be a benefit of administering the vaccine prior to chemotherapy.

3  Gene Therapy of Prostate Cancer

43

The preliminary results obtained with the poxvirus PSA/B7.1 vaccine are very encouraging and suggest that immunotherapy may bolster the effectiveness of subsequent chemotherapy. A more potent poxvirus PSA vaccine containing multiple costimulatory molecules (B7.1, ICAM-1, LFA-3) is now being evaluated in the clinic.

3.2 Cell-Based Vaccines GVAX is an “off-the-shelf” allogenic tumor vaccine comprising GM-CSF-secreting human prostate adenocarcinoma cells. The rationale for using GM-CSF as a cancer therapy is well-founded scientifically, and stems from the fact that it has demonstrated the ability to induce durable, tumoricidal, antitumor immune responses in preclinical models (Dranoff et al. 1993). GM-CSF recruits APC to immunization sites, which, in turn, activate CD4+ and CD8+ T lymphocytes by priming them with oligopeptides derived from dying cancer cells. This ultimately results in the destruction of tumor cells by both T- and B-cell mediated mechanisms. In the first trial, eight men were treated with autologous, GM-CSF-secreting tumor vaccine that was generated ex vivo by retroviral transduction of surgically harvested tumor cells (Simons et al. 1999). Patients received up to six intradermal vaccinations every 3 weeks at two dose levels (1 and 5 × 107 cells/vaccination). Patients were challenged with irradiated, untransduced autologous prostate tumor cells prior to after vaccination to examine delayed type hypersensitivity (DTH) response. The treatment was associated with minimal toxicity. Most of the adverse events were expected and included skin reactions (injection site pain, erythema, swelling, pruritis) and flu-like symptoms (mild low-grade fevers, chills, malaise). All patients demonstrated inflammatory cell infiltrates at the vaccination sites. Immune infiltrates largely comprised neutrophils and eosinophils, which increased significantly relative to prevaccination levels. Eighty-eight percent of patients demonstrated positive DTH tests after vaccination versus 25% prior to vaccination. Postvaccination biopsies were characterized by ingress of macrophages, natural killer cells, T cells, and extensive eosinophilia. Eighty percent of CD3+ T cells expressed CD45RO indicating T cell activation. An increase in antibody titers to prostate tumor antigens were observed in 3 (38%) patients. As expected following prostate resection, all patients demonstrated significant declines in serum PSA. However, all patients ultimately progressed. Owing to the difficulty in obtaining a sufficient number of autologous tumor cells to generate patient-specific tumor vaccines, subsequent trials used “off-the-shelf” allogenic tumor vaccines comprising GM-CSF-secreting human prostate adenocarcinoma cells (GVAX). In a phase 2 study, 34 patients with hormone-refractory, metastatic prostate cancer were vaccinated with a primer dose of 5 × 108 GVAX cells (Simons et  al. 2002; Small et  al. 2007; Higano et  al. 2008). Patients were subsequently administered 12 booster vaccinations every 2 weeks at two dose levels (1 or 3 × 108 cells). At 2-year follow-up, 9 of 22 (41%, two were lost to follow-up) in the

44

S.O. Freytag et al.

low-dose cohort, and 7 of 10 (70%) in the high-dose cohort, were alive indicating a trend toward increased survival with the higher dose. There was also a trend toward a longer median time to disease progression (140 vs. 85 days) in the high-dose group as determined by bone scan. A second phase 2 study used a second-generation GVAX vaccine genetically engineered to express higher levels of GM-CSF (Small et  al. 2004). Eighty patients, in three cohorts, with hormone-refractory, metastatic prostate cancer received an escalating dose of GVAX over a 24-week period. Six of 19 (32%) patients in the high-dose group exhibited PSA declines following repeat vaccinations. The proportion of patients demonstrating an antibody response at 12 weeks correlated with the vaccine dose. The majority (62%) of patients tested exhibited stable or reduced osteoclastic activity, which is positive indicator in this setting. Based on these encouraging phase 2 results, two randomized phase 3 trials were conducted in men with hormone-refractory, metastatic prostate cancer comparing the safety and efficacy of GVAX versus docetaxel and prednisone (Vital 1), and GVAX plus docetaxel versus docetaxel and prednisone (Vital 2). Enrollment in Vital 1 (n = 626) was completed. In Vital 2, an imbalance in death was noted between the two treatment arms after enrolling 408 patients triggering early termination of the study. Subsequent analyses show no significant toxicities in the GVAX plus docetaxel arm that could explain the imbalance in deaths. Eighty-five percent of the deaths in both treatment arms were due to prostate cancer, and there was no trend in the causes of death in the remaining patients. The study sponsor speculated that the decision to omit concomitant prednisone in the GVAX immunotherapy treatment arm to avoid the immunosuppressive effects of prednisone may have contributed to an unfavorable outcome compared to the combination of chemotherapy and prednisone. After the termination of Vital 2, follow-up analysis of Vital 1 showed that there was less than a 30% chance of meeting the primary endpoint of improving overall survival and, therefore, this study was terminated also.

4 Replication-Competent, Oncolytic Adenoviruses Strictly speaking, strategies utilizing oncolytic viruses lacking a therapeutic gene are not gene therapy. However, such approaches have been evaluated in the settings of locally recurrent and metastatic disease and have generated encouraging results in the localized setting. The first such trial conducted at the Johns Hopkins Medical Institute used a PSA-selective, oncolytic adenovirus (CV706) in which E1A expression, and therefore virus replication, was driven by a minimal PSA promoter (DeWeese et  al. 2001). This vector design restricts virus replication to PSA-expressing (i.e., prostate) tissues. Twenty patients with locally recurrent prostate cancer received an intraprostatic injection of CV706 up to a dose of 1 × 1013 vp. Despite this high adenovirus dose, the treatment was associated with low toxicity. Ninety-eight percent of the adverse events were mild to moderate. A minority of patients exhibited transaminitis and there was no grade 2 hepatotoxicity. Five of 20 (25%) patients exhibited an

3  Gene Therapy of Prostate Cancer

45

objective PSA response, which occurred at the two highest adenovirus dose levels suggesting a possible dose effect. One objective response lasted 11 months. A secondary, or delayed, peak of CV706 DNA was detected in patient’s circulation approximately 1 week after the adenovirus injection providing suggestive evidence of viral replication in vivo. Another oncolytic adenovirus (CG7870) related to CV706 was evaluated in the setting of hormone-refractory, metastatic disease (Small et  al. 2006). CG7870 is similar to CV706 except that both the E1A and E1B genes are under the transcriptional control of prostate-specific promoters. In contrast to CV706, CG7870 also contained the immune-modulatory genes of the adenovirus E3 region. Twenty-three patients were administered CG7870 intravenously up to a dose of 6 × 1012 vp. The treatment was well tolerated up to a dose of 3 × 1012 vp. Treatment-related adverse events included mild to moderate flu-like symptoms, hypotension, lymphopenia, thrombocytopenia, and transaminitis. At a dose of 6 × 1012 vp, a constellation of adverse events occurred in two patients halting further dosing. These events included D-dimer formation, a hallmark of disseminated intravascular coagulation, transaminitis and thrombocytopenia. Although none of these events constituted a protocol-defined DLT, accrual was, nevertheless, halted after two patients at this dose level. Five of 23 (22%) patients exhibited a 25–49% decline in PSA, most of which occurred at the higher (>6 × 1011 vp) adenovirus dose levels. Secondary, or delayed, DNA peaks were observed in 70% of patients between days 2 and 8 suggestive of viral replication.

5 Summary So what have we learned after treating over 1,000 prostate cancer patients? Two general principles have emerged, neither of which is surprising. First, when administered carefully and at the appropriate doses, gene therapy for prostate cancer is generally safe. Overall, it has been associated with little toxicity when delivered intraprostatically or systemically. Although the toxicities vary with respect to the approach taken and agent dose, the most common gene therapyrelated side effects include injection site reaction (pain or swelling), flu-like symptoms, transaminitis, and hematologic events. The vast majority of these events are mild to moderate and transient lasting less than a few days. The hepatic and hematologic events generate no noticeable symptoms to the patient and are self-limiting. As with most cancer therapies, the frequency and severity of gene therapy-related side effects increase with agent dose, and systemic approaches tend to be more toxic than local approaches. Importantly, of the strategies evaluated thus far, there do not appear to be any long-term side effects of gene therapy and none have exacerbated the most common side effects of standard prostate cancer treatments. Second, although gene therapy has clearly exhibited antitumor activity when applied as a single agent, its greatest potential may lie in its apparent ability to improve the effectiveness of standard cancer therapies. When delivered intraprostatically in

46

S.O. Freytag et al.

combination with prostate radiotherapy, adenovirus-based approaches have resulted in better-than-expected 2-year biopsy results, at least in select patient groups. It is possible that better local tumor control can be achieved across all prognostic risk groups by saturating the prostate gland with the adenovirus through the use of higher adenovirus doses. Although high-risk patients (Gleason ³8, PSA >20 ng/mL) will still require some form of systemic therapy (i.e., AST) to combat covert extraprostatic disease, here, too, the gene therapy may provide some benefit. Multiple studies have reported a slowing of disease progression (i.e., lengthening of PSADT) following the administration of gene therapy raising the possibility of an antitumor immune effect. Thus, gene therapy appears to have the potential to impact two clinical endpoints (2-year biopsy status and PSADT) that are highly prognostic for disease progression and prostate cancer-specific mortality. In advanced disease settings, poxvirus-based PSA vaccines have been shown to augment the effectiveness of subsequent chemotherapy and may have the potential to extend survival. Although the underlying basis for this effect is unknown, it is welcome news for prostate cancer is generally resistant to chemotherapy. The best available chemotherapeutic drug (Taxotere) extends survival by only 2–3 months. The poxvirus trials add to the growing body of evidence that prostate cancer may be responsive to some forms of immunotherapy. Together, we consider these preliminary results very encouraging and continue to believe that gene therapy will someday earn a place in the management of prostate cancer when the right approach is applied in the right setting. Whether any of these gene therapy strategies is robust enough to extend survival awaits the completion of prospective, randomized, controlled trials. In the meantime, it is important that we gene therapists come “full circle” and take our clinical observations back into the laboratory to better understand the molecular basis for these provocative preliminary findings.

References Arlen P, Gulley J, Todd N, Lieberman R, Steinberg S, Morin S, et al. (2005). Antiandrogen, vaccine and combination therapy in patients with non-metastatic hormone refractory prostate cancer. J Urol 174: 539–546. Arlen P, Gulley J, Parker C, Skarupa L, Pazdur M, Panicali D, et al. (2006). A randomized phase II study of concurrent docetaxel plus vaccine versus vaccine alone in metastatic androgenindependent prostate cancer. Clin Cancer Res 12: 1260–1269. Arlen P, Skarupa L, Pazdur M, Seetharam M, Tsang K, Grosenbach D, et  al. (2007). Clinical safety of a viral vector based prostate cancer vaccine strategy. J Urol 15: 15–20. Ayala G, Satoh T, Li R, Shalev M, Gdor Y, Aguilar-Cordova E, et al. (2006). Biological response determinants in HSV-tk + ganciclovir gene therapy for prostate cancer. Mol Ther 13: 716–728. Barton K, Stricker H, Brown S, Elshaikh M, Aref I, Lu M, et al. (2008). Phase I study of noninvasive imaging of adenovirus-mediated gene expression in the human prostate. Mol Ther 16: 1761–1769. Culver K, Ram Z, Wallbridge S, Ishii H, Oldfield E, and Blaese R. (1992). In vivo gene transfer with retroviral vector-producer cells for treatment of experimental brain tumors. Science 256: 1550–1552.

3  Gene Therapy of Prostate Cancer

47

DeWeese T, van der Poel H, Li S, Mikhak B, Drew R, Goemann M, et al. (2001). A Phase I trial of CV706, a replication-competent, PSA selective oncolytic adenovirus, for the treatment of locally recurrent prostate cancer following radiation therapy. Cancer Res 61: 7464–7472. Dranoff G, Jaffee E, Lazenby A, Golumbek P, Levitsky H, Brose K, et al. (1993). Vaccination with irradiated tumor cells engineered to secrete murine granulocyte-macrophage colony-stimulating factor stimulates potent, specific, and long-lasting anti-tumor immunity. Proc Natl Acad Sci U S A 90: 3539–3543. Freytag S, Rogulski K, Paielli D, Gilbert J, Kim, JH. (1998). A novel three-pronged approach to selectively kill cancer cells: concomitant viral, double suicide gene, and radiotherapy. Hum Gene Ther 9: 1323–1333. Freytag S, Khil M, Stricker H, Peabody J, Menon M, DePeralta-Venturina M, et al. (2002a). Phase I study of replication-competent adenovirus-mediated double suicide gene therapy for the treatment of locally recurrent prostate cancer. Cancer Res 62: 4968–4976. Freytag S, Paielli D, Wing M, Rogulski K, Brown S, Kolozsvary A, et al. (2002b). Efficacy and toxicity of replication-competent adenovirus-mediated double suicide gene therapy in combination with radiation therapy in an orthotopic mouse prostate cancer model. Int J Radiat Oncol Biol Phys 54: 873–886. Freytag S, Stricker H, Pegg J, Paielli D, Pradhan D, Peabody J, et al. (2003). Phase I study of replication-competent adenovirus-mediated double suicide gene therapy in combination with conventional dose three-dimensional conformal radiation therapy for the treatment of newly diagnosed, intermediate- to high-risk prostate cancer. Cancer Res 63: 7497–7506. Freytag S, Stricker H, Peabody J, Pegg J, Paielli D, Movsas B, et al. (2007a). Five-year follow-up of trial of replication-competent adenovirus-mediated suicide gene therapy for treatment of prostate cancer. Mol Ther 15: 636–642. Freytag S, Movsas B, Aref I, Stricker H, Peabody J, Pegg J, et al. (2007b). Phase I trial of replicationcompetent adenovirus-mediated suicide gene therapy with IMRT for prostate cancer. Mol Ther 15: 1016–1023. Fujita T, Teh B, Timme T, Mai W, Satoh T, Kusaka N, et al. (2006). Sustained long-term immune responses after in situ gene therapy combined with radiotherapy and hormonal therapy in prostate cancer patients. Int J Radiat Oncol Biol Phys 65: 84–90. Gulley J, Chen A, Dahut W, Arlen P, Bastian A, Steinberg S, et al. (2002). Phase I study of a vaccine using recombinant vaccinia virus expressing PSA (rV-PSA) in patients with metastatic androgen-independent prostate cancer. Prostate 53: 109–117. Gulley J, Arlen P, Bastian A, Morin S, Marte J, Beetham P, et al. (2005). Combining a recombinant cancer vaccine with standard definitive radiotherapy in patients with localized prostate cancer. Clin Cancer Res 11: 3353–3362. Gulley J, Arlen P, Tsang K, Yokokawa J, Palena C, Poole D, et al. (2008). Pilot study of vaccination with recombinant CEA-MUC-1-TRICOM pox viral-based vaccines in patients with metastatic carcinoma. Clin Cancer Res 14: 3060–3069. Herman J, Adler H, Aguilar-Cordova E, Rojas-Martinez A, Woo S, Timme T, et al. (1999). In situ gene therapy for adenocarcinoma of the prostate: a phase I clinical trial. Hum Gene Ther 10: 1239–1249. Higano C, Corman J, Smith D, Centeno A, Steidle C, Gittleman M, Simons J, Sacks N, Aimi J, Small E. (2008). Phase 1/2 dose-escalation study of a GM-CSF-secreting, allogenic, cellular immunotheraphy for metastatic hormone-refractory prostate cancer. Cancer 113: 975–984. Huber B, Austin E, Richards C, Davis S, Good, S. (1994). Metabolism of 5-fluorocytosine to 5-fluorouracil in human colorectal tumor cells transduced with the cytosine deaminase gene: significant antitumor effects when only a small percentage of tumor cells express cytosine deaminase. Proc Natl Acad Sci U S A 91: 8302–8306. Kaufman H, Wang W, Manola J, DiPaola R, Ko Y, Sweeney C, et al. (2004). Phase II randomized study of vaccine treatment of advanced prostate cancer (E7897): a trial of the Eastern Cooperative Oncology Group. J Clin Oncol 22: 2122–2132. Khil M, Kim JH, Mullen C, Kim S, Freytag S. (1995). Radiosensitization by 5-fluorocytosine of human colorectal carcinoma cells in culture transduced with cytosine deaminase gene. Clin Cancer Res 2: 53–57.

48

S.O. Freytag et al.

Kim JH, Kim S, Brown S, and Freytag S. (1994). Selective enhancement by an antiviral agent of the radiation-induced cell killing of human glioma cells transduced with HSV-tk gene. Cancer Res 54: 6053–6056. Kim JH, Kim SH, Kolozsvary A, Brown S, Kim O, Freytag S. (1995). Selective enhancement of radiation response of herpes simplex virus thymidine kinase transduced 9L gliosarcoma cells in vitro and in vivo by antiviral agents. Int J Radiat Oncol Biol Phys 33: 861–868. Kubo H, Gardner T, Wada Y, Koeneman K, Gotoh G. (2003). Phase I dose escalation clinical trial of adenovirus vector carrying osteocalcin promoter-driven herpes simplex virus thymidine kinase in localized and metastatic hormone-refractory prostate cancer. Hum Gene Ther 14: 227–241. Lattouf J, Arlen P, Pinto P, and Gulley J. (2006). A phase I feasibility study of an intraprostatic prostate-specific antigen-based vaccine in patients with prostate cancer with local failure after radiation therapy or clinical progression on androgen-deprivation therapy in the absence of local definitive therapy. Clin Genitourin Cancer 5: 89–92. Lechleider R, Arlen P, Tsang K, Steinberg S, Yokokawa J, Cereda V, et  al. (2008). Safety and immunologic response of a viral vaccine to prostate-specific antigen in combination with radiation therapy when metronomic-dose interleukin 2 is used as an adjuvant. Clin Cancer Res 14: 5284–5291. Madan R, Gulley J, Schlom J, Steinberg S, Liewehr D, Dahut W, et al. (2008). Analysis of overall survival in patients with non-metastatic castration-resistant prostate cancer treated with vaccine, nilutamide, and combination therapy. Clin Cancer Res 14: 4526–4531. Miles B, Shalev M, Aguilar-Cordova E, Timme T, Lee H, Yang G, et al. (2001). Prostate-specific antigen response and systemic T cell activation after in situ gene therapy in prostate cancer patients failing radiotherapy. Hum Gene Ther 12: 1955–1967. Paola R, Plante M, Kaufman H, Petrylak D, Israeli R, Lattime E, et al. (2006). A phase I trial of pox PSA vaccines (PROSTVAC-VF) with B7-1, ICAM-1, and LFA-3 co-stimulatory molecules (TRICOM) in patients with prostate cancer. J Transl Med 4: 1–5. Patel P, Young J, Mautner V, Ashdown D, Bonney S, Pineda R, et al. (2009). A phase I/II clinical trial in localized prostate cancer of an adenovirus expressing nitroreductase with CB1954. Mol Ther 17: 1292–1299. Rogulski K, Kim JH, Kim SH, Freytag S. (1997). Glioma cells transduced with an E. coli CD/ HSV-1 TK fusion gene exhibit enhanced metabolic suicide and radiosensitivity. Hum Gene Ther 8: 73–85. Rogulski K, Wing M, Paielli D, Gilbert J, Kim JH, Freytag, S. (2000). Double suicide gene therapy augments the therapeutic efficacy of an oncolytic adenovirus through enhanced cytotoxicity and radiosensitization. Hum Gene Ther 11: 67–76. Sanda M, Smith D, Charles L, Hwang C, Pienta K, Schlom J, et al. (1999). Recombinant vacciniaPSA (PROSTVAC) can induce a prostate-specific immune response in androgen-modulated human prostate cancer. Urology 53: 260–266. Sandler H, Dunn R, McLaughlin P, Hayman J, Sullivan M, and Taylor J. (2000). Overall survival after prostate-specific-antigen-detected recurrence following conformal radiation therapy. Int J Radiat Oncol Biol Phys 48: 629–633. Satoh T, Teh B, Timme T, Mai W, Gdor Y, Kusaka N, et al. (2004). Enhanced systemic T-cell activation after in situ gene therapy with radiotherapy in prostate cancer patients. Int J Radiat Oncol Biol Phys 59: 562–571. Simons J, Mikhak B, Chang J-F, DeMarzo A, Carducci M, Lim M, et  al. (1999). Induction of immunity to prostate cancer antigens: results of a clinical trial of vaccination with irradiated autologous prostate tumor cells engineered to secrete granulocyte-macrophage colony-stimulating factor using ex vivo gene transfer. Cancer Res 59: 5160–5168. Simons J, Nelson W, Nemuniatis J, Centeno A, Dula E, Urba W, et al. (2002). Phase II trials of a GMCSF gene-transduced prostate cancer cell line vaccine (GVAX) in hormone refractory prostate cancer. Proc Am Soc Clin Oncol 22: 172.

3  Gene Therapy of Prostate Cancer

49

Small E, Higano C, Smith D, Corman J, Centero A, Streidle C, et al. (2004). A phase 2 study of an allogeneic GM-CSF gene-transduced prostate cancer cell line vaccine in patients with metastatic hormone-refractory prostate cancer (HRPC). J Clin Oncol 22: 4565. Small E, Carducci M, Burke J, Rodriguez R, Fong L, van Ummersen L, et al. (2006). A phase I trial of intravenous CG7870, a replication-selective, prostate-specific antigen-targeted oncolytic adenovirus, for the treatment of hormone-refractory, metastatic prostate cancer. Mol Ther 14: 107–117. Small E, Sacks N, Nemunaitis J, Urba W, Dula E, Centeno A, Nelson W, Ando W, Howard C, Borellini F, Nguyen M, Hege K, Simons J. (2007). Granulocyte macrophage colony-stimulating factor-secreting allogenic cellular immunotheraphy for hormone-refractory prostate cancer. Clin Cancer Res 13: 3883–3891. Takamiya Y, Short M, Ezzeddine Z, Moolten F, Breakefield X, and Martuza R. (1992). Gene therapy of malignant brain tumors: a rat glioma line bearing the herpes simplex virus type 1-thymidine kinase gene and wild type retrovirus kills other tumor cells. J Neurosci Res 33: 493–503. Teh B, Aguilar-Cordova E, Kernen K, Chou C, Shalev M, Vlachaki M, et al. (2001). Phase I/II trial evaluating combined radiotherapy and in situ gene therapy with or without hormonal therapy in the treatment of prostate cancer- a preliminary report. Int J Radiat Oncol Biol Phys 51: 605–613. Teh B, Ayala G, Aguilar L, Mai W, Timme T, Vlachaki M, et al. (2004). Phase I-II trial evaluating combined intensity-modulated radiotherapy and in situ gene therapy with or without hormonal therapy in treatment of prostate cancer-interim report on PSA response and biopsy data. Int J Radiat Oncol Biol Phys 58: 1520–1529. Zelefsky M, Eastman J, Sartor O, and Kantoff P. (2008a). Cancer of the prostate, In DeVita V., Lawrence, T., Rosenberg, S. (eds): Cancer: Principles & Practice of Oncology, Philadelphia, JB Lippincott, pp 1392–1452. Zelefsky M, Reuter V, Fuks Z, Scardino P, and Shippy A. (2008b). Influence of local tumor control on distant metastases and cancer related mortality after external beam radiotherapy for prostate cancer. J Urol 179: 1368–1373.

Chapter 4

siRNA Versus shRNA for Personalized Cancer Therapy: Mechanisms and Applications John S. Vorhies, Donald D. Rao, Neil Senzer, and John Nemunaitis

Abstract  RNA interference (RNAi) is a natural process of gene regulation that can be harnessed to knock down gene and protein targets with high specificity and selectivity. Proteomic and genomic approaches to target identification will soon allow investigators to rapidly indentify biorelevant cancer signal transduction network hubs that are more likely to be susceptible to a therapeutically effective targeted attack by RNAi. At present, the principle methods of mediating the RNAi effect involve synthetic small interfering RNA (siRNA) oligomers and DNA vector driven expression of short hairpin RNA (shRNA). Both these methods can achieve robust and specific knockdown, but they have striking mechanistic differences with broad practical implications. shRNA can effectively target knockdown with low copy numbers and longer lasting effects than siRNA. Bifunctional design has similar benefits to standard shRNA but with greatly enhanced potency. Effective delivery and avoidance of unwanted off-target effects remain as challenges to the clinical development of siRNA and shRNA. This chapter compares and contrasts siRNA, shRNA, and bifunctional shRNA as candidates for personalized solid tumor therapeutics. Keywords  RNAi • Personalized • Cancer therapeutic

J. Nemunaitis (*) Gradalis, Inc., Dallas, TX, USA and Mary Crowley Cancer Research Centers, Dallas, TX, USA and Texas Oncology PA, Dallas, TX, USA and Baylor Sammons Cancer Center, Dallas, TX, USA e-mail: [email protected] J.A. Roth (ed.), Gene-Based Therapies for Cancer, Current Cancer Research, DOI 10.1007/978-1-4419-6102-0_4, © Springer Science+Business Media, LLC 2010

51

52

J.S. Vorhies et al.

1 Introduction Refinement of our basic understanding of cancer mechanisms is now shifting ­treatment development from traditional, broadly cytotoxic chemotherapy to more selective approaches. Tumors have long been grouped into clinical and histological subtypes, but significant variation in response to treatment still exists within these subtypes. The underlying variation in tumor gene expression patterns may explain the widely divergent responses to treatment regimens most often prescribed by histological type. Molecular subtyping has the potential to further refine patient treatment groups and to improve treatment outcomes, thereby, establishing a “so-called” personalized medicine approach. As already efficient high-throughput methods are accelerated, the possibility of personalized medicine is becoming a reality. The ultimate goal of personalized therapy is to make the drug development process, from target identification to treatment, feasible in a timescale relevant to a single patient. This would allow physicians to consider each patient’s tumor as a subtype of its own, characterizing it and delivering appropriate treatment. RNA interference (RNAi) is an evolutionarily conserved gene-silencing mechanism that occurs endogenously when small sequences of double stranded RNA, termed microRNA, suppress the translation of partially complementary posttranscriptional mRNA. When exogenously induced, it can be a powerful mechanism for targeted knockdown of over- or constitutively expressed molecular targets. It also has significant practical advantages over small molecules and antibodies in terms of production, potency, and specificity. RNAi is young as a potential therapeutic modality and there are several candidate mechanisms for inducing it. The two primary modes for inducing RNAi are through the introduction of chemically synthesized double-stranded oligomers, called small interfering RNA (siRNA) or through the introduction of a DNA vector, which expresses a short hairpin RNA (shRNA) within the target cells. These two modes have important mechanistic advantages and disadvantages relevant in terms of clinical efficacy, durability, off-target effects, and delivery (Rao et al. 2009).

2 Personalized Cancer Therapy Signal transduction networks in cancer are quite robust to random individual gene/ protein target knockout owing to the presence of functional redundancy and a scalefree interaction topology. Random pathway component failure predominantly affects targets with low connectivity within the network, thereby having limited functional impact. Highly connected information-transfer nodes are particularly vulnerable to attack and constitute weak points in the network. This property is exacerbated in cancer because oncogenic change tends to make cells more highly dependent on a specific rewired pathway (Letai 2008). Exploiting the vulnerability that such pathway dependence creates is useful for its lethality to cancer cells and its decreased

4  siRNA Versus shRNA for Personalized Cancer Therapy: Mechanisms and Applications

53

likelihood of perturbing normal cell function. Personalized RNAi-based therapeutics­ are particularly well suited to take advantage of these mechanisms. Our group is currently developing a model for personalized RNAi-based therapy: we harvested tumor and normal cells from cancer patients, comparing expression profiles for malignant versus normal tissue at the mRNA and protein level by microarray and proteomic analysis (Nemunaitis et al. 2007). The resulting expression data was further analyzed by a computational system developed by our team specifically for clinical application including, but not limited to, gene set enrichment analysis and network inference modeling platforms. This allowed us to prioritize overexpressed potential targets based on their probability of being highly connected, nonredundant points in the network. This individualized target fingerprint then served as the template for the design, synthesis, and validation of individualized therapeutic RNAi molecules with knockdown activity against these targets.

3 Mechanisms of RNAi Whether induced by shRNA or siRNA, the RNAi silencing process, as it is currently understood, converges into a final common set of pathways mediated through short (19–23  bp) oligomers of duplex RNA with 2–3 nt 3¢ overhangs on each strand. The two strands of the duplex are termed the guide (antisense) strand, which is complementary to the target mRNA sequence, and the passenger (sense) strand, which may be completely complementary to the guide strand or it may contain mismatches. Figure 1 summarizes the main entry points into this pathway by exogenous RNAi as well as the end-mechanisms of silencing.

3.1 siRNA Effective exogenous induction of RNAi was initially demonstrated by the application of RNA oligomers (Fire et al. 1998). Once in the cytoplasm, siRNA associates with several proteins that make up the RNA-interfering silencing complex (RISC). Depending on various factors, including the duplex mismatching and the nature of the RISC, RNAi can proceed through “cleavage dependent” or “cleavage independent” pathways. The major component of the RISC is the argonaute family of proteins (Ago1, Ago2, Ago3, and Ago4). Within this family, only Ago2 contains the endonuclease activity. The remaining three members of Argonaute family do not have identifiable endonuclease activity, and presumably function through a cleavageindependent manner (Farazi et al. 2008; Paroo et al. 2007). During RISC assembly in the cleavage dependent mechanism, the passenger strand is cleaved by the RNase H like activity of Ago2 and, provided thermodynamically favorable conditions,

54

J.S. Vorhies et al.

Fig. 1  Schematic of the cytoplasmic siRNA and shRNA mediated RNAi pathways. siRNA and shRNA are introduced in different ways, but they converge on a common set of pathways in the cytoplasm

the two strands of the duplex are separated. The RISC then scans mRNAs for target sites to which it binds and Ago2 cleaves the mRNA at a single site between nucleotides10 and 11 from the 5¢ end of the guide strand, thereby initiating degradation. The RISC can then dissociate and execute multiple rounds of RNAi (Paroo et al. 2007). If there are mismatches in the duplex RNA, a different, cleavage-independent RISC is assembled that lacks Ago2 endonuclease capacity. During the assembly of the cleavage-independent RISC, the passenger strand is induced to unwind and be

4  siRNA Versus shRNA for Personalized Cancer Therapy: Mechanisms and Applications

55

released by an ATP-dependent helicase. RISCs without endonucleolytic activity scan mRNAs and predominantly bind to partially complementary target sites located at the 3¢ UTR, repressing translation through mRNA sequestration in processing bodies (p-bodies). Phosphorylation of Ago 2 on Serine-387 seems to affect its localization to p-bodies. The functional implications of this are still unknown, but it may repr­ esent an important regulatory step in RNAi via p-body sequestration. The exogenously applied RNAi constructs can be designed to participate in either or both pathways (Grimm 2009; Paroo et al. 2007). Synthetic siRNA enters into the RNAi pathway at the stage of RISC assembly, but if the oligomer is longer than 19–23 bp, it requires processing by a multidomain RNase III-related endonuclease called Dicer before being loaded onto the RISC. Dicer preferentially binds to the 5¢ phosphate of 2 nt 3¢ over-hang and cleaves double-stranded RNA into 21 to 22 nucleotide siRNAs. It also forms an integral component of endogenous RNAi, processing pre-microRNA to mature miRNA and transferring the processed products to the RISC (Macrae et al. 2006; Carmell and Hannon 2004)

3.2 shRNA Several years after exogenous RNAi was discovered, it was shown that RNAi could be induced by the in vitro transcription of shRNA using a T7 RNA polymerase or a U6 promoter on a plasmid construct (Yu et al. 2002; Miyagishi and Taira 2002). shRNAs, unlike siRNAs, are synthesized in the nucleus of cells similarly to miRNA. Thus, studies on the biogenesis of miRNAs have provided the groundwork for understanding the synthesis and maturation of shRNA. shRNA is introduced as a DNA vector encoding an hairpin-like stem-loop structure. Once transcribed in the nucleus, if integrated into a miR30 scaffold, the hairpin containing the pre-shRNA-like construct is processed to a pre-shRNA by a complex containing the RNase III enzyme Drosha and the double-stranded RNA binding domain protein DGCR8 (Fig. 1). The complex measures the hairpin and allows precise processing of the long primary transcripts into individual shRNAs with a 2 nt 3¢ overhang. This processed primary transcript or an exogenous pol IIIbased stem-loop structure is then transported to the cytoplasm by Exportin V via a Ran-GTP-dependent mechanism (Grimm 2009). In the cytoplasm, the pre-shRNA undergoes a dicer-mediated endonucleolytic cleavage step, in which the loop of the hairpin is processed off to form a doublestranded siRNA with 2 nt 3¢ overhangs. Dicer interacts with the double-stranded Tat-RNA-binding protein (TRBP) or PACT (PKR-activating protein) to mediate siRNA production from shRNA (Pillai et al. 2007; Paroo et al. 2007). The activity of exogenous siRNA, unlike shRNA, does not depend on the TRBP/PACT/Dicer complex. After this last processing step, the Dicer-containing complex coordinates loading onto the RISC. Once on the RISC, shRNA and siRNA should follow the same pathways.

56

J.S. Vorhies et al.

3.2.1 Bifunctional shRNA The concept of bifunctional shRNA rests on the hypothesis that an shRNA ­construct can be designed to utilize both cleavage-dependent and cleavage-independent RISCs. The design of the bifunctional shRNA expression unit consists of two stemloop shRNA structures; one stem-loop structure composed of a fully matched passenger and guide strand duplex for cleavage-dependent RISC loading, the second stem-loop structure composed of a mismatched passenger strand (at the position 9–12) and guide strand for cleavage-independent RISC loading (Fig. 2). Simultaneous expression of both cleavage-dependant and cleavage-independent shRNAs in cells should achieve a higher level of efficacy, greater durability, and more rapid onset than either siRNA or standard shRNA. Multitarget shRNA expression systems have been validated using in vitro (Cheng et al. 2009) cancer systems. There also exists evidence to suggest that this type of functional redundancy is active within the endogenous RNAi system as well. Most mRNAs have multiple miRNA target sites, which allow for cooperative downregulation. In vitro data also suggests that miRNA sequences with the same target and even the same sequence

Fig.  2  Schematic of the bifunctional shRNA Vector design and mechanism. A construct that encodes two shRNAs for each targeted mRNA promotes translation repression through both cleavage-dependent and cleavage-independent RISCs

4  siRNA Versus shRNA for Personalized Cancer Therapy: Mechanisms and Applications

57

naturally associate with different RISCs in  vivo (Azuma-Mukai et  al. 2008; Landthaler et al. 2008).

4 SiRNA Versus shRNA 4.1 Comparative Efficacy shRNA generally has higher efficacy than siRNA when directed to the same target and in vitro. McCleary and colleagues tested shRNA and siRNA directed against firefly luciferase in HeLa cells. More effective inhibition was seen with the shRNA. In an Hepatitis-C virus (HCV) model, 19 and 25 bp shRNAs were compared with 19 and 25 bp siRNA directed against the HCV internal ribosomal entry site using a luciferase reporter in the AVA5 cell line with stable expression (Vlassov et  al. 2007). Both the shRNAs were more potent than either of the 19- or 25-bp siRNAs used. Takahashi et al. (2009) compared a luciferase-directed shRNA driven by different promoters to siRNA of the same sequence in melanoma cells and found that shRNA driven by a U6 promoter was at least 100-fold more potent and longer lasting than siRNA. Comparison of siRNA and shRNA in vivo is difficult as equivalency of strand biasing may not be assured. Several studies have used luciferase reporter systems to quantify siRNA versus shRNA potency in vivo. McAnuff and colleagues (2007) found that siRNA and shRNA are equivalent in potency at 10 mg dose; however, on a molar basis, the shRNA was 250-fold more effective than the siRNA. In a murine HCV model siRNA and shRNA constructs were directed against the nonstructural protein 5B viral polymerase coding region fused with a luciferase gene. siRNA resulted in a 75% expression reduction while shRNA produced a 92.8% average reduction over three experiments (McCaffrey et al. 2002).

4.2 Dicer/Drosha Expression in Cancer and RNAi Effector Suitability Low levels of Dicer and Drosha have been found in tumor samples from patients with ovarian cancer and breast cancer (Merritt et al. 2008; Grelier et al. 2009). In one of these studies (Merritt et al. 2008), shRNA was found to be less effective than siRNA in cells with low Dicer expression. These expression findings stand in contrast to other studies including those that have noted either no downregulation or upregulation of both Drosha and Dicer in ovarian tumors (Lin Zhang et al. 2008; Flavin et  al. 2008). Further investigations of Dicer/Drosha expression in human cancers are needed, but these findings raise the possibility that, at least in some cases, the clinical efficacy of shRNA may be affected by the expression patterns of endogenous miRNA processing machinery (Rao et al. in press).

58

J.S. Vorhies et al.

4.3 Off-Target Effects There are multiple specific and nonspecific mechanisms through which siRNA and shRNA can cause effects other than the intended mRNA suppression. Specific offtarget effects are mediated by partial sequence complementarity of the RNAi construct to mRNAs other than the intended target. Nonspecific off-target effects include a wide variety of immune- and toxicity-related effects that are intrinsic to the RNAi construct itself or its delivery vehicle.

4.3.1 Specific Off-Target Effects In vitro, siRNA creates off-target expression patterns that are unique and consistent for a given sequence. They also appear to be unrelated to target knockdown (Jackson et al. 2003). Complementarity of the mRNA 3¢UTR with nucleotides 2–7 at the 5¢ end of either the siRNA passenger or guide strands has been shown to be a key determinant in directing off-target effects. This is reminiscent of the “seed” region within miRNA, which guides silencing through complementarity with the 3¢UTR of an mRNA (Birmingham et al. 2006). Although sequence optimization to reduce specific off-target effects will benefit both siRNA and shRNA discriminative functionality, unlike shRNA, siRNA oligomers can be chemically modified to reduce direct off-target effects. Various modifications can encourage preferential strand selection, limit the construct’s association with a certain class of RISC, or discourage seed region complementarity based offtarget effects (Behlke 2008). shRNA seems to cause fewer specific off-target effects than siRNA, potentially because of its use of endogenous processing and regulatory mechanisms (Rao et al. in press). The susceptibility of siRNA to cytoplasmic degradation may also lead to more off-target effects. In one study, shRNA and siRNA of the same core sequence directed toward TP53 were applied to HCT-116 colon carcinoma cells in concentrations necessary to achieve comparable levels of target knockdown. Microarray profiling demonstrated a much higher degree of up- and downregulation of off-target transcripts in the siRNA transfected cells (M. Mehaffey, T. Ward, and M. Cleary, in prep.).

4.3.2 Nonspecific Off-Target Effects Activation of the innate immune system in the case of exogenous RNAi is likely mediated through cytoplasmic and endosomal mechanisms attuned to recognize exogenous nucleic acids from infectious agents. Introduction of dsRNA longer than 29–30  bp into mammalian cells activates receptors sensitive to exogenous nucleic acids, such as Toll-Like Receptors (TLR), and induces the innate immune system, leading to global degradation of mRNA and upregulation of interferon (IFN)-stimulated gene expression. Though siRNA constructs are less immunogenic

4  siRNA Versus shRNA for Personalized Cancer Therapy: Mechanisms and Applications

59

than longer dsRNA, both siRNA and shRNA can induce a partial IFN response (Robbins et al. 2006). Misinterpreting an immunologic effect of siRNA as a direct effect must be carefully avoided, as naked siRNA has been shown to activate the RNA-sensitive TLR-3 on the surface of vascular endothelial cells, triggering the release of IFN-g and IL-12 that mediate nonspecific antiangiogenic effects in vivo (Kleinman et al. 2008). Activation of TLR 3 is not an issue for shRNA because the construct is presented on a DNA vector. However, TLR 9 is present in the endosome and is activated by unmethylated DNA CpG motifs, necessitating careful plasmid design to avoid immunoactivation (Robbins et al. 2009). Sequence and chemical modification of siRNA (particularly the 2¢ site) can attenuate the immune response (Robbins et al. 2009). shRNA is less likely to induce an inflammatory response through cytoplasmic dsRNA receptors because it is spliced by endogenous mechanisms. In an experiment that compared liposomedelivered siRNA and shRNA in primary CD34+ progenitor-derived hematopoietic cells, it was shown that siRNA induced IFN-alpha and type I IFN genes, while the shRNA of the same sequence did not induce an immune response (Grimm and Kay 2007). Another study showed that modifying an shRNA by integration within an miR-30 scaffold could also decrease the IFN response (Bauer et al. 2009). Over-saturation of nuclear membrane Exportin V and Ago2 by shRNA (particularly at high concentrations) can cause dose-dependent liver injury as a result of downregulation of critical endogenous miRNAs, which rely on the same proteins (Grimm 2009). siRNA avoids this problem and can achieve suppression of a target gene without disrupting endogenous miRNA levels (John et  al. 2007). The oversaturation effect may be promoter related, as stable target gene suppression was subsequently demonstrated at high shRNA doses in a murine model for over one using a pol II promoter system (Grimm 2009). This indicates that selective promoter integration and careful dosing of shRNA is needed to avoid competitive inhibition of the endogenous miRNA biogenesis machinery.

5 Delivery Strategies for Clinical Translation Clinical efficacy of an RNAi cancer therapeutic is limited by the properties of its delivery vehicle. In the case of siRNA, knockdown is directly related to the quantity of the oligomer that enters the tumor cells, whereas in the case of shRNA, the expression vector must reach the nucleus for gene silencing to be achieved. Issues of safety, selective tumor targeting, pharmacokinetics, and pharmacodynamics are also affected by the delivery vehicle. These include resisting host defenses, reaching­ the tumor while avoiding normal tissue, negotiating cell penetration, and, when apropos, endocytosis then endosomal/lysosomal escape and, in the case of shRNA, penetration of the nuclear membrane. Viral vectors have received some clinical attention but concerns over efficient systemic delivery and immunogenicity may limit their clinical utility.

60

J.S. Vorhies et al.

There are three major classes of nonviral delivery vehicle systems: synthetic polymers, natural/biodegradable polymers, and lipids (Vorhies and Nemunaitis 2009; Vorhies and Nemunaitis 2007; Whitehead et  al. 2009). Hybrids of these can be effective. For instance, there is a cyclodextrin-based cationic polymer which has been used successfully to deliver siRNA targeted to RRM2 in various in vivo cancer models. The same formulation, now called CALAA-01 is currently in Phase I clinical trials (Heidel et al. 2007). Lipid-based nanoparticles are also showing potential for clinical delivery of shRNA and siRNA. Silence Therapeutics has developed a lipid-based delivery vehicle specifically designed for the delivery of siRNA Targeting Protein Kinase N3 endothelial cells. This vehicle, called AtuPLEX, contains a mix of cationic and fusogenic lipids (Aleku et al. 2008). A Phase I trial is currently recruiting to investigate an siRNA therapeutic delivered with AtuPLEX.

6 Conclusions Both siRNA and shRNA have excellent potential for clinical use within the emerging­ paradigm of scale-free biomolecular networks and their inherent integration of evolution and structure. However, several challenges remain that warrant further study at both the preclinical and clinical levels. Mechanisms influencing RNAi silencing efficacy, off-target effects, and delivery are crucial areas for further study, preclinical assessment and clinical translation. The transient effect of siRNA may be more suited to the treatment of infectious disease whereas the heightened potency and temporal and spatial control of shRNA may better suit it for the systemic treatment of malignancy. Finally, bi-shRNA represents an important therapeutic development which, by exploiting the latest advances in our understanding of RNAi mechanisms, may bring us closer to an optimized personalized cancer therapy.

References Aleku M, Schulz P, Keil O, Santel A, Schaeper U, Dieckhoff B, Janke O, Endruschat J, Durieux B, Röder N, Löffler K, Lange C, Fechtner M, Möpert K, Fisch G, Dames S, Arnold W, Jochims K, Giese K, Wiedenmann B, Scholz A, Kaufmann J. Cancer Res. 2008;68:9788–9798. Azuma-Mukai A, Oguri H, Mituyama T, Qian ZR, Asai K, Siomi H, Siomi MC. Proc. Natl. Acad. Sci. U.S.A 2008;105:7964–7969. Bauer M, Kinkl N, Meixner A, Kremmer E, Riemenschneider M, Förstl H, Gasser T, Ueffing M. Gene Ther 2009;16:142–147. Behlke MA. Oligonucleotides 2008;18:305–319. Birmingham A, Anderson EM, Reynolds A, Ilsley-Tyree D, Leake D, Fedorov Y, Baskerville S, Maksimova E, Robinson K, Karpilow J, Marshall WS, Khvorova A. Nat Methods 2006;3:199–204. Carmell MA, Hannon GJ. Nat. Struct. Mol. Biol. 2004;11:214–218.

4  siRNA Versus shRNA for Personalized Cancer Therapy: Mechanisms and Applications

61

Cheng TL, Teng CF, Tsai WH, Yeh CW, Wu MP, Hsu HC, Hung CF, Chang WT. Cancer Gene Ther. 2009;16:516–531. Farazi TA, Juranek SA, Tuschl T. Development 2008;135:1201–1214. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Nature 1998;391:806–811. Flavin RJ, Smyth PC, Finn SP, Laios A, O’Toole SA, Barrett C, Ring M, Denning KM, Li J, Aherne ST, Aziz NA, Alhadi A, Sheppard BL, Loda M, Martin C, Sheils OM, O’Leary JJ. Mod. Pathol. 2008;21:676–684. Grelier G, Voirin N, Ay A, Cox DG, Chabaud S, Treilleux I, Léon-Goddard S, Rimokh R, Mikaelian I, Venoux C, Puisieux A, Lasset C, Moyret-Lalle C. Br. J. Cancer 2009;101: 673–683. Grimm D, Kay MA. J. Clin. Invest. 2007;117:3633–3641. Grimm D. Adv. Drug Deliv. Rev. 2009;61:672–703. Heidel JD, Liu JY, Yen Y, Zhou B, Heale BSE, Rossi JJ, Bartlett DW, Davis ME. Clin. Cancer Res. 2007;13:2207–2215. Jackson AL, Bartz SR, Schelter J, Kobayashi SV, Burchard J, Mao M, Li B, Cavet G, Linsley PS. Nat. Biotechnol. 2003;21:635–637. John M, Constien R, Akinc A, Goldberg M, Moon Y, Spranger M, Hadwiger P, Soutschek J, Vornlocher H, Manoharan M, Stoffel M, Langer R, Anderson DG, Horton JD, Koteliansky V, Bumcrot D. Nature 2007;449:745–747. Kleinman ME, Yamada K, Takeda A, Chandrasekaran V, Nozaki M, Baffi JZ, Albuquerque RJC, Yamasaki S, Itaya M, Pan Y, Appukuttan B, Gibbs D, Yang Z, Karikó K, Ambati BK, Wilgus TA, DiPietro LA, Sakurai E, Zhang K, Smith JR, Taylor EW, Ambati J. Nature 2008; 452:591–597. Landthaler M, Gaidatzis D, Rothballer A, Chen PY, Soll SJ, Dinic L, Ojo T, Hafner M, Zavolan M, Tuschl T. RNA 2008;14:2580–2596. Letai AG. Nat. Rev. Cancer 2008;8:121–132. Macrae IJ, Zhou K, Li F, Repic A, Brooks AN, Cande WZ, Adams PD, Doudna JA. Science 2006;311:195–198. McAnuff MA, Rettig GR, Rice KG. J. Pharm. Sci. 2007;96:2922–2930. McCaffrey AP, Meuse L, Pham TT, Conklin DS, Hannon GJ, Kay MA. Nature 2002;418:38–39. Merritt WM, Lin YG, Han LY, Kamat AA, Spannuth WA, Schmandt R, Urbauer D, Pennacchio LA, Cheng J, Nick AM, Deavers MT, Mourad-Zeidan A, Wang H, Mueller P, Lenburg ME, Gray JW, Mok S, Birrer MJ, Lopez-Berestein G, Coleman RL, Bar-Eli M, Sood AK. N. Engl. J. Med. 2008;359:2641–2650. Miyagishi M, Taira K. Nat. Biotechnol 2002;20:497–500. Nemunaitis J, Senzer N, Khalil I, Shen Y, Kumar P, Tong A, Kuhn J, Lamont J, Nemunaitis M, Rao D, Zhang Y, Zhou Y, Vorhies J, Maples P, Hill C, Shanahan D. Cancer Gene Ther. 2007;14:686–695. Paroo Z, Liu Q, Wang X. Cell Res. 2007;17:187–194. Pillai RS, Bhattacharyya SN, Filipowicz W. Trends Cell Biol. 2007;17:118–126. Rao DD, Maples PB, Senzer N, Kumar P, Wang Z, Pappen BO, Yu Y, Haddock C, Jay C, Phadke AP, Chen S, Kuhn J, Dylewski D, Scott S, Monsma D, Webb C, Tong A, Shanahan D, Nemunaitis J. Cancer Gene Ther. (in press). Rao DD, Vorhies JS, Senzer N, Nemunaitis J. Adv. Drug Deliv. Rev. 2009;61:746–759. Robbins M, Judge A, MacLachlan I. Oligonucleotides 2009;19:89–102. Robbins MA, Li M, Leung I, Li H, Boyer DV, Song Y, Behlke MA, Rossi JJ. Nat. Biotechnol. 2006;24:566–571. Takahashi Y, Yamaoka K, Nishikawa M, Takakura Y. J. Pharm. Sci. 2009;98:74–80. Vlassov AV, Korba B, Farrar K, Mukerjee S, Seyhan AA, Ilves H, Kaspar RL, Leake D, Kazakov SA, Johnston BH. Oligonucleotides 2007;17:223–236. Vorhies JS, Nemunaitis J. Expert Rev. Anticancer Ther. 2007;7:373–382. Vorhies JS, Nemunaitis JJ. Methods Mol. Biol. 2009;480:11–29. Whitehead KA, Langer R, Anderson DG. Nat. Rev. Drug Discov. 2009;8:129–138.

62

J.S. Vorhies et al.

Yu J, DeRuiter SL, Turner DL. Proc. Natl. Acad. Sci. U.S.A 2002;99:6047–6052. Zhang L, Volinia S, Bonome T, Calin GA, Greshock J, Yang N, Liu C, Giannakakis A, Alexiou P, Hasegawa K, Johnstone CN, Megraw MS, Adams S, Lassus H, Huang J, Kaur S, Liang S, Sethupathy P, Leminen A, Simossis VA, Sandaltzopoulos R, Naomoto Y, Katsaros D, Gimotty PA, DeMichele A, Huang Q, Bützow R, Rustgi AK, Weber BL, Birrer MJ, Hatzigeorgiou AG, Croce CM, Coukos G. Proc. Natl. Acad. Sci. U.S.A. 2008;105:7004–7009.

Chapter 5

Tumor Suppressor Gene Therapy Jack A. Roth, John Nemunaitis, Lin Ji, and Rajagopal Ramesh

Abstract  Recent advances in genetics, molecular biology, and molecular pharmacology have resulted in the development of molecularly targeted therapies. Targeting specific molecular pathways essential for the survival of cancer cells would personalize treatment with the potential to improve outcome and minimize toxicities. In this chapter, we review gene-based targeted therapies for cancer. Discussion focuses on replacement therapies for abnormal p53 function and FUS1 tumor suppressor gene-mediated molecular therapy using nanoparticles for systemic gene delivery. Keywords  Gene • Molecular • Tumor suppressor • Cancer therapy The management of many common cancers has changed through the years with the development of targeted drugs such as angiogenesis and tyrosine kinase inhibitors. However, despite these recent advances, metastatic disease patients receiving frontline treatment with chemotherapy alone or in combination with angiogenesis and tyrosine kinase inhibitors have had only an incremental improvement in survival. The cancer cell has developed six alterations which contribute to malignant growth. These include self sufficiency, insensitivity to growth inhibition (including immune “escape”), independence from programmed cell death, unlimited replicative potential, sustained angiogenesis, and local and metastatic invasiveness (Hanahan and Weinberg 2000). In many instances, these alterations occur because of the inactivation of tumor suppressor genes which may regulate multiple pathways. Targeting a single pathway does not often lead to a robust therapeutic effect because the cancer cell is capable of maintaining its functional characteristics through dynamic feedback loops (Carlson and Doyle 2002; Stelling et al. 2004).

J.A. Roth (*) Anderson Cancer Center, The University of Texas M.D, Houston, TX, USA e-mail: [email protected] J.A. Roth (ed.), Gene-Based Therapies for Cancer, Current Cancer Research, DOI 10.1007/978-1-4419-6102-0_5, © Springer Science+Business Media, LLC 2010

63

64

J.A. Roth et al.

Cancer cells can survive targeted inhibition of growth factor signaling pathways by virtue of having redundant functional pathways in which different proteins have overlapping functions (Edelman and Gally 2001). Positive and negative feedback controls may allow cancer cells to bypass the growth inhibitory effects of single pathway blockade. Efforts to improve therapeutic outcomes have focused on innovative approaches involving gene replacement. Theoretically, restoration of single tumor suppressor gene function could restore pathway functions that block several of the alterations in malignant cells. The purpose of this chapter is to summarize key tumor suppressor gene replacement strategies.

1 Tumor Suppressor Gene Therapy The genetic basis of cancer is well established. Tobacco smoke, for example, contains more than 100 carcinogens that can damage DNA (Denissenko et al. 1996). Lung cancers have multiple genetic lesions which can be detected in histologically normal bronchial mucosa and premalignant lesions from individuals with history of tobacco consumption. The p53 tumor suppressor gene is mutated in over 50% of lung cancers and other epithelial cancers (Olivier et al. 2009). The p53 tumor suppressor gene appears to contribute to cancer development when it is inactivated and was the initial target of gene therapy approaches to cancer. The p53 protein monitors cellular stress and DNA damage, inducing either growth arrest to facilitate DNA repair or apoptosis (programmed cell death) if DNA damage is extensive (Burns and El-Deiry 1999). When a cell is stressed by oncogene activation, hypoxia, or DNA damage, a functioning p53 pathway may determine whether the cell will receive a signal to arrest at the G1 stage of the cell cycle, whether DNA repair will be attempted, or whether the cell will self-destruct via apoptosis. The observation that expression of a wild-type p53 gene in a cancer cell induces apoptosis provided the rationale for p53 gene therapy replacement to restore a functioning p53 pathway (Fujiwara et al. 1993). One consideration was that if gene therapy could not replace all the damaged genes in a cancer cell, there would be no therapeutic benefit. The observation that restoration of only one of the defective genes is enough to trigger apoptosis suggests that the DNA damage present in a cancer cell may prime it for an apoptotic event. It is important to note that p53 regulates many cell survival pathways and thus restoring a single gene restores the function of multiple pathways. The p53 gene product is a transcription factor that regulates many pathways including apoptosis and cell cycle progression (Raycroft et  al. 1990). p53 also downregulates the prosurvival (or antiapoptotic) genes, including the antiapoptotic genes bcl-2 and bcl-XL, and upregulates the proapoptotic genes bax, bad, bid, puma, and noxa (Adams and Cory 1998). Available transcripts of each of the pro and antiapoptotic genes with bcl2 homology-3 domains interact with one another to form heterodimers, and the relative ratio of proapoptotic to prosurvival proteins in these heterodimers determines the activity of the resulting molecule, thereby

5  Tumor Suppressor Gene Therapy

65

determining whether the cell lives or undergoes apoptosis. p53 also targets the death-receptor signaling pathway, including DR5 and Fas/CD95, and the apoptosis machinery, including caspase-6, Apaf-1, and PIDD. It also may directly mediate cytochrome c release. Regulation of the p53 pathway at the protein level is mediated by other tumor suppressor genes and by several oncogenes (Burns and El-Deiry 1999). The mdm2 protein normally binds to the N-terminal transactivating domain of p53, which prevents p53 activation and leads to its rapid degradation. In normal cells, mdm2 is inhibited by the expression of p14ARF, a tumor suppressor gene encoded by the same gene locus as p16INK4a expressed as an alternate reading frame (Kamijo et al. 1997). Deletion or mutation of the tumor suppressor gene p14ARF occurs in some cancers and results in increased levels of mdm2, which results in the inactivation of p53 causing inappropriate progression through the cell cycle. The expression of p14ARF is induced by hyperproliferative signals from oncogenes such as ras and myc. The p53 protein can mediate cell cycle arrest. This function is significant, as prolonged tumor stability has often been observed in clinical trials of p53 gene replacement, suggesting that this effect may be predominant over apoptosis in some tumors. p53 is involved in regulating cell cycle checkpoints, and p53 expression can promote cell senescence through its control of cell cycle effectors such as p21CIP1/WAF1. Loss of function in the p53 pathway is the most common alteration identified in human cancers at the present time. About 50% of common epithelial cancers have p53 mutations (Isobe et al. 1994; Martin et al. 1992; Quinlan et al. 1992). In some cancers, loss of p53 also appears to be linked to resistance to conventional DNA damaging therapies, including chemotherapy and ionizing radiation, which require apoptosis to cause cell death.

2 Gene Replacement by p53 in Laboratory Studies The findings described above suggest that expressing a wild-type p53 gene in cancer cells defective in p53 function could mediate either apoptosis or cell growth arrest, both of which would be of therapeutic benefit to a cancer patient. Initial studies showed that restoration of functional p53 using a retroviral vector suppressed the growth of some, but not all, human lung cancer cell lines (Cai et  al. 1993). The first published study of p53 gene therapy showed suppression of tumor growth in an orthotopic human lung cancer model using a retroviral expression vector (Fujiwara et al. 1994a). This was the first study to show that restoring the function of a single tumor suppressor gene could result in the regression of human cancer cells in vivo. Because of the limitations inherent in the use of retroviruses, subsequent studies of p53 gene replacement in lung cancer made use of an adenoviral vector (Ad-p53) (Zhang et al. 1994). The original adenoviral vector was a serotype five replicationdefective vector with a deleted E1 region, which has been used in all p53 clinical trials. Ad-p53 also induced apoptosis in cancer cells with nonfunctional p53 without

66

J.A. Roth et al.

significantly affecting the proliferation of normal cells (Wang et  al. 1995). Subsequent studies with Ad-p53 demonstrated inhibition of tumor growth in a mouse model of human orthotopic lung cancer (Georges et al. 1993) and induction of apoptosis and suppression of proliferation in various other cancer cell lines and in  vivo mouse xenograft tumor models (Bouvet et  al. 1998; Nielsen et  al. 1997; Spitz et al. 1996). Bystander killing (killing of nontransduced cells by transduced cells), now known to be an important phenomenon for the success of gene therapy, appears to involve regulation of angiogenesis (Dameron et al. 1994; Miyashita and Reed 1995), immune upregulation (Carroll et  al. 2001; Molinier-Frenkel et  al. 2000; Yen et  al. 2000), and secretion of soluble proapoptotic proteins (OwenSchaub et al. 1995).

3 Clinical Trials of p53 Gene Replacement Initial preclinical studies used a retrovirus to deliver the p53 gene. The first clinical trial for p53 gene-replacement utilized a replication-defective retroviral vector expressing wild-type p53 driven by a beta-actin promoter (Roth et al. 1996). The retrovirus vector was injected directly into tumors of nine patients with unresectable NSCLC that had progressed after conventional therapy. Three of the nine patients showed evidence of tumor regression. There was no vector-related toxicity, demonstrating the feasibility, safety, and antitumor activity of p53 gene therapy. Following the initial p53 retrovirus vector clinical trial, p53 clinical trials were conducted with the adenovirus p53 vector because of the ease of production, high transduction efficiency, and stability of adenovirus vectors. A phase I trial enrolled 28 NSCLC patients whose cancers had not responded to conventional treatments. Successful gene transfer was demonstrated in 80% of evaluable patients (Swisher et al. 1999). Expression of p53 was detected in 46% of patients, apoptosis was seen in all but one of the patients expressing the gene, and, importantly, no significant toxicity was observed. More than a 50% reduction in tumor size was observed in two patients, with one patient remaining free of tumor for more than a year after concluding therapy and another experiencing nearly complete regression of a chemotherapyand radiation-resistant upper lobe endobronchial tumor. Additional studies in patients with head and neck cancer evaluated Ad-p53 gene transfer as a clinically feasible strategy and showed successful gene transfer and gene expression, low toxicity, and evidence of durable tumor regression (Fig.  1). Clayman and coworkers (1998) reported 33 patients with refractory head and neck cancer treated with adenovirus p53. Li-Fraumeni syndrome (LFS) is an autosomal dominant genetic disease of the p53 gene that dramatically increases the risk of developing multiple primary cancers of differing histologies. The majority of LFS families contain a germ line mutation in the p53 tumor suppressor gene. Senzer and coworkers (2007) described the treatment of a refractory, progressive LFS embryonal carcinoma with adenovirus p53 gene therapy thus targeting the underlying molecular defect of LFS. Treatment with adenovirus p53 resulted in a complete and durable remission of the injected lesion

5  Tumor Suppressor Gene Therapy

67

Fig.  1  Durable complete response following adenovirus p53 monotherapy in a patient with refractory head and neck squamous cell carcinoma

by FDG-PET scans with symptomatic improvement (Fig.  2). This very striking response offers direct proof-of-principle for p53 gene replacement. A recent study investigated the role of p53 biomarkers that may predict the efficacy of normal p53 delivered by gene therapy in patients (Nemunaitis et  al. 2009). Tumor p53 biomarkers, including p53 protein expression determined by immunohistochemistry, and p53 mutations were evaluated in 116 patients including 29 treated with methotrexate in a Phase III randomized, controlled trial. Profiles favorable for p53 gene therapy efficacy were hypothesized to have either normal p53 gene sequences or low level p53 protein expression while unfavorable p53 inhibitor profiles were predicted to have high level expression of mutated p53 that can inhibit normal p53 protein function. A greater than threefold statistically significant increase in tumor responses was observed for patients with favorable p53 efficacy profiles compared to those with unfavorable p53 inhibitor profiles. In the Phase III trial, there was a greater than twofold statistically significant increased time to progression and survival following p53 gene therapy in patients with favorable p53 profiles compared to unfavorable p53 inhibitor profiles. In contrast, the biomarker profiles predictive of p53 gene therapy efficacy did not predict methotrexate response, time to progression or survival outcomes. Thus tumor p53 biomarker profiles may be useful for predicting p53 gene therapy efficacy in recurrent SCCHN. A novel application of adenovirus p53 is the treatment of localized precancerous lesions. Oral leukoplakia is a precancerous lesion of squamous cell carcinoma. Adenovirus p53 inhibited cell proliferation and induced apoptosis in an oral dysplastic keratinocyte cell line. Twenty-two patients with dysplastic oral leukoplakia were treated with introlesional injections of adenovirus p53 with 16 patients showing a clinical response and 5 patients showing complete regression (Li et al. 2009).

68

J.A. Roth et al.

Fig.  2  Treatment of embryonal cell ovarian carcinoma with injection of adenovirus p53 in a patient with Li-Fraumeni syndrome

4 Gene Replacement in Combination with DNA Damaging Agents Many cancers are resistant to chemotherapy and radiation therapy. The p53 gene mediates the detection of damage to DNA and either directs repair or induces apoptosis. p53 is often mutated or nonfunctional in radiation- and chemotherapy-resistant tumors. Preclinical studies of p53 gene therapy combined with cisplatin in cultured NSCLC cells and in human xenografts in nude mice showed that sequential

5  Tumor Suppressor Gene Therapy

69

administration of cisplatin and p53 gene therapy resulted in enhanced expression of the p53 gene product (Fujiwara et al. 1994b; Nguyen et al. 1996). Studies of Ad-p53 gene transfer combined with radiation therapy indicated that delivery of Ad-p53 increases the sensitivity of p53-deficient tumor cells to external beam radiation (Spitz et al. 1996). Due to Ad-p53’s low toxicity (less than a 5% incidence of serious adverse events) in initial trials, therapeutic strategies combining Ad-p53 gene replacement and conventional DNA damaging therapies were begun (Yver et al. 1999).

5 Clinical Trials of Tumor Suppressor Gene Replacement Combined with Chemotherapy Twenty-four NSCLC patients with tumors previously unresponsive to conventional treatment were enrolled in a phase I trial of intratumor injection of p53 combined with cisplatin (Nemunaitis et  al. 2000). Seventy-five percent of the patients had previously experienced tumor progression on cisplatin- or carboplatin-containing regimens. Up to six monthly courses of intravenous cisplatin, each followed 3 days later by intratumoral injection of Ad-p53, resulted in 17 patients remaining stable for at least 2 months, 2 patients achieving partial responses, 4 patients continuing to exhibit progressive disease, and 1 patient unevaluable due to progressive disease. Seventy-nine percent of tumor biopsies showed an increase in the number of apoptotic cells, 7% showed a decrease in apoptosis, and 14% showed no change. A phase II clinical trial evaluated two comparable metastatic lesions in each NSCLC patient enrolled in the study (Schuler et  al. 2001). All patients received chemotherapy, either three cycles of carboplatin plus paclitaxel or three cycles of cisplatin plus vinorelbine, and then Ad-p53 was injected directly into one lesion. Ad-p53 treatment resulted in minimal vector-related toxicity and no overall increase in chemotherapy-related adverse events. Patients receiving carboplatin plus paclitaxel, the combination of drugs that provided the greatest benefit on its own, did not realize additional benefit from Ad-p53 gene transfer which would be expected as Ad-p53 was injected in only one lesion in each patient. However, patients treated with the less-successful cisplatin and vinorelbine regimen experienced significantly greater mean local tumor regression, as measured by size, in the Ad-p53-injected lesion than in the control lesion.

6 Clinical Trials of p53 Gene Replacement Combined with Radiation Therapy Preclinical studies suggesting that p53 gene replacement might confer radiation sensitivity to some tumors (Broaddus et al. 1999; Feinmesser et al. 1999; Jasty et al. 1998; Sakakura et al. 1996; Spitz et al. 1996) led to a phase II clinical trial of p53

70

J.A. Roth et al.

gene transfer in conjunction with radiation therapy (Swisher et al. 2000). Patients with a poor performance status who could not undergo surgery and would be at high risk for combined chemotherapy and radiation received 60 Gy over 6 weeks with Ad-p53 injected on days 1, 18, and 32. Nineteen patients with localized NSCLC were treated, resulting in a complete response in 1 patient (5%), partial response in 11 patients (58%), stable disease in 3 patients (16%), and progressive disease in 2 patients (11%). Two patients (11%) were not evaluable due to tumor progression or early death. Three months after the completion of therapy, biopsies revealed no viable tumor in 12 patients (63%) and viable tumor in 3 (16%). Tumors of four patients (21%) were not biopsied because of tumor progression, early death, or weakness. The 1-year progression-free survival rate was 45.5%. Among 13 evaluable patients after 1 year, 5 (39%) had a complete response and 3 (23%) had a partial response or disease stabilization. Most treatment failures were caused by metastatic disease without local progression. Biopsies of the tumor were performed before and after treatment so that detailed studies of gene expression were possible. Ad-p53 vector-specific DNA was detected in biopsy specimens from 9 of 12 patients with paired biopsies (day 18 and 19). The ratio of copies of Ad-p53 vector DNA to copies of actin DNA was 0.15 or higher in eight of nine patients (range, 0.05–3.85), with four patients having a ratio >0.5. For 11 patients with adequate samples for both vector DNA and mRNA analysis, 8 showed a postinjection increase in mRNA expression associated with detectable vector DNA. Postinjection increases in p53 mRNA were detected in 11 of 12 paired biopsies obtained 24 h after Ad-p53 injection, with 10 of 11 increasing threefold or more. Preinjection biopsy specimens that were shown by immunohistochemistry to be negative for p53 protein expression were stained for p53 protein expression after Ad-p53 injection. Staining results confirmed that the p53 protein was expressed in the posttreatment samples in the nuclei of cancer cells. For p21 (CDKN1A) mRNA, increases of statistical significance were noted 24 h after Ad-p53 injection and during treatment, as compared with the pretreatment biopsy. MDM2 mRNA levels were higher during treatment than before treatment. Levels of FAS mRNA did not change significantly during treatment. BAK mRNA expression increased significantly 24 h after the injection of Ad-p53 and thus appeared to be the marker most acutely upregulated by Ad-p53 injection. The safety profile for intratumoral injection of Ad-p53 has been excellent. The most frequently reported adverse events related to treatment with Ad-p53 injection were fever and chills, asthenia, injection site pain, nausea, and vomiting. The vast majority of these events were mild to moderate. To date, no maximum tolerated dose for Ad-p53 injection has been established. Beginning in 1998, a similar Adenovirus p53 expressing vector was tested in China in clinical trials under the name Gendicine. A multicenter, randomized clinical trial was conducted in which Ad-p53 was administered to 135 patients with head and neck squamous cell carcinoma (Peng 2005). Of the enrolled patients, 77% had late-stage III to IV cancer and had failed in either radio- or chemotherapy or were not eligible for surgery. The majority (85%) of the patients had nasopharyngeal cancer. One group received gene therapy in combination with radiotherapy (GTRT)

5  Tumor Suppressor Gene Therapy

71

and the other group received radiotherapy alone (RT). In the GTRT group, the complete response (CR) rate determined by computed tomography was 64% with 29% partial regression (PR). The response rate in the RT group was 19% of the patients showing CR and 60% PR. There is a significant difference (p < 0.01) between the two groups in both the CR rate and the PR rate. This study served as the basis for the approval of Ad-p53 (Gendicine) by the SFDA in China, and Gendicine, thus, became the first gene therapy agent approved for human use. In another randomized clinical trial conducted in China, Ad-p53 combined with RT in 42 patients with NPC was compared with a control group of 40 patients with NPC treated with RT alone (Pan et al. 2009). In the group receiving Ad-p53 combined with RT, rAd-p53 was intratumorally injected once a week for 8 weeks. Concurrent RT (70 Gy in 35 fractions) was given to the nasopharyngeal tumor and neck lymph nodes. The complete response rate in the group receiving Ad-p53 combined with RT was 2.73 times that of the group receiving RT alone (66.7 vs. 24.4%). After 6 years, Ad-p53 significantly increased the 5-year locoregional tumor control rate by 25.3% for patients with NPC treated with irradiation. The 5-year overall survival rate and 5-year disease-free survival rate of the group receiving rAd-p53 combined with RT were 7.5% and 11.7% higher than those of the group receiving RT alone, although not statistically significant.

7 Systemic Gene Therapy for Metastases Most cancer patients die from metastatic disease, and thus the development of cancer gene therapy approaches that can treat systemic disease is critical. The development of a cancer vaccine to p53 is one approach. Although the p53 protein is expressed by normal cells, it has a short half-life and is thus present at low levels. Mutant p53 is conformationally altered and resists degradation in cancer cells. Thus, it has a prolonged half-life and is expressed at high levels in cancer cells. These differences in expression between normal and cancer cells suggest that p53 could function as a tumor antigen and vaccine target (Chada et al. 2003; Ishida et al. 1999; Mayordomo et al. 1996; Nikitina et al. 2001). Several studies have shown in cultured cells and mouse models the induction of anti-p53 cytotoxic lymphocytes that killed cancer cells but not normal cells. A strategy was developed using dendritic cells, which are the most effective antigen-presenting cells, transduced with Ad-p53 (Antonia et al. 2006). Patients with extensive-stage small-cell lung cancer (SCLC) were entered into a trial. SCLC patients with extensive stage disease have a median survival of 2–4 months untreated or 6–8 months with chemotherapy. In that trial, the patients’ autologous dendritic cells were treated ex vivo with Ad-p53, which activates the cells and results in the expression of high levels of p53 protein. Patients were first treated with conventional chemotherapy. Those who achieved at least stable disease received the vaccine biweekly for a total of three to six injections. If patients progressed, they were treated with chemotherapy. Of the 29 patients treated, 1 had a

72

J.A. Roth et al.

partial response, 7 had stable disease, and 21 had progression. Patients having progression then received second-line chemotherapy. Clinical follow-up was completed for 21 patients. Complete or partial responses to the second-line chemotherapy were observed in 61.9% of the 21 patients treated. Eleven of the patients were alive 1 year after the first vaccine treatment. These clinical responses were correlated with the induction of immune responses to the vaccine. Published objective response rates for second-line chemotherapy in extensive-stage SCLC patients range from 5 to 30%. Recently, nanoscale synthetic particles that can encapsulate plasmid DNA and deliver it to cells after intravenous injection have been developed. The efficacy of these nanoparticles was studied in mouse xenograft models of disseminated human lung cancer. P53 and other tumor suppressor genes have been delivered using this technique (Ramesh et al. 2001). Multiple 3p21.3 genes show different degrees of tumor suppression activities in various human cancers in  vitro and in preclinical animal models (Ji et al. 2002). One of the tumor suppressor genes at this locus is FUS1, which is not expressed in most lung cancers. The FUS1 gene has been found to be inactivated in primary tumors by 3p21.3 allele haploinsufficiency combined with a posttranslational modification of the gene product of the remaining allele that decreases the stability of the protein (Kondo et al. 2001; Uno et al. 2004). Only a few mutations in the FUS1 gene, including missense mutations and C-terminal deletion mutations, have been identified in primary lung cancer samples and there is no evidence of promoter methylation in lung cancers (Kondo et al. 2001; Uno et al. 2004; Zabarovsky et al. 2002). Based on this observation, it has been hypothesized that, given its lung cancer growth-suppressing properties in  vitro and in animal models, FUS1 would have to act as a TSG in a haploinsufficient manner as most lung cancers experience allele loss in this 3p21.3 region (Ji et  al. 2002). Although FUS1 mRNA transcription was detected on Northern blots of RNAs prepared from lung cancer cell lines, no endogenous FUS1 protein was detected in these lung cancer cells on Western blots using affinity-purified, anti-FUS1 peptide antibodies (Kondo et al. 2001; Uno et al. 2004; Zabarovsky et al. 2002). Uno et al. (2004) used SELDI-MS on an anti-FUS1-antibody-capture ProteinChip array to analyze the status of expression and posttranslational modifications of FUS1 protein in primary lung cancer and cancer cell lines. They identified wt-FUS1 as an N-myristoylated protein and found significant loss of expression coupled with a myristoylation defect of the FUS1 protein in primary lung cancer and cancer cell lines (Uno et al. 2004). The myristoylation defective protein of FUS1 has a greatly reduced half-life and is subject to rapid proteosomal degradation (Uno et al. 2004). To further investigate the role of the abnormal expression of FUS1 in the sequential pathogenesis of NSCLC, we studied its expression in 68 histologically normal bronchial epithelia, 120 basal cell hyperplasias, 23 squamous metaplasias, 62 squamous dysplasias, and 56 specimens of atypical adenomatous hyperplasia and adjacent normal lung tissues obtained from lung cancer patients (Prudkin et  al. 2008). Loss or reduction of expression was detected in 100% of the SCLCs and 82% of the NSCLCs. In the NSCLCs, the loss or reduction of FUS1 expression was associated with significantly worse overall patient survival. Squamous metaplasia

5  Tumor Suppressor Gene Therapy

73

and dysplasia expressed significantly lower levels of FUS1 than did normal (p = 0.014 and 0.047, respectively) and hyperplastic (p = 0.013 and 0.028) bronchial epithelia. In summary, these findings confirm the high frequency of FUS1 protein loss and its reduced expression in lung cancer and suggest that reduction of these proteins may play an important role in the early pathogenesis of lung cancer. These results also suggest a novel alternative mechanism for the inactivation of tumor suppressors in human cancer and a role for defective posttranslational modification in TSG-mediated carcinogenesis. Genes in the 3p21.3 region are associated with cell differentiation, cell proliferation, cell cycle kinetics, signal transduction, ion exchange and transportation, apoptosis, and cell death. FUS1 has the most potent tumor suppressor activity for human lung cancer cells of all the genes identified in this region as measured by the induction of apoptosis. We previously showed that enforced expression of wt-FUS1 in 3p21.3-deficient NSCLC cells significantly suppressed tumor cell growth by inducing apoptosis and altering cell cycle kinetics in vitro and in vivo (Ji et al. 2002). These results suggest that tumorigenesis might be prevented or delayed by neutralizing the effects of 3p haploinsufficiency before the premalignant lesions progress to invasive cancer, and tumor growth might be suppressed by inducing apoptosis and altering cell cycle processes after tumor onset through wt-FUS1 gene transfer. Recently, FUS1-deficient mice were generated through homologous recombination in cultured embryonic stem cells. FUS1-knockouts showed a significant increase in autoimmune-related pathology, including vasculitis and glomerulonephritis, and a marked increase in hemangiomas and hemangiosarcomas (Ivanova et al. 2007). To translate these findings to clinical applications for molecular cancer therapy, we recently developed a systemic treatment strategy by using a novel FUS1expressing plasmid vector complexed with DOTAP:cholesterol (DOTAP:Chol) liposome, termed FUS1 nanoparticle, for treating lung cancer and lung metastases (Ito et al. 2004; Uno et al. 2004). In a preclinical trial, we showed that intratumoral administration of FUS1 nanoparticles to subcutaneous NSCLC H1299 and A549 tumor xenografts resulted in significant inhibition of tumor growth. Intravenous injections of FUS1 nanoparticles into mice-bearing experimental A549 lung metastasis significantly decreased the number of metastatic tumor nodules. Lung tumorbearing animals treated with FUS1 nanoparticles survived longer (median survival time: 80 days) than control animals. These results demonstrate the potent tumor suppressive activity of the FUS1 gene, making it a promising therapeutic agent for the treatment of primary and disseminated human lung cancer (Ito et  al. 2004). Based on these studies, a phase I clinical trial with FUS1-mediated molecular therapy by systemic administration of FUS1 nanoparticles is now under way in stage IV lung cancer patients at The University of Texas M. D. Anderson Cancer Center in Houston, Texas. A cationic immunoliposome system directed by a lipid-tagged, single-chain antibody Fv fragment (scFv) against the human transferrin receptor (TfR) has been described for systemic p53 tumor suppressor gene therapy (Xu et al. 2002). The scFvcys targets the cationic liposome-DNA complex (lipoplex) to tumor cells and enhances the transfection efficiencies both in  vitro and in  vivo in a variety of human tumor

74

J.A. Roth et al.

models including breast and head and neck cancer. This scFv-immunoliposome was shown to deliver the complexed gene systemically to tumors in vivo. The investigators found that in comparison with the whole antibody or the transferrin molecule itself, the scFv had better penetration into solid tumors. This approach is now in a phase I clinical trial for patients with metastatic nonsmall cell lung cancer.

8 Summary and Conclusions Combining existing cancer treatments has reached a plateau of efficacy, and the addition of conventional cytotoxic agents is limited because of toxicity. The clinical trials summarized in this article clearly demonstrate that contrary to initial predictions that gene therapy would not be suitable for cancer, gene replacement therapy targeted to a tumor suppressor gene can cause cancer regression by the activation of known pathways with minimal toxicity. Gene expression has been documented and occurs even in the presence of an antiadenovirus immune response, clinical trials have demonstrated that direct intratumoral injection can cause tumor regression or prolonged stabilization of local disease, and the low toxicity associated with gene transfer indicates that tumor suppressor gene replacement can be readily combined with existing and future treatments. Initial concerns that the wide diversity of genetic lesions in cancer cells would prevent the application of gene therapy to cancer appear unfounded; on the contrary, correction of a single genetic lesion has resulted in significant tumor regression. Studies using the transfer of tumor suppressor genes in combination with conventional DNA damaging treatments indicate that correction of a defect in apoptosis induction can restore sensitivity to radiation and chemotherapy in some resistant tumors, and indications that sensitivity to killing might be enhanced in already sensitive tumors may eventually lead to reduced toxicity from chemotherapy and radiation therapy. The most recent laboratory data demonstrating damage to tumor suppressor genes in normal tissue and premalignant lesions suggests that these genes could someday be useful in early intervention, diagnosis, and even prevention of cancer. Preclinical studies have shown that systemic delivery of the treatment of metastases can be achieved. The ready availability of gene libraries, the ability to administer genes without the extensive reformulation required of small molecules, and their specificity make this an attractive therapeutic approach. Despite the obvious promise evident in the results of these studies, though, it is critical to recognize that there are still gaps in knowledge and technology to address. The major issues for the future development of gene therapy include: 1. Development of more efficient and less toxic gene delivery vectors for systemic gene delivery 2. Identification of the optimal genes for various tumor types 3. Optimizing combination therapy

5  Tumor Suppressor Gene Therapy

75

4 . Monitoring gene uptake and expression by cancer cells 5. Overcoming resistance pathways However, given the rapid progress in the field of gene therapy, it is likely that many of these technological problems will be solved in the near future.

References Adams, J. M. and Cory, S. (1998) The Bcl-2 protein family: arbiters of cell survival. Science 281:1322–1326 Antonia, S. J., Mirza, N., Fricke, I., Chiappori, A., Thompson, P., Williams, N., Bepler, G., Simon, G., Janssen, W., Lee, J. H., Menander, K., Chada, S., and Gabrilovich, D. I. (2006) Combination of p53 cancer vaccine with chemotherapy in patients with extensive stage small cell lung cancer. Clinical Cancer Research 12:878–887 Bouvet, M., Fang, B., Ekmekcioglu, S., Ji, L., Bucana, C. D., Hamada, K., Grimm, E. A., and Roth, J. A. (1998) Suppression of the immune response to an adenovirus vector and enhancement of intratumoral transgene expression by low-dose etoposide. Gene Therapy 5:189–195 Broaddus, W. C., Liu, Y., Steele, L. L., Gillies, G. T., Lin, P. S., Loudon, W. G., Valerie, K., Schmidt-Ullrich, R. K., and Fillmore, H. L. (1999) Enhanced radiosensitivity of malignant glioma cells after adenoviral p53 transduction. Journal of Neurosurgery 91:997–1004 Burns, T. and El-Deiry, W. (1999) The p53 pathway and apoptosis. Journal of Cellular Physiology 181:231–239 Cai, D. W., Mukhopadhyay, T., and Roth, J. A. (1993) A novel ribozyme for modification of mutated p53 pre-mrna in non-small cell lung cancer cell lines. 3rd Antisense Workshop Carlson, J. M. and Doyle, J. (2002) Complexity and robustness. Proceedings of the National Academy of Sciences of the United States of America 99 Suppl 1:2538–2545 Carroll, J. L., Nielsen, L. L., Pruett, S. B., and Mathis, J. M. (2001) The role of natural killer cells in adenovirus-mediated p53 gene therapy. Molecular Cancer Therapeutics 1:49–60 Chada, S., Mhashilkar, A., Roth, J. A., and Gabrilovich, D. (2003) Development of vaccines against self antigens:the p53 paradigm. Current Opinion in Drug Discovery and Development 6:169–173 Clayman, G. L., El-Naggar, A. K., Lippman, S. M., Henderson, Y. C., Frederick, M., Merritt, J. A., Zumstein, L. A., Timmons, T. M., Liu, T-J., Ginsberg, L., Roth, J. A., Hong, W. K., Bruso, P., and Goepfert, H. (1998) Adenovirus-mediated p53 gene transfer in patients with advanced recurrent head and neck squamous cell carcinoma. Journal of Clinical Oncology 16: 2221–2232 Dameron, K. M., Volpert, O. V., Tainsky, M. A., and Bouck, N. (1994) Control of angiogenesis in fibroblasts by p53 regulation of thrombospondin-1. Science 265:1582–1584 Denissenko, M. F., Pao, A., Tang, M., and Pfeifer, G. P. (1996) Preferential formation of benzo[a] pyrene adducts at lung cancer mutational hotspots in p53. Science 274:430–432 Edelman, G. M., and Gally, J. A. (2001) Degeneracy and complexity in biological systems. Proceedings of the National Academy of Sciences of the United States of America 98: 13763–13768 Feinmesser, M., Halpern, M., Fenig, E., Tsabari, C., Hodak, E., Sulkes, J., Brenner, B., and Okon, E. (1999) Expression of the apoptosis-related oncogenes bcl-2, bax, and p53 in Merkel cell carcinoma: Can they predict treatment response and clinical outcome? Human Pathology 30: 1367–1372 Fujiwara, T., Grimm, E. A., Mukhopadhyay, T., Cai, D. W., Owen-Schaub, L. B., and Roth, J. A. (1993) A retroviral wild-type p53 expression vector penetrates human lung cancer spheroids and inhibits growth by inducing apoptosis. Cancer Research 53:4129–4133

76

J.A. Roth et al.

Fujiwara, T., Cai, D. W., Georges, R. N., Mukhopadhyay, T., Grimm, E. A., and Roth, J. A. (1994a) Therapeutic effect of a retroviral wild-type p53 expression vector in an orthotopic lung cancer model. Journal of the National Cancer Institute 86:1458–1462 Fujiwara, T., Grimm, E. A., Mukhopadhyay, T., Zhang, W. W., Owen-Schaub, L. B., and Roth, J. A. (1994b) Induction of chemosensitivity in human lung cancer cells in  vivo by adenoviralmediated transfer of the wild-type p53 gene. Cancer Research 54:2287–2291 Georges, R. N., Mukhopadhyay, T., Zhang, Y., Yen, N., and Roth, J. A. (1993) Prevention of orthotopic human lung cancer growth by intratracheal instillation of a retroviral antisense K-ras construct. Cancer Research 53:1743–1746 Hanahan, D. and Weinberg, R. A. (2000) The hallmarks of cancer. Cell 100:57–70 Ishida, T., Chada, S., Stipanov, M., Nadaf, S., Ciernik, F. I., Gabrilovich, D. I., and Carbone, D. P. (1999) Dendritic cells transduced with wild-type p53 gene elicit potent anti-tumour immune responses. Clinical and Experimental Immunology 117:244–251 Isobe, T., Hiyama, K., Yoshida, Y., Fujiwara, Y., and Yamakido, M. (1994) Prognostic significance of p53 and ras gene abnormalities in lung adenocarcinoma patients with stage I disease after curative resection. Japanese Journal of Cancer Research 85:1240–1246 Ito, I., Ji, L., Tanaka, F., Saito, Y., Gopalan, B., Branch, C. D., Xu, K., Atkinson, E. N., Bekele, B. N., Stephens, L. C., Minna, J. D., Roth, J. A., and Ramesh, R. (2004) Liposomal vector mediated delivery of the 3p FUS1 gene demonstrates potent antitumor activity against human lung cancer in vivo. Cancer Gene Therapy 11:733–739 Ivanova, A. V., Ivanov, S. V., Pascal, V., Lumsden, J. M., Ward, J. M., Morris, N., Tessarolo, L., Anderson, S. K., and Lerman, M. I. (2007) Autoimmunity, spontaneous tumourigenesis, and IL-15 insufficiency in mice with a targeted disruption of the tumour suppressor gene Fus1. The Journal of Pathology 211:591–601 Jasty, R., Lu, J., Irwin, T., Suchard, S., Clarke, M. F., and Castle, V. P. (1998) Role of p53 in the regulation of irradiation-induced apoptosis in neuroblastoma cells. Molecular Genetics and Metabolism 65:155–164 Ji, L., Nishizaki, M., Gao, B., Burbee, D., Kondo, M., Kamibayashi, C., Xu, K., Yen, N., Atkinson, E. N., Fang, B., Lerman, M. I., Roth, J. A., and Minna, J. D. (2002) Expression of several genes in the human chromosome 3p21.3 homozygous deletion region by an adenovirus vector results in tumor suppressor activities in vitro and in vivo. Cancer Research 62:2715–2720 Kamijo, T., Zindy, F., Roussel, M. F., Quelle, D. E., Downing, J. R., Ashmun, R. A., Grosveld, G., and Sherr, C. J. (1997) Tumor suppression at the mouse INK4a locus mediated by the alternative reading frame product p19ARF. Cell 91:649–659 Kondo, M., Ji, L., Kamibayashi, C., Tomizawa, Y., Randle, D., Sekido, Y., Yokota, J., Kashuba, V., Zabarovsky, E., Kuzmin, I., Lerman, M., Roth, J., and Minna, J. D. (2001) Overexpression of candidate tumor suppressor gene FUS1 isolated from the 3p21.3 homozygous deletion region leads to G1 arrest and growth inhibition of lung cancer cells. Oncogene 20:6258–6262 Li, Y., Li, L. J., Zhang, S. T., Wang, L. J., Zhang, Z., Gao, N., Zhang, Y. Y., and Chen, Q. M. (2009) In vitro and clinical studies of gene therapy with recombinant human adenovirus-p53 injection for oral leukoplakia. Clinical Cancer Research 15:6724–6731 Martin, H. M., Filipe, M. I., Morris, R. W., Lane, D. P., and Silvestre, F. (1992) p53 expression and prognosis in gastric carcinoma. International Journal of Cancer 50:859–862 Mayordomo, J. I., Loftus, D. J., Sakamoto, H., De Cesare, C. M., Appasamy, P. M., Lotze, M. T., Storkus, W. J., Appella, E., and Deleo, A. B. (1996) Therapy of murine tumors with p53 wildtype and mutant sequence peptide-based vaccines. The Journal of Experimental Medicine 183:1357–1365 Miyashita, T. and Reed, J. C. (1995) Tumor suppressor p53 is a direct transcriptional activator of human bax gene. Cell 80:293–299 Molinier-Frenkel, V., Le Boulaire, C., Le Gal, F. A., Gahery-Segard, H., Tursz, T., Guillet, J. G., and Farace, F. (2000) Longitudinal follow-up of cellular and humoral immunity induced by recombinant adenovirus-mediated gene therapy in cancer patients. Human Gene Therapy 11: 1911–1920 Nemunaitis, J., Swisher, S. G., Timmons, T., Connors, D., Mack, M., Doerksen, L., Weill, D., Wait, J., Lawrence, D. D., Kemp, B. L., Fossella, F., Glisson, B. S., Hong, W. K., Khuri, F. R.,

5  Tumor Suppressor Gene Therapy

77

Kurie, J. M., Lee, J. J., Lee, J. S., Nguyen, D. M., Nesbitt, J. C., Perez-Soler, R., Pisters, K. M. W., Putnam, J. B., Richli, W. R., Shin, D. M., Walsh, G. L., Merritt, J., and Roth, J. A. (2000) Adenovirus-mediated p53 gene transfer in sequence with cisplatin to tumors of patients with non-small cell lung cancer. Journal of Clinical Oncology 18:609–622 Nemunaitis, J., Clayman, G., Agarwal, S. S., Hrushesky, W., Wells, J. R., Moore, C., Hamm, J., Yoo, G., Baselga, J., Murphy, B. A., Menander, K. A., Licato, L. L., Chada, S., Gibbons, R. D., Olivier, M., Hainaut, P., Roth, J. A., Sobol, R. E., and Goodwin, W. J. (2009) Biomarkers predict p53 gene therapy efficacy in recurrent, squamous cell carcinoma of the head and neck. Clinical Cancer Research 15:7719–7725 Nguyen, D. M., Spitz, F. R., Yen, N., Cristiano, R. J., and Roth, J. A. (1996) Gene therapy for lung cancer: enhancement of tumor suppression by a combination of sequential systemic cisplatin and adenovirus-mediated p53 gene transfer. The Journal of Thoracic and Cardiovascular Surgery 112:1372–1377 Nielsen, L. L., Dell, J., Maxwell, E., Armstrong, L., Maneval, D., and Catino, J. J. (1997) Efficacy of p53 adenovirus-mediated gene therapy against human breast cancer xenografts. Cancer Gene Therapy 4:129–138 Nikitina, E. Y., Clark, J. I., Van Beynen, J., Chada, S., Virmani, A. K., Carbone, D. P., and Gabrilovich, D. I. (2001) Dendritic cells transduced with full-length wild-type p53 generate antitumor cytotoxic T lymphocytes from peripheral blood of cancer patients. Clinical Cancer Research 7:127–135 Olivier, M., Petitjean, A., Marcel, V., Petre, A., Mounawar, M., Plymoth, A., de Fromentel, C. C., and Hainaut, P. (2009) Recent advances in p53 research: an interdisciplinary perspective. Cancer Gene Therapy 16:1–12 Owen-Schaub, L. B., Zhang, W., Cusack, J. C., Angelo, L. S., Santee, S. M., Fujiwara, T., Roth, J. A., Deisseroth, A. B., Zhang, W. W., Kruzel, E., and Radinsky, R. (1995) Wild-type human p53 and a temperature-sensitive mutant induce Fas/APO-1 expression. Molecular and Cellular Biology 15:3032–3040 Pan, J. J., Zhang, S. W., Chen, C. B., Xiao, S. W., Sun, Y., Liu, C. Q., Su, X., Li, D. M., Xu, G., Xu, B., and Lu, Y. Y. (2009) Effect of recombinant adenovirus-p53 combined with radiotherapy on long-term prognosis of advanced nasopharyngeal carcinoma. Journal of Clinical Oncology 27:799–804 Peng, Z. (2005) Current status of gendicine in China: recombinant human Ad-p53 agent for treatment of cancers. Human Gene Therapy 16:1016–1027 Prudkin, L., Behrens, C., Liu, D. D., Zhou, X., Ozburn, N. C., Bekele, B. N., Minna, J. D., Moran, C., Roth, J. A., Ji, L., and Wistuba, I. I. (2008) Loss and reduction of Fus1 protein expression is a frequent phenomenon in the pathogenesis of lung cancer. Clinical Cancer Research 14:41–47 Quinlan, D. C., Davidson, A. G., Summers, C. L., Warden, H. E., and Doshi, H. M. (1992) Accumulation of p53 protein correlates with a poor prognosis in human lung cancer. Cancer Research 52:4828–4831 Ramesh, R., Saeki, T., Templeton, N. S., Ji, L., Stephens, L. C., Ito, I., Wilson, D. R., Wu, Z., Branch, C. D., Minna, J. D., and Roth, J. A. (2001) Successful treatment of primary and disseminated human lung cancers by systemic delivery of tumor suppressor genes using an improved liposome vector. Molecular Therapy 3:337–350 Raycroft, L., Wu, H., and Lozano, G. (1990) Transcriptional activation by wild-type but not transforming mutants of the p53 anti-oncogene. Science 249:1049–1051 Roth, J. A., Nguyen, D., Lawrence, D. D., Kemp, B. L., Carrasco, C. H., Ferson, D. Z., Hong, W. K., Komaki, R., Lee, J. J., Nesbitt, J. C., Pisters, K. M. W., Putnam, J. B., Jr., Schea, R., Shin, D. M., Walsh, G. L., Dolormente, M. M., Han, C. I., Martin, F. D., Yen, N., Xu, K., Stephens, L. C., McDonnell, T. J., Mukhopadhyay, T., and Cai, D. (1996) Retrovirus-mediated wild-type p53 gene transfer to tumors of patients with lung cancer. Nature Medicine 2:985–991 Sakakura, C., Sweeney, E. A., Shirahama, T., Igarashi, Y., Hakomori, S., Nakatani, H., Tsujimoto, H., Imanishi, T., Ohgaki, M., Ohyama, T., Yamazaki, J., Hagiwara, A., Yamaguchi, T., Sawai, K., and Takahashi, T. (1996) Overexpression of bax sensitizes human breast cancer MCF-7 cells to radiation-induced apoptosis. International Journal of Cancer 67:101–105

78

J.A. Roth et al.

Schuler, M., Herrmann, R., De Greve, J. L., Stewart, A. K., Gatzemeier, U., Stewart, D. J., Laufman, L., Gralla, R., Kuball, J., Buhl, R., Heussel, C. P., Kommoss, F., Perruchoud, A. P., Shepherd, F. A., Fritz, M. A., Horowitz, J. A., Huber, C., and Rochlitz, C. (2001) Adenovirusmediated wild-type p53 gene transfer in patients receiving chemotherapy for advanced nonsmall-cell lung cancer: results of a multicenter phase II study. Journal of Clinical Oncology 19:1750–1758 Senzer, N., Nemunaitis, J., Nemunaitis, M., Lamont, J., Gore, M., Gabra, H., Eeles, R., Sodha, N., Lynch, F. J., Zumstein, L. A., Menander, K. B., Sobol, R. E., and Chada, S. (2007) p53 therapy in a patient with Li-Fraumeni syndrome. Molecular Cancer Therapeutics 6:1478–1482 Spitz, F. R., Nguyen, D., Skibber, J. M., Meyn, R. E., Cristiano, R. J., and Roth, J. A. (1996) Adenoviral-mediated wild-type p53 gene expression sensitizes colorectal cancer cells to ionizing radiation. Clinical Cancer Research 2:1665–1671 Stelling, J., Sauer, U., Szallasi, Z., Doyle, F. J., III, and Doyle, J. (2004) Robustness of cellular functions. Cell 118:675–685 Swisher, S. G., Roth, J. A., Nemunaitis, J., Lawrence, D. D., Kemp, B. L., Carrasco, C. H., Connors, D. G., El Naggar, A. K., Fossella, F., Glisson, B. S., Hong, W. K., Khuri, F. R., Kurie, J. M., Lee, J. J., Lee, J. S., Mack, M., Merritt, J. A., Nguyen, D. M., Nesbitt, J. C., Perez-Soler, R., Pisters, K. M. W., Putnam, J. B., Jr., Richli, W. R., Savin, M., Schrump, D. S., Shin, D. M., Shulkin, A., Walsh, G. L., Wait, J., Weill, D., and Waugh, M. K. A. (1999) Adenovirusmediated p53 gene transfer in advanced non-small cell lung cancer. Journal of the National Cancer Institute 91:763–771 Swisher, S., Roth, J. A., Komaki, R., Hicks, M., Ro, J., Dreiling, L., Yver, A. B., Stevens, C., Putnam, J. B., Smythe, W. R., Vaporciyan, A. A., and Walsh, G. L. (2000) A phase II trial of adenoviral mediated p53 gene transfer (RPR/INGN 201) in conjunction with radiation therapy in patients with localized non-small cell lung cancer (NSCLC). Proceedings of American Society Clinical Oncology 19:461a–461a Uno, F., Sasaki, J., Nishizaki, M., Carboni, G., Xu, K., Atkinson, E. N., Kondo, M., Minna, J. D., Roth, J. A., and Ji, L. (2004) Myristoylation of the FUS1 protein is required for tumor suppression in human lung cancer cells. Cancer Research 64:2969–2976 Wang, J. X., Bucana, C. D., Roth, J. A., and Zhang, W. W. (1995) Apoptosis induced in human osteosarcoma cells is one of the mechanisms for the cytocidal effect of Ad5CMV-p53. Cancer Gene Therapy 2:9–17 Xu, L., Huang, C. C., Huang, W., Tang, W. H., Rait, A., Yin, Y. Z., Cruz, I., Xiang, L. M., Pirollo, K. F., and Chang, E. H. (2002) Systemic tumor-targeted gene delivery by anti-transferrin receptor scFv-immunoliposomes. Molecular Cancer Therapeutics 1:337–346 Yen, N., Ioannides, C. G., Xu, K., Swisher, S. G., Lawrence, D. D., Kemp, B. L., El Naggar, A. K., Cristiano, R. J., Fang, B., Glisson, B. S., Hong, W. K., Khuri, F. R., Kurie, J. M., Lee, J. J., Lee, J. S., Merritt, J. A., Mukhopadhyay, T., Nesbitt, J. C., Nguyen, D., Perez-Soler, R., Pisters, K. M. W., Putnam, J. B., Jr., Schrump, D. S., Shin, D. M., Walsh, G. L., and Roth, J. A. (2000) Cellular and humoral immune responses to adenovirus and p53 protein antigens in patients following intratumor injection of an adenovirus vector expressing wild-type p53 (Adp53). Cancer Gene Therapy 7:530–536 Yver, A., Dreiling, L. K., Mohanty, S., Merritt, J., Proksch, S., Shu, C., and Tomko, L. S. (1999) Tolerance and safety of RPR/INGN 201, an adeno-viral vector containing a p53 gene, administered intratumorally in 309 patients with advanced cancer enrolled in phase I and II studies world-wide. Proceeding American Society Clinical Oncology 19:460a Zabarovsky, E. R., Lerman, M. I., and Minna, J. D. (2002) Tumor suppressor genes on chromosome 3p involved in the pathogenesis of lung and other cancers. Oncogene 21:6915–6935 Zhang, W. W., Fang, X., Mazur, W., French, B. A., Georges, R. N., and Roth, J. A. (1994) Highefficiency gene transfer and high-level expression of wild-type p53 in human lung cancer cells mediated by recombinant adenovirus. Cancer Gene Therapy 1:5–13

Chapter 6

Targeted Oncolytic Adenovirus for Human Cancer Therapy: Gene-Based Therapies for Cancer Toshiyoshi Fujiwara

Abstract  Replication-selective tumor-specific viruses represent a novel approach for the treatment of neoplastic disease. These vectors are designed to induce virusmediated lysis of tumor cells after selective viral propagation within the tumor. For targeting cancer cells, there is a need for tissue- or cell-specific promoters that are expressed in diverse tumor types, but are silent in normal cells. Telomerase activation is considered to be a critical step in carcinogenesis through the maintenance of telomeres, and its activity is closely correlated with human telomerase reverse transcriptase (hTERT) expression. We constructed an attenuated adenovirus 5 vector, in which the hTERT promoter element drives the expression of E1 genes. As only tumor cells that express telomerase activity activate this promoter, the hTERT proximal promoter allows for a preferential expression of viral genes in tumor cells, leading to selective viral replication and oncolytic cell death. This article reviews the recent findings in this rapidly evolving field; cancer therapeutic and cancer diagnostic approaches using the hTERT promoter. Keywords  Telomerase • Adenovirus • Virotherapy • Clinical trial • Molecular imaging

1 Introduction Viruses are the simplest form of life, carrying genetic materials and the capacity to efficiently enter host cells. Thus, viruses have been adapted as gene transfer vectors (Kaplan 2005; Guo et al. 2008; Kirn and Thorne 2009). Adenoviruses have been studied extensively and are well characterized. They are large, double-stranded DNA viruses with tropism for many human tissues, such as bronchial epithelia,

T. Fujiwara (*) Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan e-mail: [email protected] J.A. Roth (ed.), Gene-Based Therapies for Cancer, Current Cancer Research, DOI 10.1007/978-1-4419-6102-0_6, © Springer Science+Business Media, LLC 2010

79

80

T. Fujiwara

hepatocytes, and neurons. Furthermore, they are capable of transducing nonreplicating cells and can be grown to high titers in vitro, which allows for their potential use clinically. High titers of replication-defective adenoviruses can be produced and have been successfully used in eukaryotic gene expression (Mizuguchi and Kay 1998; Kaplan 2005; Stone and Lieber 2006). Numerous in vitro and animal studies have tested a wide variety of adenoviral gene therapy agents and reported potential beneficial effects for different target diseases, as well as their tolerability and safety (Wilson et al. 1994; Crystal et al. 1994, 1997; Sterman et al. 1998). Gene and vector-based molecular therapies for cancer encompass a wide range of treatment types that all use genetic material to modify cancer cells and/or surrounding tissues to exhibit antitumor properties. One of the most common approaches to emerge from the concept of gene therapy is the introduction of foreign therapeutic genes into target cells. A number of genes of interest with different functions, including tumor suppressor genes (Fujiwara et al. 1994a, b), proapoptotic genes (Kagawa et  al. 2000; Tsunemitsu et  al. 2004), suicide genes that cause cellular death with prodrugs (Chen et al. 1994; Sterman et al. 1998) and genes that inhibit angiogenesis (Feldman et al. 2000), have been proposed for this type of therapy. In fact, several groups have completed clinical trials of a replication-deficient adenoviral vector that delivers normally functioning p53 tumor suppressor gene to cancer cells (Ad5CMV-p53, Advexin). It has been reported that multiple courses of intratumoral injection of Ad5CMV-p53 are feasible and well tolerated in patients with advanced head and neck squamous cell carcinoma and nonsmall cell lung cancers, and appear to provide clinical benefits (Clayman et al. 1998; Swisher et al. 1999, 2003; Nemunaitis et al. 2000; Fujiwara et al. 2006b). Another rapidly growing area of molecular therapy for cancer is the use of oncolytic vectors for selective tumor cell destruction. As viruses infect cells and then induce cell lysis through their propagation, they can be used as anticancer agents by genetic engineering to replicate selectively in cancer cells while remaining innocuous to normal tissues (Hawkins et al. 2002). Clinical trials of intratumoral injection of Onyx-015, an adenovirus with the E1B 55-kDa gene deleted and engineered to selectively replicate in and lyse p53-deficient cancer cells (Bischoff et al. 1996), alone or in combination with cisplatin/5-fluorouracil have been conducted in patients with recurrent head and neck cancer (Khuri et al. 2000; Nemunaitis et al. 2001); however, the study later clarified that the capacity of Onyx-015 to replicate independently of the cell cycle is not correlated with the status of p53 (Goodrum and Ornelles 1998), but rather is determined by late viral RNA export (O’Shea et al. 2004). The optimal treatment of human cancer requires improvements in therapeutic ratio in order to increase cytotoxic efficacy against tumor cells and decrease that against normal cells. This may not be a simple task, as the majority of normal cells surrounding tumors are sensitive to cytotoxic agents. Thus, to establish reliable therapeutic strategies against human cancer, it is important to identify genetic or epigenetic targets present only in cancer cells. One of the targeting strategies involves the use of tissue-specific promoters to restrict gene expression or viral replication in specific tissues. A large number of different tissue-specific promoters have been used for virotherapy applications; for targeting tumors derived from various tissues,

6  Targeted Oncolytic Adenovirus for Human Cancer Therapy

81

however, tumor-specific, rather than tissue-specific, promoters would be more advantageous. For example, the promoter of human telomerase reverse transcriptase (hTERT) is highly active in most tumor cells, but is inactive in normal somatic cell types. This review highlights some very promising advances in cancer therapeutic and diagnostic technologies using the hTERT promoter.

2 Telomerase Activity for Transcriptional Cancer Targeting One of the hallmarks of cancer is unregulated proliferation of a certain cell population, which eventually affects normal cellular function in the human body, and this almost universally correlates with the reactivation of telomerase. Tumor cells are able to maintain telomere length predominantly due to telomerase, and its activity is detected in about 85% of malignant tumors (Shay and Bacchetti 1997), whereas telomerase is absent in most normal somatic tissues (Dong et al. 2005), with a few exceptions, including peripheral blood leukocytes and certain stem cell populations (Hiyama et al. 1995; Tahara et al. 1999). There is also an increasing trend in telomerase activity between early- and late-stage tumors. The strong association between telomerase activity and malignant tissue suggests that telomerase is a plausible target for the diagnosis and treatment of cancer (Shay and Wright 2002), although an alternative mechanism for telomere elongation, the so-called alternative lengthening of telomeres (ALT), is present in a certain proportion of telomerase-negative human tumors (Muntoni and Reddel 2005). The enzyme telomerase is a ribonucleoprotein complex responsible for the addition of TTAGGG repeats to the telomeric ends of chromosomes, and contains three components: the RNA subunit (known as hTR, hTER, or hTERC) (Feng et  al. 1995); the telomerase-associated protein (hTEP1) (Harrington et al. 1997); and the catalytic subunit (hTERT) (Meyerson et al. 1997; Nakamura et al. 1997). Both hTR and hTERT are required for the reconstitution of telomerase activity in  vitro (Nakayama et al. 1998) and therefore represent the minimal catalytic core of telomerase in humans (Beattie et al. 1998). However, while hTR is widely expressed in embryonic and somatic tissues, hTERT is tightly regulated and is not detectable in most somatic cells. Thus, the hTERT promoter region can be used as a fine-tuning molecular switch that works exclusively in tumor cells.

3 Telomerase-Specific Oncolytic Adenovirus for Cancer Therapeutics 3.1 Structure of hTERT Promoter-Driven Oncolytic Adenovirus The use of modified adenoviruses that replicate and complete their lytic cycles preferentially in cancer cells is a promising strategy for the treatment of cancer.

82

T. Fujiwara

One approach to achieve tumor specificity of viral replication is based on the transcriptional control of genes that are critical for virus replication, such as E1A or E4. As described above, telomerase, particularly its catalytic subunit hTERT, is expressed in the majority of human cancers and the hTERT promoter is preferentially activated in human cancer cells (Shay and Bacchetti 1997). Thus, the broadly applicable hTERT promoter may be a suitable regulator of adenoviral replication. Indeed, it has previously been reported that the transcriptional control of E1A expression via the hTERT promoter restricts adenoviral replication to telomerase-positive tumor cells and efficiently lyses tumor cells (Wirth et  al. 2003; Lanson et  al. 2003; Irving et  al. 2004; Kim et  al. 2003). Furthermore, Kuppuswamy et  al. (2005) recently developed a novel oncolytic adenovirus (VRX-011), in which the replication of the vector targets cancer cells by replacing adenovirus E4 promoter with the hTERT promoter. VRX-011 is also able to overexpress the adenovirus death protein (ADP) (also known as E3-11.6K), which is required for efficient cell lysis and the release of virions from cells at late stages of infection. The adenovirus E1B gene is expressed early in viral infection and its gene product inhibits E1A-induced p53-dependent apoptosis, which in turn promotes the cytoplasmic accumulation of late viral mRNA, leading to a shutdown of host cell protein synthesis. In most vectors that replicate under the transcriptional control of the E1A gene, including hTERT-specific oncolytic adenoviruses, the E1B gene is driven by the endogenous adenovirus E1B promoter. However, Li et  al. (2001) demonstrated that transcriptional control of both E1A and E1B genes by the a-fetoprotein (AFP) promoter with the use of IRES significantly improved the specificity and therapeutic index in hepatocellular carcinoma cells. Based on the above information, we developed Telomelysin (OBP-301), in which the tumor-specific hTERT promoter regulates both the E1A and E1B genes (Fig. 1). Telomelysin is expected to control viral replication more stringently, thereby providing better therapeutic effects in tumor cells, as well as attenuated toxicity in normal tissues (Kawashima et al. 2004).

3.2 Preclinical Studies of hTERT Promoter-Driven Oncolytic Adenovirus The majority of human cancer cells acquire immortality and unregulated proliferation by the expression of hTERT (Shay and Bacchetti 1997); therefore, hTERTspecific Telomelysin may possess broad-spectrum antineoplastic activity against a variety of human tumors (Kawashima et al. 2004; Taki et al. 2005). Telomelysin induced selective E1A and E1B expression in cancer cells, which resulted in a viral replication at 5–6 logs by 3 days after infection; on the other hand, Telomelysin replication was attenuated up to two logs in cultured normal cells (Kawashima et al. 2004; Taki et al. 2005). In vitro cytotoxicity assays demonstrated that Telomelysin are able to efficiently kill various human cancer cell lines, including head and neck

6  Targeted Oncolytic Adenovirus for Human Cancer Therapy

83

Fig.  1  Structures of telomerase-specific oncolytic adenoviruses. Telomelysin (OBP-301), in which the hTERT promoter element drives the expression of E1A and E1B linked with IRES. TelomeScan (OBP-401) is a telomerase-specific replication-competent adenovirus variant, in which GFP is inserted under the CMV promoter into the E3 region for monitoring viral replication. Telomelysin-RGD (OBP-405) is also a telomerase-specific replication-competent adenovirus with mutant fiber-containing RGD peptide, CDCRGDCFC, in the HI loop of the fiber knob. Left panel, schematic representation depicting the major structural components of Telomelysin (hexon, penton base, and fiber) and transmission electron photomicrograph

cancer, lung cancer, esophageal cancer, gastric cancer, colorectal cancer, breast cancer, pancreas cancer, hepatic cancer, prostate cancer, cervical cancer, melanoma, sarcoma, and mesothelioma in a dose-dependent manner (Hashimoto et al. 2008). These data clearly demonstrate that Telomelysin exhibits desirable features for use as an oncolytic therapeutic agent, as the proportion of cancers potentially treatable by Telomelysin is extremely high (Fig. 2). The in  vivo antitumor effects of Telomelysin were also investigated by using athymic mice carrying xenografts. Intratumoral injection of Telomelysin into human tumor xenografts resulted in significant inhibition of tumor growth and enhancement of survival (Kawashima et al. 2004; Taki et al. 2005). Macroscopically, massive ulceration was noted on the tumor surface after injecting high-dose Telomelysin, indicating that Telomelysin induced intratumoral necrosis due to direct lysis of tumor cells by viral replication in vivo (Fujiwara et al. 2007). Head and neck cancer is characterized by locoregional spread, and it is clinically accessible, making it an

84

T. Fujiwara

Fig. 2  Antitumor mechanisms of Telomelysin. Telomelysin exhibits antitumor effects both as a direct cytotoxic drug and as an immunostimulatory agent that induces specific cytotoxic T-lymphocytes (CTL) and antiangiogenic properties through the production of endogenous antiangiogenic factors such as INF-g

attractive target for intratumoral virotherapy. Thus, an orthotopic nude mouse model of human tongue squamous cell carcinoma was also used to explore the in  vivo antitumor effects of Telomelysin. Intratumoral injection of Telomelysin significantly shrunk tongue tumor volume, which in turn increased the body weight of mice by enabling oral ingestion (Kurihara et al. 2009). As the body weight loss due to feeding problems in this orthotopic tongue cancer model resembles the disease progression in head and neck cancer patients, the finding that Telomelysin increased the body weight of mice suggests that telomerase-specific virotherapy can potentially improve the quality of life in advanced head and neck cancer patients (Fujiwara 2009). For effective treatment of distant metastatic tumors, intravenously infused chemotherapeutic drugs must be distributed in sufficient concentrations at tumor sites; oncolytic viruses, however, can still replicate in the tumor, cause oncolysis, and then release virus particles that could reach distant metastatic lesions. Therefore, intratumoral administration that causes the release of newly formed viruses from infected tumor cells is theoretically suitable for oncolytic viruses. In addition, regionally administered viruses may be capable of trafficking to the regional draining lymph nodes. We demonstrated that injection of telomerase-specific oncolytic adenovirus expressing green fluorescent protein (GFP) into primary tumors allows lymphatic spread, which in turn induces viral replication in metastatic lymph nodes, allowing direct imaging of micrometastases (Kishimoto et al. 2006; Kurihara et al. 2009). These data support the possible application of Telomelysin as a lymphotropic agent in the treatment of lymph node metastasis.

6  Targeted Oncolytic Adenovirus for Human Cancer Therapy

85

3.3 Immune Activation by hTERT Promoter-Driven Oncolytic Adenovirus Chemotherapeutic drugs kill tumor cells mainly by inducing apoptosis, which is characterized by chromosome condensation and nuclear shrinkage and fragmentation. The nuclear morphology of cells infected with Telomelysin, however, is distinct from apoptosis. Apoptosis in mammalian cells is mediated by a family of cysteine proteases known as caspases, which are the executioners of apoptosis and are essential for disassembly of the cell. In contrast, cells infected with Telomelysin showed no changes in procaspase-3 levels and no expression of the cleaved form of caspase-3, suggesting that oncolytic cells are distinct from apoptotic cells. Moreover, flow cytometric analysis demonstrated that Telomelysin infection does not modulate cell cycle distribution (Watanabe et al. 2006; Fujiwara et al. 2006a). Recently, Ito et al. (2006) reported that hTERT-specific oncolytic adenovirus causes autophagic cell death, which is another type of programmed cell death different from apoptosis, in malignant glioma cells via inhibition of the mTOR signal. We also identified the partial involvement of autophagy in the cell death machinery triggered by Telomelysin infection (Endo et al. 2008; Yokoyama et al. 2008). Dendritic cells (DCs) are the most potent antigen-presenting cells and acquire cellular antigens and danger signals from dying cells to initiate antitumor immune responses via direct cell-to-cell interaction and cytokine production. The optimal forms of tumor cell death for priming DCs to release danger signals are not fully understood. We demonstrated that Telomelysin replication produces uric acid, an endogenous danger signaling molecule, by infected human tumor cells, which in turn stimulated DCs to produce interferon-g (IFN-g) and interleukin-12 (IL-12). Subsequently, IFN-g release upregulated endogenous expression of PA28, a proteasome activator in tumor cells and resulted in the induction of cytotoxic T-lymphocytes (CTL) (Endo et al. 2008). These results suggest that virus-mediated oncolysis is an effective stimulus for immature DCs to induce specific activity against human cancer cells (Fig. 2). Soluble factors in the tumor microenvironment may also influence the process of angiogenesis, which is essential for the growth and progression of malignant tumors. The finding that dying tumor cells infected with Telomelysin promoted DCs to produce Th1 cytokines such as IFN-g, which is one of the most potent antiangiogenic factors, suggests that local administration of Telomelysin affects the tumor microenvironment, thus explaining the potential therapeutic benefits on tumor angiogenesis. In fact, we found that the growth of subcutaneous murine colon tumors in syngenic mice is significantly inhibited due to reduced vascularity by intratumoral injection of Telmelysin, which was also confirmed by in vitro angiogenesis assay (Ikeda et  al. 2009). Thus, Telomelysin exerts antiangiogenic properties through the stimulation of host immune cells to produce endogenous antiangiogenic factors such as INF-g (Fig. 2).

86

T. Fujiwara

4 Telomerase-Specific Oncolytic Adenovirus for Cancer Diagnostics 4.1 hTERT Promoter-Driven GFP-Expressing Oncolytic Adenovirus Various imaging technologies have been investigated as tools for cancer diagnosis, detection, and treatment monitoring; a limiting factor in structural and anatomical imaging, however, is the inability to specifically identify malignant tissues. Although positron emission tomography (PET) using the glucose analogue 18F-2-deoxy-d-glucose (FDG) has high detection sensitivity, it is associated with limitations such as difficulty in distinguishing between proliferating tumor cells and inflammation, and its unsuitability for real-time detection of tumor tissues. This paradigm requires an appropriate “marker” that can facilitate visualization of physiological or molecular events that occur in tumor cells but not normal cells. GFP, which was originally obtained from the jellyfish Aequorea victoria, is an attractive molecular marker for imaging of live tissues because of the relatively noninvasive nature of the fluorescent (Hoffman 2005). Substantial tumor specificity resulting from the transcriptional targeting described above can be applied to cancer diagnosis by combining the replication-restricted adenovirus with GFP technology. To efficiently label and uniformly target tumor cells with green fluorescence, we modified Telomelysin to contain the GFP gene driven by the cytomegalovirus (CMV) promoter in the E3 deleted region (Fig.1). The resultant adenovirus was designated TelomeScan or OBP-401 (Watanabe et al. 2006; Fujiwara et al. 2006a). Similarly to Telomelysin, TelomeScan replicated in human cancer cell lines and coordinately induced GFP expression; TelomeScan replication, however, was attenuated in normal human fibroblasts without GFP expression.

4.2 Ex vivo Imaging of Human Circulating Tumor Cells with GFP Fluorescence The presence of circulating tumor cells (CTC) in the peripheral blood is associated with short survival; therefore, the detection of CTC is clinically useful as a prognostic factor of disease outcome and/or surrogate marker of treatment response (Cristofanilli et al. 2004, 2005). Recent technical advances in immunocytometric analysis and quantitative real-time PCR have made it possible to detect few CTC in the blood; however, there is no sensitive assay for detecting viable CTC. We developed a simple ex vivo method for detecting viable human CTC among millions of peripheral blood leukocytes by using TelomeScan (Kojima et al. 2009). The detection method involves a three-step procedure including the lysis of red blood cells, the subsequent addition of TelomeScan to cell pellets, and an automated scan

6  Targeted Oncolytic Adenovirus for Human Cancer Therapy

87

under a fluorescent microscope at 24 h after infection. TelomeScan increases the signal-to-background ratio as a tumor-specific probe, because the fluorescent signal is amplified by viral replication only in viable human tumor cells. Early and accurate evaluation of therapeutic efficacy is the hallmark of successful cancer treatment. Viable CTC may be a less invasive, repeatable biomarker for monitoring tumor response to various types of therapy, although its clinical significance remains uncertain and debatable. Our patient data demonstrate that enumeration of CTC reflects tumor burden, as CTC counts decrease upon complete surgical removal of primary tumors. In addition, cancer patients who favorably respond to systemic chemotherapy exhibit a gradual reduction in CTC counts in parallel with decreased levels of tumor markers, whereas a radiographically nonresponding patient shows an increased CTC count. This technology has the potential to allow physicians to assess response to treatment as a relevant clinical parameter, particularly in patients without elevated levels of tumor markers.

4.3 In Vivo Imaging of Lymph Node Micrometastasis with GFP Fluorescence Tumor-specific imaging is of considerable value in the treatment of human cancer, as it can define the location and tumor areas without microscopic analysis. In particular, if tumors too small for direct visual detection and therefore not detectable by direct inspection could be imaged in situ, surgeons could precisely excise tumors with appropriate surgical margins. Lymphatic invasion is one of the major routes for cancer metastasis, and adequate resection of locoregional lymph nodes is required for curative treatment in patients with advanced malignancies. Therefore, the utility of TelomeScan in real-time imaging of tumor tissues in  vivo offers a practical, safe, and cost-effective alternative to the traditional, cumbersome procedures of histopathological examination. Following intratumoral injection of TelomeScan into orthotopically implanted human colorectal or tongue tumors in mice, para-aortic or neck lymph node metastasis could be visualized under a CCD camera (Kishimoto et  al. 2006; Kurihara et al. 2009). Histopathological analysis confirmed the presence of metastatic tumor cells in lymph nodes exhibiting fluorescence, whereas GFP-negative lymph nodes contained no tumor cells. Of particular interest, metastatic lymph nodes were imaged in spots with GFP fluorescence, which was in agreement with histologically confirmed micrometastasis. These data indicate that TelomeScan causes viral spread into the regional lymphatic area and selectively replicates in neoplastic lesions, resulting in GFP expression in metastatic lymph nodes. This experiment mimics the clinical scenario where patients with gastrointestinal or head and neck malignancies undergo surgery, and the data suggest that surgeons may be able to excise primary tumors as well as metastatic lymph nodes precisely with appropriate margins by using this novel surgical navigation system with TelomeScan.

88

T. Fujiwara

5 Clinical Application of Telomerase-Specific Oncolytic Adenovirus Preclinical models have suggested that Telomelysin can selectively kill a variety of human cancer cells in vitro and in vivo via intracellular viral replication regulated by hTERT transcriptional activity. Pharmacological and toxicological studies in mice and cotton rats confirmed that none of the animals treated with Telomelysin showed signs of viral distress (e.g., ruffled fur, weight loss, lethargy or agitation) or histopathological changes in any organs at autopsy. These promising data led us to design a phase I clinical trial of Telomelysin as a monotherapy. The protocol “A phase I dose-escalation study of intratumoral injection with telomerase-specific replication-competent oncolytic adenovirus, Telomelysin (OBP-301) for various solid tumors” sponsored by Oncolys BioPharma, Inc., is an open-label, phase I, three cohort dose-escalation study (Nemunaitis et al. 2010). The trial commenced following approval of the US Food and Drug Administration (FDA) in October 2006. The study was completed to assess the safety, tolerability, and feasibility of intratumoral injection of the agent in patients with advanced solid cancer. The doses of Telomelysin were escalated from low to high virus particles (VP) in one-log increments. Patients were treated with a single intratumoral injection of Telomelysin and were then monitored over 1 month. All patients received Telomelysin without dose-limiting toxicity. Data on pharmacokinetics and biodistribution of Telomelysin may be of interest. Clinical trials of intratumoral and intravenous administration of CG7870, a replication-selective oncolytic adenovirus genetically engineered to replicate preferentially in prostate tissue, demonstrated a second virus genome peak in the plasma (DeWeese et  al. 2001; Small et al. 2006), thus suggesting active viral replication and shedding into the bloodstream. In fact, circulating Telomelysin viral genome became detectable in the plasma within 24 h of injection. This dose-dependent initial peak in circulating virus was followed by a rapid decline; however, some patients receiving 1010 and 1012 VP showed a second peak of circulating viral DNA on day 14, thus suggesting Telomelysin replication in primary tumors. All of the first nine patients had stable disease at the day-28 assessment, although six patients showed 6.6 to 34% tumor size reduction. Thus, Telomelysin is well tolerated and warrants further clinical studies for solid cancer.

6 Conclusions and Perspectives There have been substantial advances in our understanding of the molecular aspects of human cancer and in the development of technologies for the genetic modification of viral genomes. Transcriptional targeting is a powerful tool for tumor selectivity in cancer therapy and diagnosis, and the hTERT-specific oncolytic adenovirus achieves stricter targeting potential due to the amplified effects of viral replication.

6  Targeted Oncolytic Adenovirus for Human Cancer Therapy

89

Several independent studies using different regions of the hTERT promoter and different sites of the adenoviral genome responsible for viral replication, have shown that the hTERT promoter allows adenoviral replication as a molecular switch and induces selective cytopathic effects in a variety of human tumor cells (Wirth et al. 2003; Lanson et al. 2003; Irving et al. 2004; Kawashima et al. 2004; Taki et al. 2005; Hashimoto et al. 2008). Among these viral constructs, to the best of our knowledge, Telomelysin appears to be the first hTERT-dependent oncolytic adenovirus that has been used in a clinical trial based on preclinical pharmacological and toxicological studies. Thus, telomerase-specific targeted oncolytic adenovirus holds promise for the treatment of human cancer. Nevertheless, many ethical and technical hurdles remain to be tackled and must be overcome before virotherapy reaches routine clinical application. Safety considerations in virus manufacture and clinical protocols are among the most important issues to be studied. Another important issue is to find ways of improving virus cell binding and entry. Although Telomelysin showed a broad and profound antitumor effect in human cancer originating from various organs, one weakness of Telomelysin is that virus infection efficiency depends on coxsackie-adenovirusreceptor (CAR) expression, which may not be highly expressed on the cell surface of some types of human cancer cells. Thus, tumors that have lost CAR expression might be refractory to infection with Telomelysin. As modification of fiber proteins is an attractive strategy for overcoming the limitations imposed by the CAR dependence of Telomelysin infection, we modified the fiber of Telomelysin to contain RGD (Arg-Gly-Asp) peptide, which binds with high affinity to integrins (avb3 and avb5) on the cell surface, on the HI loop of the fiber protein (Fig. 1). The resultant adenovirus, termed Telomelysin-RGD or OBP-405, mediated not only CARdependent virus entry but also CAR-independent, RGD-integrin-dependent virus entry (Taki et al. 2005; Yokoyama et al. 2008). Telomelysin-RGD had an apparent oncolytic effect on human cancer cell lines with extremely low CAR expression. These data suggest that fiber-modified Telomelysin-RGD exhibits a broad target range by increasing infection efficiency, although increased toxicity is a concern, as hematopoietic cell populations such as DCs can be efficiently infected with RGD-modified adenovirus (Okada et al. 2001). A possible future direction for Telomelysin includes combination therapy with conventional therapies such as chemotherapy, radiotherapy, surgery, immunotherapy, and new modalities such as antiangiogenic therapy. As clinical activities observed by the intratumoral injection of Telomelysin suggest that even a partial elimination of the tumor could be clinically beneficial, combination approaches may lead to the development of more advanced biological therapies for human cancer. The combination of systemic chemotherapy and local injection of Telomelysin has previously shown to be effective (Fujiwara et al. 2006a; Watanabe et al. 2006; Liu et al. 2009). Peri or postoperative administration of Telomelysin may be also valuable as adjuvant therapy in areas of microscopic residual disease at tumor margins to prevent tumor recurrence or regrowth. The field of targeted oncolytic virotherapy is progressing considerably and rapidly gaining medical and scientific acceptance. Although many technical and conceptual

90

T. Fujiwara

problems remain to be solved, ongoing and future clinical studies will no doubt continue to provide important clues that may allow substantial progress in human cancer therapy.

References Beattie TL, Zhou W, Robinson MO, Harrington L (1998). Reconstitution of human telomerase activity in vitro. Curr Biol 8:177–180. Bischoff JR, Kirn DH, Williams A, Heise C, Horn S, Muna M, Ng L, Nye JA, Sampson-Johannes A, Fattaey A, McCormick F (1996). An adenovirus mutant that replicates selectively in p53deficient human tumor cells. Science 274:373–376. Chen SH, Shine HD, Goodman JC, Grossman RG, Woo SL (1994). Gene therapy for brain tumors: regression of experimental gliomas by adenovirus-mediated gene transfer in vivo. Proc Natl Acad Sci U S A 91:3054–3057. Clayman GL, el-Naggar AK, Lippman SM, Henderson YC, Frederick M, Merritt JA, Zumstein LA, Timmons TM, Liu TJ, Ginsberg L, Roth JA, Hong WK, Bruso P, Goepfert H (1998). Adenovirus-mediated p53 gene transfer in patients with advanced recurrent head and neck squamous cell carcinoma. J Clin Oncol 16:2221–2232. Cristofanilli M, Budd GT, Ellis MJ, Stopeck A, Matera J, Miller MC, Reuben JM, Doyle GV, Allard WJ, Terstappen LW, Hayes DF (2004). Circulating tumor cells, disease progression, and survival in metastatic breast cancer. N Engl J Med 351:781–791. Cristofanilli M, Hayes DF, Budd GT, Ellis MJ, Stopeck A, Reuben JM, Doyle GV, Matera J, Allard WJ, Miller MC, Fritsche HA, Hortobagyi GN, Terstappen LW (2005). Circulating tumor cells: a novel prognostic factor for newly diagnosed metastatic breast cancer. J Clin Oncol 23:1420–1430. Crystal RG, Hirschowitz E, Lieberman M, Daly J, Kazam E, Henschke C, Yankelevitz D, Kemeny N, Silverstein R, Ohwada A, Russi T, Mastrangeli A, Sanders A, Cooke J, Harvey BG (1997). Phase I study of direct administration of a replication deficient adenovirus vector containing the E. coli cytosine deaminase gene to metastatic colon carcinoma of the liver in association with the oral administration of the pro-drug 5-fluorocytosine. Hum Gene Ther 8:985–1001. Crystal RG, McElvaney NG, Rosenfeld MA, Chu CS, Mastrangeli A, Hay JG, Brody SL, Jaffe HA, Eissa NT, Danel C (1994). Administration of an adenovirus containing the human CFTR cDNA to the respiratory tract of individuals with cystic fibrosis. Nat Genet 8:42–51. DeWeese TL, van der PH, Li S, Mikhak B, Drew R, Goemann M, Hamper U, DeJong R, Detorie N, Rodriguez R, Haulk T, DeMarzo AM, Piantadosi S, Yu DC, Chen Y, Henderson DR, Carducci MA, Nelson WG, Simons JW (2001). A phase I trial of CV706, a replication-competent, PSA selective oncolytic adenovirus, for the treatment of locally recurrent prostate cancer following radiation therapy. Cancer Res 61:7464–7472. Dong CK, Masutomi K, Hahn WC (2005). Telomerase: regulation, function and transformation. Crit Rev Oncol Hematol 54:85–93. Endo Y, Sakai R, Ouchi M, Onimatsu H, Hioki M, Kagawa S, Uno F, Watanabe Y, Urata Y, Tanaka N, Fujiwara T (2008). Virus-mediated oncolysis induces danger signal and stimulates cytotoxic T-lymphocyte activity via proteasome activator upregulation. Oncogene 27:2375–2381. Feldman AL, Restifo NP, Alexander HR, Bartlett DL, Hwu P, Seth P, Libutti SK (2000). Antiangiogenic gene therapy of cancer utilizing a recombinant adenovirus to elevate systemic endostatin levels in mice. Cancer Res 60:1503–1506. Feng J, Funk WD, Wang SS, Weinrich SL, Avilion AA, Chiu CP, Adams RR, Chang E, Allsopp RC, Yu J (1995). The RNA component of human telomerase. Science 269:1236–1241. Fujiwara T (2009). Telomerase-specific virotherapy for human squamous cell carcinoma. Expert Opin Biol Ther 9:321–329.

6  Targeted Oncolytic Adenovirus for Human Cancer Therapy

91

Fujiwara T, Cai DW, Georges RN, Mukhopadhyay T, Grimm EA, Roth JA (1994a). Therapeutic effect of a retroviral wild-type p53 expression vector in an orthotopic lung cancer model. J Natl Cancer Inst 86:1458–1462. Fujiwara T, Grimm EA, Mukhopadhyay T, Zhang WW, Owen-Schaub LB, Roth JA (1994b). Induction of chemosensitivity in human lung cancer cells in  vivo by adenovirus-mediated transfer of the wild-type p53 gene. Cancer Res 54:2287–2291. Fujiwara T, Kagawa S, Kishimoto H, Endo Y, Hioki M, Ikeda Y, Sakai R, Urata Y, Tanaka N, Fujiwara T (2006a). Enhanced antitumor efficacy of telomerase-selective oncolytic adenoviral agent OBP-401 with docetaxel: Preclinical evaluation of chemovirotherapy. Int J Cancer 119:432–440. Fujiwara T, Tanaka N, Kanazawa S, Ohtani S, Saijo Y, Nukiwa T, Yoshimura K, Sato T, Eto Y, Chada S, Nakamura H, Kato H (2006b). Multicenter phase I study of repeated intratumoral delivery of adenoviral p53 in patients with advanced non-small-cell lung cancer. J Clin Oncol 24:1689–1699. Fujiwara T, Urata Y, Tanaka N (2007). Telomerase-specific oncolytic virotherapy for human cancer with the hTERT promoter. Curr Cancer Drug Targets 7:191–201. Goodrum FD, Ornelles DA (1998). p53 status does not determine outcome of E1B 55-kilodalton mutant adenovirus lytic infection. J Virol 72:9479–9490. Guo ZS, Thorne SH, Bartlett DL (2008). Oncolytic virotherapy: molecular targets in tumorselective replication and carrier cell-mediated delivery of oncolytic viruses. Biochim Biophys Acta 1785:217–231. Harrington L, McPhail T, Mar V, Zhou W, Oulton R, Bass MB, Arruda I, Robinson MO (1997). A mammalian telomerase-associated protein. Science 275:973–977. Hashimoto Y, Watanabe Y, Shirakiya Y, Uno F, Kagawa S, Kawamura H, Nagai K, Tanaka N, Kumon H, Urata Y, Fujiwara T (2008). Establishment of biological and pharmacokinetic assays of telomerase-specific replication-selective adenovirus. Cancer Sci 99:385–390. Hawkins LK, Lemoine NR, Kirn D (2002). Oncolytic biotherapy: a novel therapeutic plafform. Lancet Oncol 3:17–26. Hiyama K, Hirai Y, Kyoizumi S, Akiyama M, Hiyama E, Piatyszek MA, Shay JW, Ishioka S, Yamakido M (1995). Activation of telomerase in human lymphocytes and hematopoietic progenitor cells. J Immunol 155:3711–3715. Hoffman RM (2005). The multiple uses of fluorescent proteins to visualize cancer in vivo. Nat Rev Cancer 5:796–806. Ikeda Y, Kojima T, Kuroda S, Endo Y, Sakai R, Hioki M, Kishimoto H, Uno F, Kagawa S, Watanabe Y, Hashimoto Y, Urata Y, Tanaka N, Fujiwara T (2009). A novel antiangiogenic effect for telomerase-specific virotherapy through host immune system. J Immunol 182:1763–1769. Irving J, Wang Z, Powell S, O’Sullivan C, Mok M, Murphy B, Cardoza L, Lebkowski JS, Majumdar AS (2004). Conditionally replicative adenovirus driven by the human telomerase promoter provides broad-spectrum antitumor activity without liver toxicity. Cancer Gene Ther 11:174–185. Ito H, Aoki H, Kuhnel F, Kondo Y, Kubicka S, Wirth T, Iwado E, Iwamaru A, Fujiwara K, Hess KR, Lang FF, Sawaya R, Kondo S (2006). Autophagic cell death of malignant glioma cells induced by a conditionally replicating adenovirus. J Natl Cancer Inst 98:625–636. Kagawa S, Gu J, Swisher SG, Ji L, Roth JA, Lai D, Stephens LC, Fang B (2000). Antitumor effect of adenovirus-mediated Bax gene transfer on p53-sensitive and p53-resistant cancer lines. Cancer Res 60:1157–1161. Kaplan JM (2005). Adenovirus-based cancer gene therapy. Curr Gene Ther 5:595–605. Kawashima T, Kagawa S, Kobayashi N, Shirakiya Y, Umeoka T, Teraishi F, Taki M, Kyo S, Tanaka N, Fujiwara T (2004). Telomerase-specific replication-selective virotherapy for human cancer. Clin Cancer Res 10:285–292. Khuri FR, Nemunaitis J, Ganly I, Arseneau J, Tannock IF, Romel L, Gore M, Ironside J, MacDougall RH, Heise C, Randlev B, Gillenwater AM, Bruso P, Kaye SB, Hong WK, Kirn DH (2000). A controlled trial of intratumoral ONYX-015, a selectively-replicating adenovirus, in combination with cisplatin and 5-fluorouracil in patients with recurrent head and neck cancer. Nat Med 6:879–885.

92

T. Fujiwara

Kim E, Kim JH, Shin HY, Lee H, Yang JM, Kim J, Sohn JH, Kim H, Yun CO (2003). Ad-mTERTdelta19, a conditional replication-competent adenovirus driven by the human telomerase promoter, selectively replicates in and elicits cytopathic effect in a cancer cell-specific manner. Hum Gene Ther 14:1415–1428. Kirn DH, Thorne SH (2009). Targeted and armed oncolytic poxviruses: a novel multi-mechanistic therapeutic class for cancer. Nat Rev Cancer 9:64–71. Kishimoto H, Kojima T, Watanabe Y, Kagawa S, Fujiwara T, Uno F, Teraishi F, Kyo S, Mizuguchi H, Hashimoto Y, Urata Y, Tanaka N, Fujiwara T (2006). In vivo imaging of lymph node metastasis with telomerase-specific replication-selective adenovirus. Nat Med 12:1213–1219. Kojima T, Hashimoto Y, Watanabe Y, Kagawa S, Uno F, Kuroda S, Tazawa H, Kyo S, Hizuguchi H, Urata Y, Tanaka N, Fujiwara T (2009). A simple biological imaging system for viable human circulating tumor cells. J Clin Invest 119:3172–3181. Kuppuswamy M, Spencer JF, Doronin K, Tollefson AE, Wold WS, Toth K (2005). Oncolytic adenovirus that overproduces ADP and replicates selectively in tumors due to hTERT promoter-regulated E4 gene expression. Gene Ther 12:1608–1617. Kurihara Y, Watanabe Y, Onimatsu H, Kojima T, Shirota T, Hatori M, Liu D, Kyo S, Mizuguchi H, Urata Y, Shintani S, Fujiwara T (2009). Telomerase-specific virotheranostics for human head and neck cancer. Clin Cancer Res 15:2335–2343. Lanson NA, Jr., Friedlander PL, Schwarzenberger P, Kolls JK, Wang G (2003). Replication of an adenoviral vector controlled by the human telomerase reverse transcriptase promoter causes tumor-selective tumor lysis. Cancer Res 63:7936–7941. Li Y, Yu DC, Chen Y, Amin P, Zhang H, Nguyen N, Henderson DR (2001). A hepatocellular carcinoma-specific adenovirus variant, CV890, eliminates distant human liver tumors in combination with doxorubicin. Cancer Res 61:6428–6436. Liu D, Kojima T, Ouchi M, Kuroda S, Watanabe Y, Hashimoto Y, Onimatsu H, Urata Y, Fujiwara T (2009). Preclinical evaluation of synergistic effect of telomerase-specific oncolytic virotherapy and gemcitabine for human lung cancer. Mol Cancer Ther 8:980–987. Meyerson M, Counter CM, Eaton EN, Ellisen LW, Steiner P, Caddle SD, Ziaugra L, Beijersbergen RL, Davidoff MJ, Liu Q, Bacchetti S, Haber DA, Weinberg RA (1997). hEST2, the putative human telomerase catalytic subunit gene, is up-regulated in tumor cells and during immortalization. Cell 90:785–795. Mizuguchi H, Kay MA (1998). Efficient construction of a recombinant adenovirus vector by an improved in vitro ligation method. Hum Gene Ther 9:2577–2583. Muntoni A, Reddel RR (2005). The first molecular details of ALT in human tumor cells. Hum Mol Genet 14 Spec No. 2:R191–R196. Nakamura TM, Morin GB, Chapman KB, Weinrich SL, Andrews WH, Lingner J, Harley CB, Cech TR (1997). Telomerase catalytic subunit homologs from fission yeast and human. Science 277:955–959. Nakayama J, Tahara H, Tahara E, Saito M, Ito K, Nakamura H, Nakanishi T, Tahara E, Ide T, Ishikawa F (1998). Telomerase activation by hTRT in human normal fibroblasts and hepatocellular carcinomas. Nat Genet 18:65–68. Nemunaitis J, Khuri F, Ganly I, Arseneau J, Posner M, Vokes E, Kuhn J, McCarty T, Landers S, Blackburn A, Romel L, Randlev B, Kaye S, Kirn D (2001). Phase II trial of intratumoral administration of ONYX-015, a replication-selective adenovirus, in patients with refractory head and neck cancer. J Clin Oncol 19:289–298. Nemunaitis J, Swisher SG, Timmons T, Connors D, Mack M, Doerksen L, Weill D, Wait J, Lawrence DD, Kemp BL, Fossella F, Glisson BS, Hong WK, Khuri FR, Kurie JM, Lee JJ, Lee JS, Nguyen DM, Nesbitt JC, Perez-Soler R, Pisters KM, Putnam JB, Richli WR, Shin DM, Walsh GL, Merritt J, Roth J (2000). Adenovirus-mediated p53 gene transfer in sequence with cisplatin to tumors of patients with non-small-cell lung cancer. J Clin Oncol 18:609–622. Nemunaitis J, Tong AW, Nemunaitis M, Senzer N, Phadke AP, Bedell C, Adams N, Zhang YA, Maple PB, Chen S, Pappen B, Burke J, Ichimaru D, Urata Y, Fujiwara T (2010). A phase I study of telomerase specific replication competent oncolytic adenovirus (Telomelysin) for various solid tumors. Mol Ther 18:429-434.

6  Targeted Oncolytic Adenovirus for Human Cancer Therapy

93

O’Shea CC, Johnson L, Bagus B, Choi S, Nicholas C, Shen A, Boyle L, Pandey K, Soria C, Kunich J, Shen Y, Habets G, Ginzinger D, McCormick F (2004). Late viral RNA export, rather than p53 inactivation, determines ONYX-015 tumor selectivity. Cancer Cell 6:611–623. Okada N, Tsukada Y, Nakagawa S, Mizuguchi H, Mori K, Saito T, Fujita T, Yamamoto A, Hayakawa T, Mayumi T (2001). Efficient gene delivery into dendritic cells by fiber-mutant adenovirus vectors. Biochem Biophys Res Commun 282:173–179. Shay JW, Bacchetti S (1997). A survey of telomerase activity in human cancer. Eur J Cancer 33:787–791. Shay JW, Wright WE (2002). Telomerase: a target for cancer therapeutics. Cancer Cell 2: 257–265. Small EJ, Carducci MA, Burke JM, Rodriguez R, Fong L, van UL, Yu DC, Aimi J, Ando D, Working P, Kirn D, Wilding G (2006). A phase I trial of intravenous CG7870, a replicationselective, prostate-specific antigen-targeted oncolytic adenovirus, for the treatment of hormone-refractory, metastatic prostate cancer. Mol Ther 14:107–117. Sterman DH, Treat J, Litzky LA, Amin KM, Coonrod L, Molnar-Kimber K, Recio A, Knox L, Wilson JM, Albelda SM, Kaiser LR (1998). Adenovirus-mediated herpes simplex virus thymidine kinase/ganciclovir gene therapy in patients with localized malignancy: results of a phase I clinical trial in malignant mesothelioma. Hum Gene Ther 9:1083–1092. Stone D, Lieber A (2006). New serotypes of adenoviral vectors. Curr Opin Mol Ther 8:423–431. Swisher SG, Roth JA, Komaki R, Gu J, Lee JJ, Hicks M, Ro JY, Hong WK, Merritt JA, Ahrar K, Atkinson NE, Correa AM, Dolormente M, Dreiling L, El-Naggar AK, Fossella F, Francisco R, Glisson B, Grammer S, Herbst R, Huaringa A, Kemp B, Khuri FR, Kurie JM, Liao Z, McDonnell TJ, Morice R, Morello F, Munden R, Papadimitrakopoulou V, Pisters KM, Putnam JB, Jr., Sarabia AJ, Shelton T, Stevens C, Shin DM, Smythe WR, Vaporciyan AA, Walsh GL, Yin M (2003). Induction of p53-regulated genes and tumor regression in lung cancer patients after intratumoral delivery of adenoviral p53 (INGN 201) and radiation therapy. Clin Cancer Res 9:93–101. Swisher SG, Roth JA, Nemunaitis J, Lawrence DD, Kemp BL, Carrasco CH, Connors DG, El-Naggar AK, Fossella F, Glisson BS, Hong WK, Khuri FR, Kurie JM, Lee JJ, Lee JS, Mack M, Merritt JA, Nguyen DM, Nesbitt JC, Perez-Soler R, Pisters KM, Putnam JB, Jr., Richli WR, Savin M, Schrump DS, Shin DM, Shulkin A, Walsh GL, Wait J, Weill D, Waugh MK (1999). Adenovirus-mediated p53 gene transfer in advanced non-small-cell lung cancer. J Natl Cancer Inst 91:763–771. Tahara H, Yasui W, Tahara E, Fujimoto J, Ito K, Tamai K, Nakayama J, Ishikawa F, Tahara E, Ide T (1999). Immuno-histochemical detection of human telomerase catalytic component, hTERT, in human colorectal tumor and non-tumor tissue sections. Oncogene 18:1561–1567. Taki M, Kagawa S, Nishizaki M, Mizuguchi H, Hayakawa T, Kyo S, Nagai K, Urata Y, Tanaka N, Fujiwara T (2005). Enhanced oncolysis by a tropism-modified telomerase-specific replicationselective adenoviral agent OBP-405 (‘Telomelysin-RGD’). Oncogene 24:3130–3140. Tsunemitsu Y, Kagawa S, Tokunaga N, Otani S, Umeoka T, Roth JA, Fang B, Tanaka N, Fujiwara T (2004). Molecular therapy for peritoneal dissemination of xenotransplanted human MKN-45 gastric cancer cells with adenovirus mediated Bax gene transfer. Gut 53:554–560. Watanabe T, Hioki M, Fujiwara T, Nishizaki M, Kagawa S, Taki M, Kishimoto H, Endo Y, Urata Y, Tanaka N, Fujiwara T (2006). Histone deacetylase inhibitor FR901228 enhances the antitumor effect of telomerase-specific replication-selective adenoviral agent OBP-301 in human lung cancer cells. Exp Cell Res 312:256–265. Wilson JM, Engelhardt JF, Grossman M, Simon RH, Yang Y (1994). Gene therapy of cystic fibrosis lung disease using E1 deleted adenoviruses: a phase I trial. Hum Gene Ther 5:501–519. Wirth T, Zender L, Schulte B, Mundt B, Plentz R, Rudolph KL, Manns M, Kubicka S, Kuhnel F (2003). A telomerase-dependent conditionally replicating adenovirus for selective treatment of cancer. Cancer Res 63:3181–3188. Yokoyama T, Iwado E, Kondo Y, Aoki H, Hayashi Y, Georgescu MM, Sawaya R, Hess KR, Mills GB, Kawamura H, Hashimoto Y, Urata Y, Fujiwara T, Kondo S (2008). Autophagy-inducing agents augment the antitumor effect of telerase-selve oncolytic adenovirus OBP-405 on glioblastoma cells. Gene Ther 15:1233–1239.

Chapter 7

Gene Therapy for Malignant Pleural Mesothelioma Edmund K. Moon, Sunil Singhal, Andrew R. Haas, Daniel H. Sterman, and Steven M. Albelda

Abstract  Malignant pleural mesothelioma (MPM) is a neoplasm of the intrathoracic cavity associated with asbestos exposure that presents with symptoms of dyspnea, a pleural effusion, and nonpleuritic chest pain. MPM is associated with a poor prognosis with current treatment regimens having only a modest effect on its progressive course. However, a number of preclinical and early clinical studies have been performed investigating novel gene therapy strategies that have the potential of positively impacting the disease course. These strategies include induction of apoptosis, angiogenesis blockade, suicide gene expression, immunogene therapy, and viral oncolysis. Keywords  Malignant pleural mesothelioma • Gene therapy • Apoptosis • Antiangiogenesis • Suicide gene therapy • Immunogene therapy • Viral oncalysis

1 Background Malignant mesothelioma (MM) is an insidious neoplasm originating from the mesothelial surface which lines the pleural and peritoneal cavities, the tunica vaginalis, and pericardium. Malignant pleural mesothelioma (MPM) accounts for 80% of mesothelioma cases and usually presents in the fifth to seventh decade of life with dyspnea, a pleural effusion, and nonpleuritic chest pain in the context of an asbestos exposure history (Robinson and Lake 2005; Sterman and Albelda 2005). MPM portends a dismal prognosis (6–18 months median survival) unless it can be completely resected (a rare occurrence), and the disease course is affected only minimally by current treatments (Sugarbaker et  al. 1995; Robinson and Lake 2005). This chapter focuses on novel gene therapy approaches for MPM.

S.M. Albelda (*) Pulmonary, Allergy, and Critical Care Division, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA e-mail: [email protected] J.A. Roth (ed.), Gene-Based Therapies for Cancer, Current Cancer Research, DOI 10.1007/978-1-4419-6102-0_7, © Springer Science+Business Media, LLC 2010

95

96

E.K. Moon et al.

2 MPM as a Target for Gene Therapy The nature of MPM makes it a disease especially attractive as a target for gene therapy. There is: (1) a lack of effective treatment strategies, (2) a thin layer of mesothelial and malignant cells that offer a large surface area for efficient, rapid, and diffuse gene transfer, and (3) relatively easy access of the pleural space for biopsy, delivery of study vector/gene, and fluid sampling to document the successful gene transfer. Access and assessment of the pleural space have been greatly enhanced by an indwelling tunneled pleural catheter system (Pollak 2002).

3 Preclinical Investigations Numerous gene therapy strategies for malignant pleural disease have been investigated with the aid of cell culture and animal models (Table 1).

3.1 Induction of Apoptosis A common gene therapy strategy has involved the insertion of normal copies of absent or mutated tumor suppressor genes with the goal of inducing target cell apoptosis. In light of p53 mutations accounting for the majority of solid tumor genetic abnormalities, wild type p53 insertion has been the most commonly explored solid tumor gene therapy strategy. Most MPMs have a wild type p53 gene;

Table 1  Gene therapy approaches for malignant pleural disease Approach Examples Induction of apoptosis p53, p16INK4A, p14(ARF), Bak, antisense SV40-T antigen, REIC/Dkk-3 Antiangiogenesis Soluble form of the VEGF receptor (Flt-1), antiangiogenic pigment epithelium-derived factor Suicide gene therapy Herpes simplex thymidine kinase gene plus ganciclovir Cytosine deaminase gene plus 5-flurocytosine Immunogene therapy   Cytokine therapy Interleukin-2, interleukin-12, type 1 and type 2 interferons, GM-CSF   Nonspecific induction of Liposome/DNA complexes, mycobacterial heat shock innate and acquired immunity protein gene (HSP-65), anti-CD40 ligand Tumor-selective replicating Herpes virus, vaccinia virus, adenovirus, measles viruses virus

7  Gene Therapy for Malignant Pleural Mesothelioma

97

h­ owever, there is functional pathway impairment due to inhibiting factors such as MDM-2 and simian virus 40 (SV40) large T antigen (Tag). Thus, inducing wild type p53 overexpression may be one option of approaching MPMs as demonstrated by Giuliano and colleagues (2000). In their series of experiments, human mesothelioma cell transduction with a replication-deficient adenovirus (Ad) vector carrying the wild-type p53 gene led to significant inhibition of in vitro tumor cell growth via apoptotic pathways. The effect was also demonstrated in immunodeficient mice with established human mesothelioma xenografts (Giuliano et al. 2000). Another important gene in apoptotic pathways is p16INK4A. Mutations in p16INK4A result in unchecked cell cycle progression even in the presence of other normally expressing apoptotic genes (e.g., pRB, p53). Thus, attempts to restore normal expression of this cyclin-dependent kinase inhibitor have been another MPM gene therapy strategy. Frizelle and colleagues (1998) demonstrated that p16INK4A reexpression in mesothelioma cells in vitro and in vivo resulted in cell cycle arrest, cell growth inhibition, apoptosis, and tumor reduction. In an athymic mouse model with established human mesothelioma xenografts, increased survival was shown with repeated administration of an adenoviral vector expressing wild-type p16INK4A (Frizelle et  al. 2000). The same investigative group demonstrated the ability to restore p16INK4A function in mesothelioma cells in vitro and in vivo using the transactivator protein (TAT) human immunodeficiency virus type-1 (HIV-1) delivery system, a relatively new method of protein transduction bypassing the need for virus (Frizelle et al. 2008). Another common MPM genetic abnormality is the loss of the INK4a/ARF gene that encodes p14(ARF), a protein complex that promotes degradation of the p53 inhibitor MDM-2. Yang and colleagues (2000) have targeted this locus by transfecting human mesothelioma cells in vitro with an adenoviral vector encoding human p14(ARF) complementary DNA. Intracellular p53 levels were enhanced and tumor cell growth was inhibited via cell cycle arrest and apoptosis induction. An alternative apoptosis induction strategy for mesothelioma cells is to introduce “downstream” apoptosis inducers. An example of such an inducer is Bak, a member of the proapoptotic Bcl2 gene family. After in vitro Bak overexpression via adenoviral vector cell transduction, mesothelioma cell viability was significantly decreased in both p53-dependent and p53-independent cell lines (Pataer et al. 2001). A controversial potential MM carcinogen is SV40 large T-antigen (Tag) (Klein et al. 2002). SV40 sequences have been identified in MPM tumor specimens and have been postulated as a possible causative factor in MM oncogenesis and proliferation. SV40 is a nonhuman polyomavirus that contaminated some polio vaccines in the mid-twentieth century. SV40 can transform normal cells by way of its prooncogenic Tag, as has been demonstrated intrapleurally and intraperitoneally in hamsters (Cicala et al. 1993). Schrump and colleagues (2001) demonstrated in vitro that antisense oligonucleotides engineered to prevent SV40 Tag expression induced apoptosis and sensitized mesothelioma cells to chemotherapeutic drugs. More recently, another tumor suppressor gene was identified, REIC/Dickkopf-3 (Dkk-3) (Tsuji et  al. 2001), which induces apoptosis from phosphorylation of

98

E.K. Moon et al.

c-Jun-NH2 kinase (JNK) and may be another possible therapeutic MPM gene therapy tool. Adenoviral mediated REIC/Dkk-3 overexpression induced mesothelioma cell apoptosis in vitro and in the pleural cavity of an orthotopic MPM mouse model (Kashiwakura et al. 2008). Attempts at increasing vector specificity to induce apoptosis in mesothelioma but not normal cells, have been made by Fukazawa and colleagues (2008). Taking advantage of the CREBBP/EP300 inhibitory protein 1 (CRI1) gene, which is specifically expressed in MPM, the investigators were able to engineer a recombinant adenoviral vector expressing the proapoptotic BH3-interacting death agonist driven by the CRI1 promoter. This vector induced cell death in MM cells but not in normal cells. Antitumor effects were also demonstrated in a mesothelioma xenograft mouse model. Despite the value of these studies in learning about various apoptosis candidate genes in MPM cells, these approaches have an important intrinsic limitation: the lack of a strong bystander effect (that is, killing of cells not transduced with the vector). Without bystander effects, cell death induction will be restricted only to transduced cells. Given our current ability to transduce only a minority of tumor cells in vivo, this lack of bystander effect is likely to ultimately limit therapeutic efficacy.

3.2 Antiangiogenesis Another area of active research interest has been to develop ways to limit the supply of blood, and ultimately nutrients and oxygen, to tumor cells. This approach could bypass some of the “bystander effect” limitations mentioned above. Studies of tumor blood vessel growth inhibition as a therapeutic avenue have resulted in a number of approved therapies such as anti-vascular endothelial growth factor (VEGF) antibody. Soluble VEGF receptor (Flt-1) and the antiangiogenic pigment epitheliumderived factor have been somewhat successful in inhibiting tumor progression in animal models of lung cancer and MPM, respectively, when delivered intrapleurally using adenoviral vector (Mae and Crystal 2002; Merritt et al. 2004). Here, the relatively short gene expression duration using adenoviral vectors will likely limit the efficacy of this strategy clinically, but transducing antiangiogenic factors using adeno-associated virus (AAV) vectors (De et  al. 2004) or lentiviral vectors may overcome this limitation, thereby allowing an effective strategy as primary treatment or chemotherapy adjuvant in the future.

3.3 Suicide-Gene Therapy Taking into account the importance of bystander effects, one of the first approaches for mesothelioma was the use of “suicide gene therapy”. Tumor cells were transduced

7  Gene Therapy for Malignant Pleural Mesothelioma

99

with a cDNA encoding for a specific enzyme that caused them to be sensitive to an otherwise benign agent. In essence, a “prodrug” is transformed into a toxic metabolite by the enzyme introduced into the cells with subsequent accumulation leading to tumor cell death or “suicide” (Tiberghien 1994). Often, nonhuman suicide genes are utilized in an effort to limit unwanted effects on normal human tissues (Hoganson et al. 1996). Examples include the Escherichia coli cytosine deaminase gene which converts 5-fluorocytosine to the toxic 5-fluorouracil (Huber et al. 1994) and the herpes simplex virus-1 thymidine kinase (HSVtk) gene which make transduced cells sensitive to the nucleoside analog ganciclovir (GCV). GCV is metabolized poorly by mammalian cells and thus it is usually nontoxic. However, after conversion to GCV-monophosphate by HSVtk, it is metabolized rapidly by endogenous kinases to GCV-triphosphate which acts as a potent inhibitor of DNA polymerase and competes with normal mammalian nucleosides for DNA replication (Matthews and Boehme 1988; Tiberghien 1994). HSVtk’s effect in the presence of GCV is enhanced by the “bystander effect” that was first observed when tumor regression in in  vivo HSVtk experiments resulted in the absence of ubiquitous transgene expression (Smythe et  al. 1994, 1995; Esandi et al. 1997; Qiao et al. 2000). The passage of toxic GCV metabolites from transduced to nontransduced cells is possible via gap junctions and/or apoptotic vesicles, leading to cell killing in nontransduced or “bystander” cells. Subsequent studies have revealed that in addition to this direct transfer of toxic metabolites, an antitumor immune response induced by HSVtk/GCV “immunogenic killing” is important in bystander killing. Ganciclovir can induce apoptosis and necrosis in tumor cells transduced with Ad.HSVtk with important immunological consequences (Vile et al. 1997). Ganciclovir-induced killing stimulates endogenous “danger signals” (such as heat shock proteins and protein high mobility group box  1 protein) that activate both the adaptive and innate immune system (Melcher et al. 1998; Candolfi et al. 2009). Macrophages are rapidly attracted to the site of release of these local danger signals (Gough et al. 2001). In addition, these stressed dying cells provide tumor-associated antigens for dendritic cell loading to trigger specific T cell activation (Todryk et  al. 1999; Xing et  al. 2008). These immune activating pathways increase natural killer cell and lymphocyte infiltration (Neves et  al. 2009), augment T-lymphocyte cytotoxicity (Okada et  al. 2001), increase MHC I expression on tumor cells (Okada et  al. 2001), and result in increased antitumor cytokine (i.e., RANTES) production (Felzmann et  al. 1997; Neves et al. 2009). It also appears that neutrophils infiltrate tumors dying by suicide gene therapy, although the exact phenotype and the pro- versus antitumor nature of these cells is still unknown (Sanchez-Perez et al. 2007). In our mesothelioma trial (discussed below), a delayed and progressive response in some patients and the appearance of antibody responses against tumor cell lines suggest that these immunostimulatory effects also occur after suicide gene therapy in humans. HSVtk DNA transfer has been performed to target pleural and peritoneal tumors by way of a variety of vector systems including carrier cells (Schwarzenberger et  al. 1998b; Schwarzenberger et  al. 1998c), liposomes (Aoki et  al. 1997;

100

E.K. Moon et al.

Nagamachi et al. 1999), plasmid DNA:polyethylenimine complexes (Aoki et al. 1997), and AAV2 vectors (Berlinghoff et al. 2004). Among the delivery systems, however, the adenoviral vector appears most effective. Initial investigations of replicationdeficient Ad.HSVtk demonstrated efficient transduction of mesothelioma cells in both tissue explants and animal models that resulted in HSVtk-mediated MM cell killing, even in the presence of relatively low GCV concentrations (Smythe et al. 1995). Furthermore, Ad.HSVtk/GCV therapy successfully treated intraperitoneal human mesothelioma and lung cancer xenografts in immunodeficient murine (Hwang et al. 1995) and rat models (Elshami et al. 1996; Esandi et al. 1997) resulting in increased survival. These preclinical studies led to a series of clinical trials using Ad.HSVtk (see below).

3.4 Immunogene Therapy Innate and adaptive antitumoral immune responses can be elicited by delivering nonspecific immunostimulatory genes. Delivery of the protein gene-65, a mycobacterial heat shock protein, resulted in significant antitumoral responses in a syngeneic MM murine model. However, this effect appeared to be associated with nonspecific lipid-pDNA complex effects likely secondary to the unmethylated cytosine-guanine (CpG) motifs of the prokaryotic DNA in the vector plasmids. These motifs appeared sufficient to activate “danger signals” and to induce innate and adaptive antitumoral immune responses (Lanuti et al. 2000; Rudginsky et al. 2001). Similar findings were published by Lukacs and colleagues (1999) who studied intraperitoneal b-galactosidase gene delivery in immunocompetent mice-bearing intra-abdominal MM. Tumor cell transfection with plasmid-liposome complexes or replication-incompetent retroviruses encoding for b-galactosidase resulted in significant decreases in intra-abdominal MM tumor burden. This effect was not seen in immunodeficient mice. Tumor-specific cytotoxic T lymphocyte (CTL) generation in MM-bearing mice was seen and was likely related to the antitumoral effect demonstrated. As opposed to nonspecific immunostimulatory gene delivery as just discussed, specific cytokine delivery can generate directed immunologic and antitumor responses. Cytokines, such as Type I interferons, have both direct antiproliferative and apoptotic effects upon MM in addition to activating intrapleural and intratumoral immune effector cells (e.g., cytotoxic T lymphocytes and natural killer cells) in vivo (Fig. 1). Tumor cell cytokine gene expression results in high levels of intratumoral cytokines generated in a paracrine manner, theoretically leading to powerful local cytokine effects without significant systemic toxicity. Odaka and colleagues (2001) eradicated tumor and prolonged survival in an established murine MM tumor model with a single intraperitoneal injection of a recombinant adenovirus expressing the murine IFN-b gene (Ad.muIFN-b). Interestingly, marked responses were also seen in distant subcutaneous tumors confirming generation of a systemic antitumor immune response. This Ad.muIFN-b

7  Gene Therapy for Malignant Pleural Mesothelioma

101

Direct inhibition of tumor cell proliferation and survival Direct killing of human tumor cells (primarily apoptotic cell death)

Indirect anti-tumor immune response by activating natural killer cells, macrophages and cytotoxic T lymphocytes

Potential antiangiogenic activity inhibiting tumor growth by decreasing blood supply to solid tumor Inhibition of Tumor Cell Growth Tumor Cell Death

Fig. 1  Multi-pronged effect of IFN-b cytokine on malignant mesothelioma cells leading to tumor cell growth inhibition and tumor cell death

antitumor effect was mediated by CD8+ T lymphocyte induction and recruitment (Odaka et al. 2002). Other animal studies have shown good antimesothelioma effects using other proinflammatory cytokines including interleukin-2 (IL-2) (Leong et  al. 1997), interleukin-12 (IL-12) (Caminschi et  al. 1999), granulocyte-monocyte-colony stimulating factor (GMC-SF) (Mukherjee et  al. 2001; Triozzi et  al. 2005), interferon-g (IFN-g) (Gattacceca et al. 2002; Cordier Kellerman et al. 2003), ­interleukin-24 (mda-7) (Cao et al. 2002), and CD40-ligand (Friedlander et al. 2003; Nowak et al. 2003).

3.5 Replicating, Tumor-Selective Oncolytic Viral Vectors One strategy of increasing tumor-specific targeting has been to modify or engineer viruses so that they preferentially replicate within tumor, but not normal cells (i.e., oncolytic viruses). Furthermore, these tumor-selective viruses can be genetically modified with specific transgenes (i.e., a suicide gene or cytokine). This strategy has largely been incorporated utilizing mutated adenoviruses (e.g., ONYX-015, a replication-restricted adenovirus that lacks functional p53) and has led to a number of clinical trials in solid tumors, such as head and neck tumors and prostate cancers. To date, its application in MPM has been limited to in vitro models (Yang et al. 2001; Zhu et al. 2005). Herpes viruses (e.g., NV1020, NV1066, G207, HSV1716) have also been modified to increase tumor-selectivity with successful reduction of tumor burden and survival

102

E.K. Moon et al.

prolongation in murine MM models (Kucharczuk et  al. 1997; Adusumilli et  al. 2006) and metastatic pleural cancer (Stiles et al. 2006). A fairly recent strategy to increase oncolytic herpes virus efficacy has been to combine oncolytic virotherapy with radiotherapy. Adusumilli and colleagues (2007) applied the oncolytic herpes virus NV1066 to a MPM cell line in the presence of 1–5  Gy radiation. Growth Arrest and DNA Damage Protein 34 (GADD34)dependent synergism was demonstrated by increased cell killing to potentially minimize dosage and toxicity of each agent. Live-attenuated measles virus was also investigated as another potential oncolytic viral agent by Gauvrit and colleagues (2008). Its effect was studied on a panel of MM cells derived from MPM pleural effusions. MPM cells were preferentially infected and killed versus nontransformed mesothelial cells with the generation of an autologous antitumor response. After the induction of apoptotic cell death of infected mesothelioma cells, tumor-specific cytotoxic T cell proliferation occurred via dendritic cell antigen presentation (Gauvrit et al. 2008). Kelly and colleagues (2008) have recently reported the use of a replicationcompetent engineered vaccinia virus (GLV-1h68) to treat MPM in vitro and in vivo. The virus successfully infected six different human MPM cell lines as measured by b-galactosidase expression. There was greater than 50% cell killing of five of the seven MPM cell lines by day 7 after infection with one cell line totally eradicated by day 4. Intrapleural GLV-1h68 administration had significant effects in an orthotopic murine MPM model as measured by prevention of cachexia and tumor-related morbidity, reduced tumor burden, and cure in some (Kelly et al. 2008).

4 Clinical Investigations 4.1 Suicide Gene Therapy A Phase 1 trial using irradiated ovarian carcinoma cells retrovirally transfected with HSVtk (PA1-STK cells) was conducted based on preclinical data showing prolonged survival using these carrier cells in syngeneic murine MM models (Schwarzenberger et al. 1998c; Schwarzenberger et al. 1998b). PA1-STK cells were instilled intrapleurally followed by GCV for 7 days. Minimal side effects were seen and 99Tc radiolabeled PA1-STK cells demonstrated preferential adhesion to the tumor lining the chest wall. There were also some posttreatment increases in the percentage of CD8+ T lymphocytes in the pleural fluid. However, no significant clinical responses were seen (Schwarzenberger et al. 1998b; Harrison et al. 2000). Based on the preclinical murine model data discussed above, Sterman and ­colleagues (1998) initiated a Phase 1 clinical trial of first-generation Ad.HSVtk/ GCV gene therapy in advanced MPM patients to assess toxicity, gene transfer efficiency, and immune response induction (Treat et al. 1996). Subsequent to a single intrapleural administration of Ad.HSVtk vector, GCV was given intraveneously twice daily for 2 weeks.

7  Gene Therapy for Malignant Pleural Mesothelioma

103

Dose-related intratumoral HSVtk gene transfer was demonstrated in 17 of 25 patients with those treated at a dose of equal to or greater than 3.2 × 1011 particle forming units (pfu) having evidence of HSVtk protein expression at tumor surfaces and up to 30–50 cell layers deep by immunohistochemical assessment (Fig.  2). Overall, the therapy was well tolerated with minimal side effects (e.g., reversible transaminitis, anemia, fever, bullous skin eruption at instillation site, and transient and fully reversible hypotension and hypoxemia in those who received the highest dose) and dose-limiting toxicity was not reached. Antitumor antibodies and antiadenoviral immune responses, including high titers of anti-adenoviral neutralizing antibody and proliferative T-cell responses were generated in both serum and pleural

Fig.  2  HSVtk protein expression in pleural biopsies was determined 72  h after instillation of Ad.HSVtk. Red staining represents HSVtk expression. While pretreatment samples showed no staining, clear nuclear and cytoplasmic staining could be seen (a) on tumor surface (b) and in deeper layers as assessed by immunohistochemistry

104

E.K. Moon et al.

fluid. These immune responses did not seem to be affected by the administration of intravenous corticosteroids at the time of vector instillation (Sterman et al. 2000). A number of clinical responses (i.e., survival of more than 3 years) were seen at the higher dose levels (Sterman et al. 2005). A second Phase 1 clinical trial by Sterman and colleagues was initiated in 1998 using an advanced-generation (E1/E4-deleted) adenoviral vector that allowed increased vector doses due to decreased contamination with high levels of replicationcompetent adenovirus (1.5 and 5.0 × 1013 viral particles were the two dose levels administered). Diminished cytopathic effects and reduced cellular immune responses were additional theoretical advantages over the older generation vector (Gao et al. 1996). The five patients treated under this protocol experienced similar side effects as seen in the first phase 1 trial (Sterman et al. 1999). Dose-related gene transfer was detected in all patients by immunohistochemistry. Again, significant humoral responses to the rAd.HSVtk virus were detected in all subjects with high serum titers of total and neutralizing antibodies. Of the five patients treated in this second phase 1 trial, there were two patients with long periods of survival (one 7 years and one still alive after 9 years), each enrolled with a diagnosis of stage I epithelioid MPM, and each treated at the higher vector dose. One of the two surviving patients had a demonstrable reduction of tumor metabolic activity as assessed by serial 18-fluorodeoxyglucose positron emission tomography (18FDG PET) scans over several months. This relatively long response period was likely due to the induction of a secondary immune bystander effect of the Ad.HSVtk/GCV instillation (see discussion of mechanisms above).

4.2 Cytokine Gene Therapy Several Phase 1 and Phase 2 clinical trials have been published documenting antitumor responses in MM after intrapleural infusion of interleukin-2 (IL-2), interferon-beta (IFN-b), and interferon-gamma (IFN-g) proteins (Boutin et  al. 1991, 1994; Astoul et al. 1993, 1998; Christmas et al. 1993; Goey et al. 1995). One major concern with these approaches has been the toxicity and need for repeat administration of the soluble agent into the pleural space to maintain efficacy. In Perth, Australia, Robinson and colleagues conducted the first clinical trial of intratumoral cytokine gene delivery in MPM patients using a recombinant partially replication-restricted vaccinia virus (VV) that expressed the human IL-2 gene. Serial VV-IL-2 vector injections over a period of 12 weeks into chest wall lesions of six patients with advanced MPM resulted in minimal toxicity with no demonstrable evidence of vector spread to patient contacts. Though no significant regression of tumor was seen, modest intratumoral T-cell infiltration was detected on posttreatment biopsy specimens. As measured by reverse transcriptase polymerase chain reaction (rtPCR), VV-IL-2 mRNA was detected in biopsy specimens for up to 6 days postinjection (though declined to low levels by day 8) despite the generation of significant levels of anti-VV-neutralizing antibodies (Mukherjee et al. 2000).

7  Gene Therapy for Malignant Pleural Mesothelioma

105

Vero cells, which are immortalized monkey fibroblasts capable of expressing human proteins, have also been studied as a cytokine-delivering vector in humans. Fourteen patients received four courses of injections of Vero cells expressing IL-2. The treatment was well tolerated with no significant adverse effects. Levels of circulating IL-2 were detected in half of the patients with one patient demonstrating transient tumor regression and one resulting in disease stabilization for 4 months. The results were reported in abstract form and, to our knowledge, this approach is not being pursued further (Pitako et al. 2003). Based on preclinical data by Odaka and colleagues (described above) (2001, 2002), a Phase 1 Ad.IFN-b dose escalation trial in MPM patients (seven patients) and metastatic pleural malignancies (three patients) was undertaken (Sterman et al. 2006, 2007). The first three patients received 9 × 1011 Ad.IFN-b viral particles and had mild to moderate toxicities (e.g., transient lymphopenia, chest pain, coryza, fever, anemia, transaminase elevation). Two of four patients with MPM who were subsequently enrolled at 3 × 1012 viral particles experienced dose-limiting toxicities (transient hypoxia and grade 3 transaminase elevations, both of which returned to baseline levels.) Thus the remaining three patients were treated at the previous dose level of 9 × 1011 viral particles without serious adverse events. Gene transfer was detected in seven of the ten patients by the measurement of pleural fluid IFN-b mRNA or protein. Antitumor immune responses, including humoral responses to known tumor antigens (e.g., SV40 Virus Tag, mesothelin) and unknown tumor antigens were elicited in seven of the ten patients (Fig.  3). Four patients demonstrated meaningful clinical responses defined as disease stability and/or regression on 18FDG-PET and CT imaging at Day 60 postvector administration. Two patients are still alive, surviving for longer than 3 years after intrapleural Ad.IFN-b gene therapy. Based on preclinical studies showing enhanced effects after two doses, a second Phase 1 trial involving two administrations of Ad.IFN-b (levels ranging from 3 × 1011 to 3 × 1012 viral particles) via an indwelling pleural catheter separated by 1–2 weeks was conducted in seventeen patients (10 with MPM and 7 with malignant pleural effusions.) Again, overall treatment was well tolerated and antitumor humoral immune responses similar to that seen in the initial trials were induced. Several patients had meaningful clinical responses (mixed and/or partial responses) as determined by pre- and postvector delivery PET/CT scans (Fig. 4). However, high antiadenoviral neutralizing antibodies titers were detected after either a 1- or 2-week period, inhibiting effective gene transfer of the second dose. Patients are being enrolled into a third Phase 1 trial of Ad.IFN (a instead of b, solely as a result of changes in corporate sponsorship), with a modified protocol: the two Ad.IFN-a vector doses are being administered 3 days apart. Preliminary results show high IFN-a protein expression with IFN-a production also seen from the second dose, probably due to the fact that neutralizing antibodies are not induced within a three-day time frame. Based on preclinical studies showing synergy between Ad.IFN and systemic chemotherapy, our group plans to administer Ad.IFN vector in combination with

106

E.K. Moon et al.

SV40 Serum #107 Serum #107 Pre Post Ren SV40 Ren

SV40

64Kd 51Kd 39Kd

Cell line

Patient 112

Pt 106 - M30

Pre 6wk 4m 6m 97kd

97Kd

Mesothelin

191kd Serum 1:1500

97kd

64kd

64kd

51kd

51kd

97kd

97Kd

Anti-mesothelin

64Kd

39kd

64kd

51Kd

28kd 51kd

39Kd SV40 mab

19kd Pre

6 wk 4 months

Fig.  3  Examples of antitumor humoral immune responses from patients in the Ad.IFN-b trial. Serum was taken from patients before and after (6 weeks to 4 months) gene transfer. This serum (diluted 1:1,500) was used in immunoblotting of gels on which known tumor antigens (i.e., SV40 large Tag or mesothelin) had been run and transferred to nitrocellulose (left and middle gels) or on which a mesothelioma cell line extract had been run and transferred. Marked increases in antibodies to SV40 Tag, mesothelin, or unknown tumor antigens (arrows) were seen in posttreatment serum

chemotherapy for MPM patients. Additionally, in light of preclinical studies demonstrating a benefit of debulking surgery in combination with immunotherapy (Kruklitis et al. 2004), a neoadjuvant surgery trial involving vector administration to MPM patients followed by maximal cytoreduction and adjuvant chemo-radiotherapy is also being planned.

5 Summary Thus far, MPM continues to loom as an ominous disease due to the lack of effective treatment. In the past two decades, substantial progress has been made in both preclinical and clinical settings and there is potential for novel gene therapy strategies to make a positive impact. Initial efforts to genetically target tumor and augment immune mechanisms against MPM have been promising. A multi-modality surgical, chemotherapeutic, and gene therapy approach has the potential to further enhance our ability to treat this devastating disease. Oncolytic viruses also hold promise. As investigations increase, so does the confidence that gene therapy will have a significant role in the future of MPM treatment.

7  Gene Therapy for Malignant Pleural Mesothelioma

107

Fig. 4  CT Response to intrapleural instillation of Ad.INFb. Upper panel shows pretreatment CT scan with large amount of tumor in the right base (white arrow). MPM tumor burden in posterior aspect of right thoracic cavity eradicated by day 60 postvector instillation (white arrow). Clear area in the liver is a cyst

References Adusumilli PS, Stiles BM, Chan MK et al. (2006). Imaging and therapy of malignant pleural mesothelioma using replication-competent herpes simplex viruses. J Gene Med 8(5): 603–15. Adusumilli PS, Chan MK, Hezel M et al. (2007). Radiation-induced cellular DNA damage repair response enhances viral gene therapy efficacy in the treatment of malignant pleural mesothelioma. Ann Surg Oncol 14(1): 258–69. Aoki K, Yoshida T, Matsumoto N et  al. (1997). Gene therapy for peritoneal dissemination of pancreatic cancer by liposome-mediated transfer of herpes simplex virus thymidine kinase gene. Hum Gene Ther 8(9): 1105–13.

108

E.K. Moon et al.

Astoul P, Viallat JR, Laurent JC et al. (1993). Intrapleural recombinant IL-2 in passive immunotherapy for malignant pleural effusion. Chest 103(1): 209–13. Astoul P, Picat-Joossen D, Viallat JR et al. (1998). Intrapleural administration of interleukin-2 for the treatment of patients with malignant pleural mesothelioma: a Phase II study. Cancer 83(10): 2099–104. Berlinghoff S, Veldwijk MR, Laufs S et al. (2004). Susceptibility of mesothelioma cell lines to adeno-associated virus 2 vector-based suicide gene therapy. Lung Cancer 46(2): 179–86. Boutin C, Viallat JR, Van Zandwijk N et al. (1991). Activity of intrapleural recombinant gammainterferon in malignant mesothelioma. Cancer 67(8): 2033–7. Boutin C, Nussbaum E, Monnet I et al. (1994). Intrapleural treatment with recombinant gammainterferon in early stage malignant pleural mesothelioma. Cancer 74(9): 2460–7. Caminschi I, Venetsanakos E, Leong CC et al. (1999). Cytokine gene therapy of mesothelioma. Immune and antitumor effects of transfected interleukin-12. Am J Respir Cell Mol Biol 21(3): 347–56. Candolfi M, Yagiz K, Foulad D et  al. (2009). Release of HMGB1 in response to proapoptotic glioma killing strategies: efficacy and neurotoxicity. Clin Cancer Res 15(13): 4401–14. Cao XX, Mohuiddin I, Chada S et  al. (2002). Adenoviral transfer of mda-7 leads to BAX upregulation and apoptosis in mesothelioma cells, and is abrogated by over-expression of BCL-XL. Mol Med 8(12): 869–76. Christmas TI, Manning LS, Garlepp MJ et al. (1993). Effect of interferon-alpha 2a on malignant mesothelioma. J Interferon Res 13(1): 9–12. Cicala C, Pompetti F and Carbone M (1993). SV40 induces mesotheliomas in hamsters. Am J Pathol 142(5): 1524–33. Cordier Kellerman L, Valeyrie L, Fernandez N et al. (2003). Regression of AK7 malignant mesothelioma established in immunocompetent mice following intratumoral gene transfer of interferon gamma. Cancer Gene Ther 10(6): 481–90. De B, Heguy A, Leopold PL et  al. (2004). Intrapleural administration of a serotype 5 adenoassociated virus coding for alpha1-antitrypsin mediates persistent, high lung and serum levels of alpha1-antitrypsin. Mol Ther 10(6): 1003–10. Elshami AA, Kucharczuk JC, Zhang HB et al. (1996). Treatment of pleural mesothelioma in an immunocompetent rat model utilizing adenoviral transfer of the herpes simplex virus thymidine kinase gene. Hum Gene Ther 7(2): 141–8. Esandi MC, van Someren GD, Vincent AJ et al. (1997). Gene therapy of experimental malignant mesothelioma using adenovirus vectors encoding the HSVtk gene. Gene Ther 4(4): 280–7. Felzmann T, Ramsey WJ and Blaese RM (1997). Characterization of the antitumor immune response generated by treatment of murine tumors with recombinant adenoviruses expressing HSVtk, IL-2, IL-6 or B7-1. Gene Ther 4(12): 1322–9. Friedlander PL, Delaune CL, Abadie JM et al. (2003). Efficacy of CD40 ligand gene therapy in malignant mesothelioma. Am J Respir Cell Mol Biol 29(3 Pt 1): 321–30. Frizelle SP, Grim J, Zhou J et al. (1998). Re-expression of p16INK4a in mesothelioma cells results in cell cycle arrest, cell death, tumor suppression and tumor regression. Oncogene 16(24): 3087–95. Frizelle SP, Rubins JB, Zhou JX et al. (2000). Gene therapy of established mesothelioma xenografts with recombinant p16INK4a adenovirus. Cancer Gene Ther 7(11): 1421–5. Frizelle SP, Kratzke MG, Carreon RR et al. (2008). Inhibition of both mesothelioma cell growth and Cdk4 activity following treatment with a TATp16INK4a peptide. Anticancer Res 28(1A): 1–7. Fukazawa T, Matsuoka J, Naomoto Y et  al. (2008). Malignant pleural mesothelioma-targeted CREBBP/EP300 inhibitory protein 1 promoter system for gene therapy and virotherapy. Cancer Res 68(17): 7120–9. Gao GP, Yang Y and Wilson JM (1996). Biology of adenovirus vectors with E1 and E4 deletions for liver-directed gene therapy. J Virol 70(12): 8934–43. Gattacceca F, Pilatte Y, Billard C et al. (2002). Ad-IFN gamma induces antiproliferative and antitumoral responses in malignant mesothelioma. Clin Cancer Res 8(10): 3298–304. Gauvrit A, Brandler S, Sapede-Peroz C et al. (2008). Measles virus induces oncolysis of mesothelioma cells and allows dendritic cells to cross-prime tumor-specific CD8 response. Cancer Res 68(12): 4882–92.

7  Gene Therapy for Malignant Pleural Mesothelioma

109

Giuliano M, Catalano A, Strizzi L et al. (2000). Adenovirus-mediated wild-type p53 overexpression reverts tumourigenicity of human mesothelioma cells. Int J Mol Med 5(6): 591–6. Goey SH, Eggermont AM, Punt CJ et al. (1995). Intrapleural administration of interleukin 2 in pleural mesothelioma: a phase I-II study. Br J Cancer 72(5): 1283–8. Gough MJ, Melcher AA, Ahmed A et al. (2001). Macrophages orchestrate the immune response to tumor cell death. Cancer Res 61(19): 7240–7. Harrison LH, Jr., Schwarzenberger PO, Byrne PS et  al. (2000). Gene-modified PA1-STK cells home to tumor sites in patients with malignant pleural mesothelioma. Ann Thorac Surg 70(2): 407–11. Hoganson DK, Batra RK, Olsen JC et al. (1996). Comparison of the effects of three different toxin genes and their levels of expression on cell growth and bystander effect in lung adenocarcinoma. Cancer Res 56(6): 1315–23. Huber BE, Austin EA, Richards CA et al. (1994). Metabolism of 5-fluorocytosine to 5-fluorouracil in human colorectal tumor cells transduced with the cytosine deaminase gene: significant antitumor effects when only a small percentage of tumor cells express cytosine deaminase. Proc Natl Acad Sci U S A 91(17): 8302–6. Hwang HC, Smythe WR, Elshami AA et al. (1995). Gene therapy using adenovirus carrying the herpes simplex-thymidine kinase gene to treat in vivo models of human malignant mesothelioma and lung cancer. Am J Respir Cell Mol Biol 13(1): 7–16. Kashiwakura Y, Ochiai K, Watanabe M et al. (2008). Down-regulation of inhibition of differentiation-1 via activation of activating transcription factor 3 and Smad regulates REIC/Dickkopf-3induced apoptosis. Cancer Res 68(20): 8333–41. Kelly KJ, Woo Y, Brader P et  al. (2008). Novel oncolytic agent GLV-1h68 is effective against malignant pleural mesothelioma. Hum Gene Ther 19(8): 774–82. Klein G, Powers A and Croce C (2002). Association of SV40 with human tumors. Oncogene 21(8): 1141–9. Kruklitis RJ, Singhal S, Delong P et al. (2004). Immuno-gene therapy with interferon-beta before surgical debulking delays recurrence and improves survival in a murine model of malignant mesothelioma. J Thorac Cardiovasc Surg 127(1): 123–30. Kucharczuk JC, Randazzo B, Chang MY et  al. (1997). Use of a “replication-restricted” herpes virus to treat experimental human malignant mesothelioma. Cancer Res 57(3): 466–71. Lanuti M, Rudginsky S, Force SD et al. (2000). Cationic lipid:bacterial DNA complexes elicit adaptive cellular immunity in murine intraperitoneal tumor models. Cancer Res 60(11): 2955–63. Leong CC, Marley JV, Loh S et al. (1997). Transfection of the gene for B7-1 but not B7-2 can induce immunity to murine malignant mesothelioma. Int J Cancer 71(3): 476–82. Lukacs KV, Porter CD, Pardo OE et al. (1999). In vivo transfer of bacterial marker genes results in differing levels of gene expression and tumor progression in immunocompetent and immunodeficient mice. Hum Gene Ther 10(14): 2373–9. Mae M and Crystal RG (2002). Gene transfer to the pleural mesothelium as a strategy to deliver proteins to the lung parenchyma. Hum Gene Ther 13(12): 1471–82. Matthews T and Boehme R (1988). Antiviral activity and mechanism of action of ganciclovir. Rev Infect Dis 10 Suppl 3: S490–4. Melcher A, Todryk S, Hardwick N et  al. (1998). Tumor immunogenicity is determined by the mechanism of cell death via induction of heat shock protein expression. Nat Med 4(5): 581–7. Merritt RE, Yamada RE, Wasif N et al. (2004). Effect of inhibition of multiple steps of angiogenesis in syngeneic murine pleural mesothelioma. Ann Thorac Surg 78(3): 1042–51; discussion 1042–51. Mukherjee S, Haenel T, Himbeck R et  al. (2000). Replication-restricted vaccinia as a cytokine gene therapy vector in cancer: persistent transgene expression despite antibody generation. Cancer Gene Ther 7(5): 663–70. Mukherjee S, Nelson D, Loh S et al. (2001). The immune anti-tumor effects of GM-CSF and B7-1 gene transfection are enhanced by surgical debulking of tumor. Cancer Gene Ther 8(8): 580–8. Nagamachi Y, Tani M, Shimizu K et al. (1999). Suicidal gene therapy for pleural metastasis of lung cancer by liposome-mediated transfer of herpes simplex virus thymidine kinase gene. Cancer Gene Ther 6(6): 546–53.

110

E.K. Moon et al.

Neves S, Faneca H, Bertin S et al. (2009). Transferrin lipoplex-mediated suicide gene therapy of oral squamous cell carcinoma in an immunocompetent murine model and mechanisms involved in the antitumoral response. Cancer Gene Ther 16(1): 91–101. Nowak AK, Robinson BW and Lake RA (2003). Synergy between chemotherapy and immunotherapy in the treatment of established murine solid tumors. Cancer Res 63(15): 4490–6. Odaka M, Sterman DH, Wiewrodt R et al. (2001). Eradication of intraperitoneal and distant tumor by adenovirus–mediated interferon–beta gene therapy is attributable to induction of systemic immunity. Cancer Res 61(16): 6201–12. Odaka M, Wiewrodt R, DeLong P et al. (2002). Analysis of the immunologic response generated by Ad.IFN-beta during successful intraperitoneal tumor gene therapy. Mol Ther 6(2): 210–8. Okada T, Shah M, Higginbotham JN et  al. (2001). AV.TK-mediated killing of subcutaneous tumors in situ results in effective immunization against established secondary intracranial tumor deposits. Gene Ther 8(17): 1315–22. Pataer A, Smythe WR, Yu R et al. (2001). Adenovirus-mediated Bak gene transfer induces apoptosis in mesothelioma cell lines. J Thorac Cardiovasc Surg 121(1): 61–7. Pitako J, Squiban P, Acres B et  al. (2003). A randomized phase II single center study of gene transfer-based non-specific immunotherapy of malignant mesothelioma (MM) by intratumoral injections of an interleukin-2 producing vero cells. Proc American Society Clinical Oncology 2003(22): abstract 920. Pollak JS (2002). Malignant pleural effusions: treatment with tunneled long-term drainage catheters. Curr Opin Pulm Med 8(4): 302–7. Qiao J, Black ME and Caruso M (2000). Enhanced ganciclovir killing and bystander effect of human tumor cells transduced with a retroviral vector carrying a herpes simplex virus thymidine kinase gene mutant. Hum Gene Ther 11(11): 1569–76. Robinson BW and Lake RA (2005). Advances in malignant mesothelioma. N Engl J Med 353(15): 1591–603. Rudginsky S, Siders W, Ingram L et al. (2001). Antitumor activity of cationic lipid complexed with immunostimulatory DNA. Mol Ther 4(4): 347–55. Sanchez-Perez L, Gough M, Qiao J et al. (2007). Synergy of adoptive T-cell therapy and intratumoral suicide gene therapy is mediated by host NK cells. Gene Ther 14(13): 998–1009. Schrump DS and Waheed I (2001). Strategies to circumvent SV40 oncoprotein expression in malignant pleural mesotheliomas. Semin Cancer Biol 11(1): 73–80. Schwarzenberger P, Harrison L, Weinacker A et al. (1998). Gene therapy for malignant mesothelioma: a novel approach for an incurable cancer with increased incidence in Louisiana. J La State Med Soc 150(4): 168–74. Schwarzenberger P, Harrison L, Weinacker A et al. (1998). The treatment of malignant mesothelioma with a gene modified cancer cell line: a phase I study. Hum Gene Ther 9(17): 2641–9. Schwarzenberger P, Lei D, Freeman SM et al. (1998). Antitumor activity with the HSV-tk-genemodified cell line PA-1-STK in malignant mesothelioma. Am J Respir Cell Mol Biol 19(2): 333–7. Smythe WR, Hwang HC, Amin KM et al. (1994). Use of recombinant adenovirus to transfer the herpes simplex virus thymidine kinase (HSVtk) gene to thoracic neoplasms: an effective in vitro drug sensitization system. Cancer Res 54(8): 2055–9. Smythe WR, Hwang HC, Elshami AA et al. (1995). Treatment of experimental human mesothelioma using adenovirus transfer of the herpes simplex thymidine kinase gene. Ann Surg 222(1): 78–86. Sterman DH and Albelda SM (2005). Advances in the diagnosis, evaluation, and management of malignant pleural mesothelioma. Respirology 10(3): 266–83. Sterman DH, Treat J, Litzky LA et al. (1998). Adenovirus-mediated herpes simplex virus thymidine kinase/ganciclovir gene therapy in patients with localized malignancy: results of a phase I clinical trial in malignant mesothelioma. Hum Gene Ther 9(7): 1083–92. Sterman DH, Recio A and Molnar-Kimber K (1999). Herpes simplex virus thymidine kinase (HSVtk) gene therapy utilizing an E1/E4-deleted adenoviral vector: preliminary results of a phase I clinical trial for pleural mesothelioma. Am J Respir Crit Care Med 159: A237.

7  Gene Therapy for Malignant Pleural Mesothelioma

111

Sterman DH, Molnar-Kimber K, Iyengar T et al. (2000). A pilot study of systemic corticosteroid administration in conjunction with intrapleural adenoviral vector administration in patients with malignant pleural mesothelioma. Cancer Gene Ther 7(12): 1511–8. Sterman DH, Recio A, Vachani A et al. (2005). Long-term follow-up of patients with malignant pleural mesothelioma receiving high-dose adenovirus herpes simplex thymidine kinase/ ganciclovir suicide gene therapy. Clin Cancer Res 11(20): 7444–53. Sterman DH, Gillespie CT, Carroll RG et al. (2006). Interferon beta adenoviral gene therapy in a patient with ovarian cancer. Nat Clin Pract Oncol 3(11): 633–9. Sterman DH, Recio A, Carroll RG et al. (2007). A phase I clinical trial of single-dose intrapleural IFN-beta gene transfer for malignant pleural mesothelioma and metastatic pleural effusions: high rate of antitumor immune responses. Clin Cancer Res 13(15 Pt 1): 4456–66. Stiles BM, Adusumilli PS, Bhargava A et al. (2006). Minimally invasive localization of oncolytic herpes simplex viral therapy of metastatic pleural cancer. Cancer Gene Ther 13(1): 53–64. Sugarbaker DJ, Jaklitsch MT and Liptay MJ (1995). Mesothelioma and radical multimodality therapy: who benefits? Chest 107(6 Suppl): 345S-50S. Tiberghien P (1994). Use of suicide genes in gene therapy. J Leukoc Biol 56(2): 203–9. Todryk S, Melcher AA, Hardwick N et al. (1999). Heat shock protein 70 induced during tumor cell killing induces Th1 cytokines and targets immature dendritic cell precursors to enhance antigen uptake. J Immunol 163(3): 1398–408. Treat J, Kaiser LR, Sterman DH et  al. (1996). Treatment of advanced mesothelioma with the recombinant adenovirus H5.010RSVTK: a phase 1 trial (BB-IND 6274). Hum Gene Ther 7(16): 2047–57. Triozzi PL, Aldrich W, Allen KO et al. (2005). Antitumor activity of the intratumoral injection of fowlpox vectors expressing a triad of costimulatory molecules and granulocyte/macrophage colony stimulating factor in mesothelioma. Int J Cancer 113(3): 406–14. Tsuji T, Nozaki I, Miyazaki M et  al. (2001). Antiproliferative activity of REIC/Dkk-3 and its significant down-regulation in non-small-cell lung carcinomas. Biochem Biophys Res Commun 289(1): 257–63. Vile RG, Castleden S, Marshall J et al. (1997). Generation of an anti-tumour immune response in a non-immunogenic tumour: HSVtk killing in vivo stimulates a mononuclear cell infiltrate and a Th1-like profile of intratumoural cytokine expression. Int J Cancer 71(2): 267–74. Xing W, Wu S, Yuan X et al. (2008). The anti-tumor effect of human monocyte-derived dendritic cells loaded with HSV-TK/GCV induced dying cells. Cell Immunol 254(2): 135–41. Yang CT, You L, Yeh CC et al. (2000). Adenovirus-mediated p14(ARF) gene transfer in human mesothelioma cells. J Natl Cancer Inst 92(8): 636–41. Yang CT, You L, Uematsu K et al. (2001). p14(ARF) modulates the cytolytic effect of ONYX-015 in mesothelioma cells with wild-type p53. Cancer Res 61(16): 5959–63. Zhu ZB, Makhija SK, Lu B et  al. (2005). Incorporating the survivin promoter in an infectivity enhanced CRAd-analysis of oncolysis and anti-tumor effects in vitro and in vivo. Int J Oncol 27(1): 237–46.

Chapter 8

Mesenchymal Stem/Stromal Cells as Cellular Vehicles for Tumor Targeting Frank Marini, Matus Studeny, Jennifer Dembinski, Keri L. Watson, Shannon Kidd, Erika Spaeth, Zhizong Zeng, Xiaoyang Ling, Ann Klopp, Fredrick Lang, Brett Hall, and Michael Andreeff

Abstract  Data published over the last 10 years suggest that mesenchymal stem/ stromal cells (MSC) possess the innate capacity to home to sites of inflammation, including tumors and wounding microenvironments. Evidence suggests that the increased production of inflammatory mediators found at these sites is potential attractants for recruitment and engraftment. This innate homing response can be exploited by using MSC as a cellular delivery vehicle to deliver anticancer agents directly to tumors. The high-level intratumoral production of these agents controls tumor growth and prolongs survival in numerous animal models. In this review, we examine the ability of MSC to selectively home to and engraft within the tumor microenvironment and the multiple gene products delivered by MSC when used as delivery vehicles for anticancer therapies. Keywords  Carcinoma-Associated Fibroblast (CAF)  •  Tumor Microenvironment •  Tumor homing  •  Tumor stroma  •  Interferon-beta

1 Introduction 1.1 Tumor Cell-Centric View on Tumor Development The development of cancer is thought to be a progressive multistep process ­during which time a cell accumulates consecutive genetic alteration that ­ultimately leads in a phenotypic change in the cell and results in uncontrolled proliferation and

F. Marini (*) Department of Stem Cell Transplant and Cellular Therapy, Section of Molecular hematology, 1515 Holcombe Boulevard, Houston, TX 77030, USA e-mail: [email protected] J.A. Roth (ed.), Gene-Based Therapies for Cancer, Current Cancer Research, DOI 10.1007/978-1-4419-6102-0_8, © Springer Science+Business Media, LLC 2010

113

114

F. Marini et al.

growth (Palmero et al. 2010; Cho 2009). This tumor cell-­centered view of cancer progression has resulted in many important discoveries that have furthered the understanding of cancer. However, the typical phenotypic changes that permit ­cancer cells to grow into a tumor are incompletely explained by any tumor-centric analysis. This is primarily a result of the analysis being restricted to the cellular level not tissue level, which is a much more complex structure than has been previously modeled in cell culture. During the past few years, the concept of tumor “stem cells,” a stem cell-like mother cell possessing the capacity to generate the bulk of the tumor cells and containing increased tumor-initiation potential, have been described as sufficient to generate tumors in animal models (Reya et al. 2001; Dalerba et  al. 2007; Gilbert and Ross 2009; Schatton et  al. 2009; Weaver et  al. 1995), however, absent from most cancer stem cell work has been the understanding­ of the complex interplay between the various cell types required to generate tumors, such as tumorigenic epithelia cells, and the non-neoplastic microenvironmental cells such as those required to generate matrix, vasculature, and modulate the immune system (Bissell and Labarge 2005; Nelson and Bissell 2006; Kenny et al. 2007). Importantly, although a tremendous number of alterations have been described resulting in malignant development of tumors, the connective tissue component of solid tumor malignancies remains consistent. Initially, this microenvironment was described as a nourishing and supportive neighbor for the tumor cells; it was considered an innocent bystander, a passive result of tumor development (Park et  al. 2000). However, recently a new view of the significance of stroma has emerged, with the acknowledgment of the fact that tumors do not exist in isolation, and increasing experimental evidence has emphasized the importance and criticality of the microenvironment in tumor progression, suggesting that tumor cells themselves, even though they may possess genetic alterations, are not sufficient to generate a malignant tumor but that a tumor-supportive permissive stroma is required as well (Zipori et al. 1985; Lorusso and Rüegg 2008; Mahadevan and Von Hoff 2007; Kopfstein and Christofori 2006).

1.2 Stroma is a Common Ground for Numerous Cancers Solid tumors are complex tissues that are supported by dynamic interactions between tumor parenchyma (the neoplastic cells) and non-neoplastic microenvironmental cells (such as vasculature, inflammatory cells, fibroblasts, and extracellular matrix) frequently referred to as tumor stroma (Petrulio et  al. 2006) (Fig.  8.1). These interactions appear critical to tumor development and progression and have been reviewed extensively (Liao et al. 2009a; Anton and Glod 2009; Guturu et al. 2009; Zumsteg and Christofori 2009). Stroma provides architectural and organizational support, the blood supply for nutrient refreshment, paracrine and juxtacrine growth factor production, and removes waste (Pollard 2008; Kidd et  al. 2008; Radisky and Radisky 2007). The current hypothesis is that premalignant cells are kept in check by “normal” stromal cells, and upon a transition to a more reactive

8  Mesenchymal Stem/Stromal Cells as Cellular Vehicles for Tumor Targeting

115

Fig.  8.1  Interactions of neoplastic tumor cells and the tumor microenvironment. Tumor cells influence and direct stromal fibroblasts through both paracrine and juxtacrine production of factors. In concert with the tumor cells, the tumor stroma produce a number of tumor supportive factors, such as the production of extracellular matrix and paracrine growth factors, for example hepatocyte growth factors/scatter factor (HGF/SF), stromal-derived factor-1 (SDF1), plateletsderived growth factor (PDGF), and vascular endothelium growth factor (VEGF). The outcome of this protumor supportive microenvironment often leads to increased angiogenesis tumor size increases and metastasis

environment (such as an increased inflammatory stimulus) tissue remodeling ­initiates, and tumor progression starts (Biswas et al. 2004). This model is supported in numerous knockout animal models in which key paracrine regulators, one such example is when TGF-b signaling specifically in fibroblasts is abrogated (Bierie and Moses 2006). Mice with functional TGF-b receptors suppressed the development of gastric tumors suggesting the role that environmental fibroblasts play in controlling neighboring epithelia via TGF-b signaling. In contrast to this is that the tumors alter their microenvironments to recruit additional stromal cells that support progression, as well as keep engrafted stromal cells activated in a protumor supportive state (Cheng et al. 2005). This event occurs in small tumors as studies have demonstrated their requirement for angiogenesis, to demonstrate the complexity of this interaction, one needs to consider the complex cellular and molecular cooperation between the tumor cells and the neighboring tissues, such as the recruited fibroblasts, endothelial cells, and inflammatory cells required to build supportive vasculature (Ding et  al. 2008). Under inflammatory situations, such as during tumor development or wound healing, local fibroblasts are recruited from neighboring tissues, and start to divide to form fibrovascular structures (Nyberg et al. 2008). Additionally, recruited endothelial cells are activated, and they start to divide and

116

F. Marini et al.

construct new blood vessels­(Erickson and Barcellos-Hoff 2003). Tumor-associated macrophages (TAMs) and tumor-associated fibroblasts (TAFs) produce numerous vascular growth factors, such as VEGF, that are frequently associated with tumorinduced angiogenesis (Pittet 2009; Lin et  al. 2007). Both TAFs and TAMs are profusely detected in tumors and have been demonstrated with tumor growth support (Korc 2007; Silzle et al. 2003). Of importance to this review is that TAFs are considered homogeneous but very functionally heterogeneous group of mesenchymal cells, these TAFs can take on matrix degrading, or matrix synthesizing roles (Sugimoto et  al. 2006). Additionally, circulating structural cells (fibrocytes) (Kalluri and Zeisberg 2006), contractile cells (myofibroblasts) (Grande and LópezNovoa 2009), or pericytes (vessel incorporating) (Zeisberg et al. 2007) are required to complete the structural elements of the tumor stroma. Lastly, TAFs have been shown to produce a myriad of tumor-promoting growth factors, cytokines, chemokines and have been suggested to modulate the local immune response against the tumor (De Wever et al. 2008). Given their critical role in tumor stroma construction, one can envision the innate tropism of these cells for tumor and wounding environments. The invasion of cancer cells into surrounding tissue is the hallmark of malignancy, and the invasion process is associated with considerable destruction and regeneration of intercellular elements. Newly synthesized stroma, known as “­cancer induced stroma,” is composed of inflammatory cells (including lymphocytes, granulocytes, and macrophages), the endothelial cells of blood and lymph ­vessels, pericytes, and fibroblasts. Although inflammatory and endothelial cells have been reported to impact tumor immunity (Johnson et al. 2009) and neoangiogenesis (Moserle et al. 2009; Gerber et al. 2009), the role of TAFs, which also participate in cancer progression, has not yet been fully elucidated. The “desmoplastic reaction” is a kinetic sequence of events during tumor progression that describes the presence of activated tumor fibroblasts. Fibroblasts, depending on their tissue origin, represent a cell type of limited diversity that have been observed to express subtle biochemical and phenotypic differences in response to different cytokines and extracellular matrix components (Keeley et  al. 2009; Schmid et al. 2009). These observations suggest the need for other cell types in the formation of tumor stroma. While the cellular origin of TAFs remains unclear, accumulating evidence ­suggests that TAFs originally stem from resident organ fibroblasts. While these studies have indicated that organ fibroblasts in the proximity of the developing tumor became TAFs, these data do not preclude the possibility that circulating mesenchymal stem/stromal cells (MSC) or MPCs (mesenchymal progenitor cells) contribute to the heterogeneous organ specific fibroblastic population or to the TAF cell pool directly. A recent set of data also suggests that tumors can generate their own intrinsic mesenchyme through an event termed epithelial-mesenchymal transition (EMT). EMT is a normally latent embryonic program that plays a role during cancer progression. During EMT, epithelial cells lose cell–cell contact and cell polarity and acquire mesenchymal morphology by undergoing major changes in their cytoskeleton (Jayanthi et al. 2005; Okamoto et al. 2009; Duband et al. 2009)

8  Mesenchymal Stem/Stromal Cells as Cellular Vehicles for Tumor Targeting

117

Recently, Mani et  al. described that the induction of EMT in differentiated adult human mammary epithelial cells (HMECs) results in the acquisition of not only mesenchymal cell-like traits, but also confers the EMT cells with stem cell-like properties (Mani et al. 2008). Taken together, these data suggest that the epithelial cells induced to undergo EMT exhibit molecular features and functional behavior similar to BM-derived mesenchyme. These similarities suggest that, along with being specially adapted to metastasize, cells that have undergone EMT may act like TAFs in the tumor and have significant paracrine effects in the tumor microenvironment­(Fig. 8.2).

1.3 Role of Fibroblasts and Stromal Precursors It has been demonstrated that TAFs provide organization of the tumor stroma by the production of extracellular matrix components. In this context, activated fibroblasts are a rich source of growth factors, such as TGF-b, IGF-1, and bFGF

Tumorgenerat ed EMTMSC

From tumor cell to EMT-MSC

Bone marrow (BM)

Tumor cells

Tumor tissue

Bone marrow MSC

MSC from BM

Bone

Fig. 8.2  The recruitment and intratumoral production of supportive stromal elements. Data published by a number of groups suggest that tumors recruit stromal elements from both local neighboring tissues as well as systemic sources. Systemic sources include, immune cells such as macrophages, and fibrocytes, as well as bone marrow-originated mesenchymal cell populations have been shown to engraft and proliferate in tumors. Recent data from Mani et al. (2008) suggest that tumors also can generate their own intrinsic stroma (mesenchyme) via transformation of epithelial cells to mesenchyme cell transition

118

F. Marini et al.

Fig. 8.3  Detection and staining of bone marrow derived mesenchyme in ovarian carcinomas in mouse models. Briefly, human MSC were injected systemically in SCID mice bearing xenograft SKOV3 ovarian carcinomas. After 80 days, the tumors were harvested and sections were reacted with antibodies specific to human alpha-smooth muscle actin. As shown in panels (a) and (b), unique fibrovascular structures can be observed in both the center (a) and the periphery of the ovarian carcinomas. Suggesting the recruitment, engraftment, and subsequent proliferation of the MSC in the tumor microenvironment

(Wang et al. 2008a; Denys et al. 2008). Solid tumor growth cannot be sustained unless the tumor cells attract and stimulate fibroblasts. Based on our current understanding of tumor stroma development (Castelló-Cros and Cukierman 2009), we propose that stromagenesis occurs as a multistep process involving the recruitment of local tissue fibroblasts and the concomitant recruitment of circulating BM-derived stem cells or MSC from the bone marrow into the tumor. Once recruited, bone marrow-derived cells proliferate in response to tumor microenvironmental cues (Bagley et  al. 2009) (Fig.  8.3). These cell types phenotypically resemble TAFs, myofibroblasts, and may differentiate into fibroblast-like cells that produce ECM components and/or contribute to perivascular or vascular structures (vessel containing cells) (Sund and Kalluri 2009). Further support for this model stems from similarities between reactive tumor stroma and stroma involved in wound repair (Dvorak 1987). Many of the biological processes in wound repair, including stromal cell acquisition of a myofibroblast phenotype, deposition of type I collagen, and induction of angiogenesis, are observed in reactive stroma during cancer progression (Brown et al. 1989).

2 Tropism of MSC for Wounds and Tumors Homing of MSC after systemic or local infusion has been studied in a variety of animal models using numerous experimental scenarios (Shake et al. 2002; Herrera et al. 2004). In a study by Pereira et al. (1995), they reported the systemic infusion of marker gene expressing MSC in irradiated syngeneic mice. After 30 days, they detected marker gene expression in 5% of lung cells and 8% of bone marrow cells.

8  Mesenchymal Stem/Stromal Cells as Cellular Vehicles for Tumor Targeting

119

Next, baboons receiving systemically injected GFP-marked MSC had low levels of detected GFP signal in the bone marrow more than 500 days post transplantation (Devine et al. 2001). It appears that the innate ability of MSC is to adhere to matrix components, thereby favoring their preferential residence homing to lung and bone when injected systemically. As an aside, a few reports now suggest that conditioning regimens prior to cell transplantation (such as chemotherapy, focal or total body irradiation treatment) enhance the efficiency of MSC homing to sites of engraftment (Allers et  al. 2004; Erices et  al. 2003; Klopp et  al. 2007). In this regard, a number of reports have established that under different pathological conditions, MSC selectively homed to sites of injury irrespective of tissue or organ (Kumamoto et al. 2009; Semedo et al. 2009). This innate tropism for sites of wounding has been confirmed in experiments with wound healing (Satoh et  al. 2004; Falanga et al. 2007), tissue repair and regeneration (Mansilla et al. 2006; Le Blanc 2006; Kähler et al. 2007), and brain injury (van Velthoven et al., 2010; Liao et al. 2009b). One can speculate that higher concentrations of chemoattractant mediators produced at the site of damage is requisite for MSC to migrate, engraft, and proliferate to replace the damaged niche. Thus, this innate tropism for inflammatory sites such as tumors, and the ability to home to and engraft in these pathological sites (and deliver therapeutics) represents an additional attractive feature of these cells and complements the native properties of MSC making them favorable for cell-based strategy. The ability of MSC to home to tumor sites could be hypothesized since evidence suggests that tumors can be considered sites of tissue “damage,” or “wounds that never heal” Dvorak (1986).

2.1 But Which Cell in the Stroma to Target Elegant work by Ischii et al. (2005) implicates the role of BM-derived cells in the development of tumors. Briefly, SCID mice recipients were reconstituted with bone marrow cells from Bgal+ and RAG-1 deficient double-mutant mice (also expressing a different MHC). Once the mice demonstrated engraftment, pancreatic tumors were implanted, and allowed to progress. During the next days, tumors were removed and contributions of Bgal+ cells in the tumor microenvironment were assessed. Their data revealed that a small proportion (13%) of H-2Kb/a-SMA double-positive myofibroblasts around the developing malignancy 2 weeks after tumor xenotransplantation. These results suggested that a small number of BM-derived myofibroblasts are incorporated into cancer stroma during even in the early stage of tumor growth. By 4 weeks after tumor implantation, about 40% of the myofibroblasts in the cancer-induced stroma were of BM origin. Immunohistochemical staining of serial sections revealed that non-BM-derived myofibroblasts exist adjacent to the cancer nest, whereas BM-derived myofibroblasts were on the outside of non-BMderived myofibroblasts. These data indicated that BM-derived myofibroblasts were incorporated into cancer-induced stroma mainly in the late stage of tumor development. Taking these results into consideration, it was suggested that BM-derived

120

F. Marini et al.

myofibroblasts might contribute to the pathogenesis of cancer-induced desmoplastic reaction and might change the “microenvironment” that influences tumor growth. Furthermore, the results of their study suggest that the local tissue reaction starts at the circumference of a neoplasm in the early stage of tumor development. However, due to limitation in the experiment, it is unclear from this data whether the tumor-resident Bgal+ myofibroblasts were generated from a circulating stem cells originating from an adherent BM population, such as MSC, or endothelial precursor, or from a circulating “hematopoietic” population. However, their findings directly demonstrate the specific contribution of BM-derived cells to the formation of tumor stroma, and strongly suggests that BM-derived cells are able to target tumors due to physiological requests by the tumor (Sangai et al. 2005; Reddy et al. 2008; Studeny et al. 2002; Gonda et al. 2010; Davis and Desai 2008; Guest et al. 2010; Gao and Mittal 2009). Ischii’s results support our initial observations (Studeny et al. 2002) that tumors recruit cells from circulation and that if one could isolate such a population of these “stromal-precursor” cells, one would have unique access to growing tumors and their metastases and could potentially target novel therapies to prevent or slow the local and metastatic tumor growth. Since our original report, a number of other groups have reported similar findings in various tumor types, including prostate, glioma, lung, ovarian, breast tumors, sarcomas, and hematological malignancies (Alison et al. 2009; Gu et al. 2010; Chanda et al. 2009; Bexell et al. 2009; Sasportas et al. 2009a; Zischek et al. 2009; Xin et al. 2009a, b; Xiang et al. 2009; Menon et al. 2009a; You et al. 2009; Loebinger et al. 2009a; Sun et al. 2009; Uchibori et al. 2009; Duan et al. 2009; Jiang et al. 2010; Kallifatidis et al. 2008; Kyriakou et al. 2006; Rachakatla et al. 2007a).

2.2 Rationale for Targeting Tumors Using Stromal Precusor Cells Targeting the tumor microenvironment would potentially increase the therapeutic effectiveness of anticancer treatments (Hamada et al. 2005). Our results raise the possibility of using BM-derived myofibroblast progenitor cells as carriers of novel therapies to prevent or slow the local and metastatic growth of cancer cells. There are advantages in using BM-derived stem cells as cellular vehicles. First, it is likely that most invasive cancers cause a desmoplastic reaction to some extent, thereby providing a common target for the treatment of many types of cancers. Second, MSC are normal cells with a low intrinsic mutation rate, and therefore are less likely to acquire a drug-resistant phenotype than the genetically unstable cancer cells. While sarcomas have been reported in mice implanted with high passage-number MSC (Tolar et  al. 2007), this result has not been reported in perhaps thousands of patients who have received MSC to date mostly in the context of tissue repair studies or for the treatment of graft vs. host disease (Le Blanc et al. 2008).

8  Mesenchymal Stem/Stromal Cells as Cellular Vehicles for Tumor Targeting

121

2.3 Migratory Factors A number of factors have been implicated in homing of bone marrow cells to sites of injury. We have speculated that upregulation within the tumor microenvironment of the same factors involved in wound healing may initiate the homing process (Spaeth et al. 2008). In wound repair, as in cancer, cells that usually divide infrequently are induced to proliferate rapidly, extracellular matrix is invaded, connective tissues are remodeled, epithelial and stromal cells migrate, and new blood vessels are formed. Hepatocyte growth factor/scatter factor (HGF/SF) and keratinocyte growth factor (KGF) are regulators of the healing process (Hall et al. 2007a). Mesenchymal fibroblasts produce HGF/SF, which is a paracrine mediator of epithelial function in many tissues (Wang et  al. 2006), and has been implicated in stimulating motility by binding to its receptor c-Met. KGF is a member of the heparin-binding fibroblast growth factor family (FGF-7), and acts in a similar fashion (Hofer et  al. 2005). Both HGF/SF and KGF are produced by fibroblasts in response to c-Kit/stem cell factor, which is upregulated following injury. Additionally, HGF/SF promotes CXCR-4 upregulation and stromal cell-derived factor-1 (SDF-1) mediated directional migration of human CD34+ progenitors (Muehlberg et al. 2009). Other possible key players, also involved in wound repair, include growth factors such as transforming growth factor-beta (TGFb), platelet derived growth factor (PDGF), epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF). MSC have been shown to express cell surface receptors for each of these factors. As suggested earlier in this review, TGFb is a growth factor with both positive and negative tumorigenic effects; it is produced by a number of cells, including epithelial cells and mesenchymal fibroblasts. It serves to stimulate the migration and proliferation of mesenchymal cells, and its receptors TGFb-RII and CD105 (endoglin), which are abundant on the surface of MSC (Wang et al. 2004). TGFb has also previously been shown to stimulate the chemotactic migration of human fibroblasts (Ponte et al. 2007). In tumors, PDGF is also suggested to take part in the stroma conversion, and therefore may be one of the factors involved in the recruitment of TAFs and/or MSC (Hata et al. 2010). Epidermal growth factor receptor (EGFR) has been shown to regulate migration in a variety of cells through multiple mechanisms. There are many ligands for EGFR, including EGF, heparin-binding EGF (HB-EGF), and transforming growth factor-alpha (TGFa) (Wang et  al. 2008b). In wound-healing models, EGF, TGFa, and HB-EGF play essential roles in enhancing migration of fibroblasts following their binding to EGFR. HB-EGF, a mitogenic and chemotactic molecule that partakes in tissue repair, tumor growth and other tissue-modeling events, such as angiogenesis and fibrogenesis, also enhances ex-vivo MSC proliferation (Dembinski and Krauss 2009). MSC often express EGFR and have a role in processes where HB-EGF is involved. HB-EGF may be implicated in recruitment/migration and proliferation of MSC to tumor and/or sites of injury (Krampera et  al. 2005). bFGF (FGF-2) is another important molecule known to be involved in stromal

122

F. Marini et al.

cell induction. Ovarian tumor cell lines express bFGF, which leads to the ­recruitment of human primary fibroblasts during tumor growth. Additionally, our group showed the chemotactic potential of PDGF, EGF, and FGF on MSC in vitro and in vivo (Yong et al. 2009). To demonstrate additional chemoattractant mediators besides cytokines and chemokines, a recent publication from our group demonstrated that the antibacterial peptide – leucine leucine 37 – (LL-37), the C-terminal peptide of human cationic antimicrobial protein 18, was a potential migratory attractant for both MSC in vitro and in vivo, as neutralization of systemic LL-37 through antibody treatment resulted in a marked reduction in MSC migration to tumors, but importantly resulted in a disorganization of the tumor stroma within the ovarian tumors (Coffelt et al. 2009) suggesting its role in MSC recruitment, as well as structural organization.

3 Use of Stem Cells as Cellular Vehicles to Target Tumors Aboody et  al. (2000) demonstrated that neural stem cells (NSC) administered intracranially possess extensive tropism for experimental glioma and significant migratory behavior. NSC distribute throughout the primary tumor bed and migrate together with widely outgrowing tumor microsatellites after intratumoral implantation. Moreover, when NSC are implanted intracranially at sites distant from the tumor, they migrate through the normal parenchyma and localize in the tumor. This behavior of NSC has been exploited as a tumor-targeting strategy for antiglioma gene therapy. The authors demonstrated potent antitumor effects following intracranial administration of gene-modified NSC expressing interleukin (IL)-4 (Brown et  al. 2003), or tumor necrosis factor-related apoptosis inducing ligand (TRAIL) (Kim et al. 2006) in experimental glioma models. While these data demonstrate the tropism of stem cell for tumor microenvironments, the isolation and application of NSC for clinical application is currently technically challenging, as a result one can consider other sources of stem cells, such as MSC. The potential use for MSC as cellular gene delivery vehicles has been suggested for a number of years, and some studies have begun to evaluate the feasibility, safety, and practicality of the therapeutic potential of MSC in tissue regeneration or cell replacement therapies (Fig. 8.4). While the natural prevalence of MSC within the tumor microenvironment is unknown, many observations support the concept of utilizing MSC as delivery vehicles for antitumor proteins. We and others have observed dynamic interactions between bone marrow-derived MSC and various tumor cell lines in  vitro and in vivo, we have shown that human MSC engraft and persist within existing tumor microenvironments. Further support includes the observation that MSC phenotypically resemble TAF in the presence of TGF-b1 or in coculture with tumor cells, and independent groups have shown that murine bone marrow fibroblasts contribute to the developing tumor stromal cell populations in mouse models.

8  Mesenchymal Stem/Stromal Cells as Cellular Vehicles for Tumor Targeting

123

Fig. 8.4  Cartoon Depicting MSC tropism for the tumor microenvironment

Potential concerns in utilizing MSC as gene delivery vehicles stem from how little we know about the homeostatic maintenance of this cell population in vivo and the possibility that MSC themselves may enhance or initiate tumor growth. However, unlike homeostatic MSC niches, tumor microenvironments are pathologically altered tissues that resemble unresolving wounds, therefore a better understanding of the impact of MSC on the structure and function of the tumor microenvironment will be more important for MSC-based strategies. In regards to the safety of utilizing MSC as gene delivery vehicles for antitumor proteins, studies that demonstrated MSC possess tumorigenic properties utilized extensively passaged and/or gene-manipulated MSC. We and others have shown that lower passage MSC do not form tumors in vivo (Thu et al. 2009). A number of studies from our group (Studeny et al. 2002; Alison et al. 2009; Bexell et al. 2009; Sasportas et al. 2009a; Rachakatla et al. 2007a; Studeny et al. 2004; Nakamizo et  al. 2005; Hall et  al. 2007b) have reported that exogenously administered MSC could migrate and preferentially survive and proliferate within tumor masses. Once in the tumor microenvironment, MSC incorporated into the tumor architecture and served as precursors for stromal elements, predominately fibroblasts. The preferential distribution of MSC in lung tumor nodules, but not in lung parenchyma, was demonstrated after systemic MSC administration in mice bearing melanoma xenografts, and when the ability of systemically injected MSC to integrate into subcutaneously established tumors was investigated,

124

F. Marini et al.

­ SC-derived fibroblasts were consistently identified in tumors and not found in M other organs (Nakamizo et al. 2005). Next, we examined the therapeutic potential of MSC as a cellular delivery system for interferon-beta (IFNb) into the tumor microenvironment. Our data demonstrated that IFNb exerts its effect through local paracrine effects following delivery into tumors by cell carriers (“cellular mini-pumps”), thus emphasizing the importance of tumor-targeted delivery of a cytokine. The same MSC, producing systemic IFNb at sites distant from tumor, as well as recombinant IFNb protein delivered systemically without carrier, did not show any benefit in survival (Hall et al. 2007b). Of interest, it appears that MSC may have similar capabilities to specifically home to and selectively engraft into established gliomas in vivo. Data from our group demonstrated that intracarotid injected MSC can specifically target U87 glioma, and that the MSC can negotiate from either the contralateral or the ipsilateral carotid artery, suggesting that blood flow or a “perfusion” effect is not directly responsible for getting the MSC to the tumor (Ayala et al. 2009). Additionally, when MSC were armed to secrete IFNb, we observed statistically significant decreases in tumor survival, suggesting that MSC producing IFNb were able to control tumor growth kinetics. Additional work from Matsumoto et al. (1999) has demonstrated that gene modified MSC (expressing IL-2) injected intratumorally into established gliomas can also control tumor development, again suggesting that intratumor production of this immunostimulatory cytokine is responsible for controlling tumor kinetics. Thus, cultured bone marrow adherent cells contained cells with extraordinarily high proliferative capacity that can contribute to the maintenance of connective tissue in organs remote from the bone marrow as well as the stromal component of tumors (Spaeth et al. 2009). At the same time, the successful engraftment of MSC in tissues would most likely take place only in a state of increased cell turnover triggered by tissue damage or by tumor growth. These properties of MSC make this cell population very attractive candidate for the cell-based delivery­ of therapeutics to tumor sites.

3.1 MSC as Cell Vehicles for Cancer Over the past 10 years, since our initial discovery and report that MSC display innate tropism for tumors, approximately 90 papers (as of Dec2009) have been published from other groups reporting tumor-targeted delivery of MSC, most report selective tumor targeting and impact on various tumor growth kinetics (see (Gonda et al. 2010; Davis and Desai 2008; Guest et  al. 2010; Gao and Mittal 2009; Alison et  al. 2009; Gu et al. 2010; Chanda et al. 2009; Bexell et al. 2009; Sasportas et al. 2009a; Zischek et al. 2009; Xin et al. 2009a, b; Xiang et al. 2009; Menon et al. 2009a; You et al. 2009; Loebinger et al. 2009a; Sun et al. 2009; Uchibori et al. 2009; Duan et al. 2009; Jiang et al. 2010)). Detailed investigations into MSC migration and potential chemoattractant factors have supported our initial observations, and now numerous studies demonstrating that genetically modified MSC either to over express

8  Mesenchymal Stem/Stromal Cells as Cellular Vehicles for Tumor Targeting

125

t­herapeutic gene products or to enhance tumor tropism can be directed toward a very promising step in cellular therapy that allows targeted treatment of cancer. In the next few paragraphs, we summarize the various therapeutic gene products that have been successfully delivered to tumors using MSC as cellular vehicles.

4 Interferons Interferons have a wide biological activity that includes direct cytotoxic effect and immune stimulatory effects. Gene therapy utilizing beta interferon (IFNb) has shown that tumor regression can be achieved when as little as 1% of the tumor mass expresses the protein (Vitale et al. 2007). However, clinical IFNb has been hampered by poor pharmacokinetics (biological half life in the order of minutes) and high toxicity when administered at clinical doses (Markowitz 2007). Human and murine MSC engineered to express interferon beta were the first demonstration that MSC expressing a therapeutic protein could be effective against primary and metastatic tumors. Studies reported by Studeny et al. (2002) demonstrated that breast and melanoma xenografts could be controlled in vivo by weekly systemic injections of MSC-IFNb in contrast to daily injections of recombinant IFNb protein. Importantly, the poor pharmacokinetics that systemic IFNb protein possess were overcome by using MSC as intratumoral “cellular minipumps” to selectively engraft in the tumor microenvironment, and produce high levels of protein in situ. Additionally, IV injected MSC-IFNb significantly prolonged the survival of animals with establish lung metastases of MDA231 breast carcinoma and A375 melanoma and this effect was due primarily to the local production of high levels of IFNb within the tumor. Importantly, the systemic toxicity associated with high dose IFNb was also ameliorated. Next, Nakamizo et al. demonstrated that MSC express tropism for gliomas after intravascular injection. In this study, MSCIFNb were injected into either contralateral or ipsilateral arteries or into neighboring tissue (locoregional delivery) into mice bearing U87 human glioma xenografts (Nakamizo et al. 2005). In all three examples, MSC demonstrated selective engraftment into the tumor microenvironment and when expressing IFNb, significantly extended the survival of animals bearing the intracranial tumors. In a related study, Ren et al. evaluated the potential of MSC also expressing IFNb in controlling tumor growth in a prostate cancer lung metastases model (Markowitz 2007). Tumor-tropic MSCproduced IFNb was observed at the tumor sites throughout the lungs in animal bearing established TRAMP-C2 pulmonary metastases, and importantly, gene-modified MSC controlled tumor growth (Fig. 8.5). Next, Ren et al. (2008) demonstrated the immunomodulatory effects of IFNa, a multifunctional regulatory cytokine, on murine melanoma in an immunocompetent mouse model of cancer. Briefly, murine B16 melanoma was systemically injected in mice to form lung metastases; next, MSC modified by an adeno-associated virus expressing IFNa were injected systemically and shown to home to lung metastases. Ren reported a significant reduction in lung tumor colonies after MSC-IFNa ­treatment, as well as an increase in life span compared to MSC-control animals.

126

F. Marini et al.

Fig. 8.5  Colocalization of murine MSC (mMSC) to establish 4T1 breast carcinomas in a mouse model. Renilla luciferase mMSC were injected systemically in mice harboring 3-week-old established 4T1 breast carcinomas (stably expressing firefly luciferase). Three days after injection, using a renilla luciferase specific substrate, mMSC were imaged by in vivo bioluminescent imaging. Three hours after the renilla imaging, an injection of a firefly luciferase specific substrate allows detection of the 4T1 tumor. As shown in this figure, MSC appear to colocalize with the established tumors, suggesting their specificity for the tumor microenvironment

5 Interleukins MSC have also been engineered to express interleukins with the rationale of improving antitumor surveillance by activating a host of immune cells, such as natural killers, dendritic cells and cytotoxic lymphocytes. In a report by Chen et al. (2008), MSC transduced with adenoviruses expressing interleukin 12 were utilized to prophylactically inhibit the establishment of subcutaneous B16 melanoma, Lewis lung carcinoma, and hepatomas in immunocompetent mice. While the B16 melanoma was successfully prevented by MSC-IL12 (12 out of 12 animals had no tumors), the heptoma and lung tumor bearing animals had limited success (1 out of

8  Mesenchymal Stem/Stromal Cells as Cellular Vehicles for Tumor Targeting

127

12 animals and 2 out of 12 animals, respectively). Another study from the same group investigated immunotherapy against pre-established metastases of more advanced tumors, such as B16 melanoma, 4T1 breast carcinoma and HCA hepatoma in immune competent mice. After each tumor displayed metastasis to organs or lymph nodes, the treatment of MSC-IL12 is commenced. The progression of tumors in all cases was prevented without systemic or toxic effects. Next, a study by Elzaouk et  al. evaluated the antitumor effects of human MSC expressing rat IL12 against a mouse B16 melanoma, while they reported successful treatment of the tumors of interest was that human MSC displayed tropism for murine tumors and that rat IL12 was effective in a mouse setting (Chen et al. 2006). Studies by Stagg et  al. (2004) and Nakamura et  al. (2004) demonstrated the effective use of IL-2 to exert a potent immune response against either the poorly immunogenic B16 melanoma or Rat 9L gliomas, respectively. In both cases, tumor growth was significantly delayed in a dose dependent manner. Of interest in Staggs’s work was the MSC-IL2 were encapsulated in a matrix, whereby creating an IL2 secreting organoid which when placed in proximity to the tumor afforded a potent immune response to the neighboring lesion.

6 Conditionally Replication Adenoviral Vectors Replication competent tumor-tropic adenoviruses have been utilized in locoregional delivery for the treatment of cancers (Breau and Clayman 1996). While these CRads have demonstrated benefit, poor tumor targeting complicates their application and their nonspecific uptake in liver and spleen increases systemic toxicities. To increase tumor tropism and to shield the CRads from immune surveillance, Komarova et al. utilized MSC as a cellular carrier for CRads and demonstrated that within MSC CRads replicate, and once at the tumor site escape and lateralize with high efficiency within the tumor. Their data demonstrates tumor growth control in a peritoneal model of ovarian xenograft tumors in animal treated with MSC-CRad as compared to animals bearing tumors treated with MSC-controls. A similar strategy was used by Stoff-Khalili et  al. (2007) in which systemically administered MSC-CRads displayed high tumor tropism for lung metastases of MDA231 breast carcinomas in SCID mice. The effects of human MSC-loaded with CRads enhanced the oncolytic effects and increased survival of the tumor bearing animals. These results confirmed that MSC could be used as a cellular carrier for CRads to both local and distant tumors and mediate CRads specific oncolysis. Next, a report from Hakkarainen et  al. (2007) investigated the efficacy of capsid-modified CRads to replicate within MSC and after systemic injection distribute oncolytic virus to tumors. While the authors reported antitumor effects of the MSC carrying capsidmodified CRads, their report suggested that MSC did NOT display tumor tropism, and that CRads released after MSC lysis circulated into tumors. While their observed lack of tumor tropism of MSC is at odds with numerous other publications­, their data did show that MSC loaded with CRads increased their ­bioavailability and

128

F. Marini et al.

afforded systemic lateralization of CRads. Lastly, a report from our group ­demonstrates that MSC carrying CRads reduced the nonspecific organ infection after intraperitoneal injection, such as those observed in the liver, and spleen after systemic injection of CRads (Dembinski et  al. 2009). Taken together, these data suggest that the use of MSC as cellular carriers for replication viruses increases tumor tropism, increases the CRad bioavailability for infecting tumor cells and reduces the nontarget organ infection of CRads improving their pharmacology and increasing their therapeutic benefit.

7 Chemokines and Growth Factor Antagonists A report by Xin et al. (2007, 2009c) utilized MSC expressing the immunostimulatory chemokines CX3CL1-fractalkine as a treatment for lung metastases of C26 colon carcinoma and B16 melanoma in immunocompetent mice. Essentially, fractalkine is a member of the CX3CL family and a soluble form of CX3Cl1 that induces the migration of cells expressing the cognate receptor CXC3R1. Systemic administration of MSC-CX3Cl1 robustly inhibited the development of lung metastases and prolongs the survival of tumor bearing mice. Next, a study by Kanehira et al. (2007) examined the ability of MSC to express NK4, an antagonist of HGF. HGF is a potent inducer of tumor growth, and has been shown to increase angiogenesis and lymphangiogenesis. Systemically injected MSC-NK4 only migrated to sites of lung tumors, and was not found in nontumor bearing tissues. Additionally, NK4 efficiently inhibited C26 tumor progression to the lung and prolonged survival in tumor bearing animals; their data suggested that the antitumor effects were due to the inhibition of angiogenesis within the developing tumors.

8 Suicide Genes The use of gene direct enzyme prodrug therapy is a highly investigated technique for gene therapy of cancer. Essentially, this therapy involves delivery of gene products into tumor cells that confer sensitivity to or convert nontoxic prodrugs into toxic metabolites within the cells that are gene-modified. A common side effect of suicide gene/prodrug therapy is the release of toxic metabolites into neighboring cells eliciting a near neighbor effect of killing adjacent tumor cells through a “bystander effect.” The three suicide gene that were evaluated in clinical trials were Herpes simplex virus thymidine kinase (HSVtk) and the antiherpetic drug ganciclovir­ (GCV), cytosine deaminase (CD) and the antifungal agent 5-fluorocytosine, and the carboxylesterase gene which metabolizes camptothecin (CPT-11). In ­preliminary studies by Kucerova et  al. (2008), they demonstrated that MSC expressing the CD gene product(MSC-CD/AT) in combination with systemic 5-FC administration amplified the cytotoxic effects of 5-FC on HT-29 tumor cell in vitro.

8  Mesenchymal Stem/Stromal Cells as Cellular Vehicles for Tumor Targeting

129

Next, ­engineered MSC-CD demonstrated that systemically administered MSC expressing yeast CD was capable of selective engraftment into tumors and were able to suppress tumor growth of melanoma breast and colon carcinomas in animal models. They noted the effects were due primarily to the bystander effects from the MSC-CD after prodrug administration.

9 Tumor Necrosis Factor-Related Apoptosis Inducing Ligand TRAIL is a member of the tumor necrosis factor-alpha family. This molecule is known to induce apoptosis in various tumor types that express the appropriate death receptors, by initiating caspase-mediated cell death. A number of publications have demonstrated that MSC engineered to express TRAIL are capable of inducing neighboring tumor cell death after systemic infusion (Menon et al. 2009b; Kim et al. 2008). Of importance is that MSC appears refractory to TRAIL-mediated cell killing, and can therefore produce TRAIL at high concentration without any deleterious effects (Loebinger et al. 2009b) A report by Mohr et al. (2008) demonstrated that MSC-TRAIL was active against lung cancer after systemic injection, as well as had high activity in suppressing tumor growth in a refractory epithelial lung cancer tumor grown subcutaneously. Recent work by Sasportas et  al. (2009b) and Yang et al. (2009) demonstrated that engineered MSC-TRAIL appears more potent than wild type TRAIL and is actively secreted into the tumor milieu at high concentration. As a counter to this work, a publication by Luetzkendorf et al. suggests that tumor suppression mediated by MSC-TRAIL is only observed when very high numbers of MSC are present in the tumor, suggesting the need for high level expression of TRAIL and greater distribution of MSC throughout the tumor bed (Luetzkendorf et al. 2009). Lastly, in regards to TRAIL one report demonstrated that TRAIL was a potent tumor-tropic migratory factor produced at the tumor site and facilitated tumor-specific homing of MSC. These data hint to the fact that MSC are responding to multiple attractants produced at the tumor sites; cytokines, chemokines, and various tumor produced ligands, but also confuses the tumor-specific migration question, as this data also suggests that MSC secreting TRAIL may be rendered confused and unresponsive to migrate by their engineered production of TRAIL from within (Secchiero et al. 2008; Szegezdi et al. 2009).

10 Alternative Mesenchymal Tissues as Sources for Anticancer Therapies It is now well described that various tissues throughout the body possess residual adult MSC that appear similar to BM-derived MSC (Akavia et al. 2008). Alternative sources of MSC-like cells have been identified and isolated from fetal lung, adipose tissue, cardiac muscle, and throughout the placenta and umbilical cord (Maijenburg

130

F. Marini et al.

et al. 2010; Quirici et al. 2009; Troyer and Weiss 2008). These mesenchymal cells share common characteristics with BM-MSC, including cellular morphology and phenotypic surface marker expression, multilineage differentiation potential, extensive proliferative potential, and importantly tumor tropism. In fact, adipose-derived MSC have been gene modified to express suicide genes (such as HSVtk and CD) and were shown to effectively inhibit glioblastoma and prostate cancer in animal models after systemic injection (Matuskova et  al. 2010; Cavarretta et  al. 2010). Work from our collaborator demonstrated that placenta derived Wharton jelly-isolated MSC (termed Wharton jelly cells or cord matrix cells) possess tumor tropism for MDA231 breast carcinomas in mouse models and importantly, when modified to express IFNb were effective at suppressing tumor metastases in vivo, an effect amplified by the addition of chemotherapy (Rachakatla et al. 2007b, 2008).

11 Conclusions In this review, we have illustrated the recent progress on the development of MSC populations from bone marrow and other sources, MSC and their potential use as cellular delivery vehicles, demonstrating specific tropisms for solid tumors and their microenvironments. We believe that this tropism for tumors is based on their innate physiological ability to home to sites of inflammations and tissue repair, i.e., “wounding environments.” The most obvious advantage offered by the MSC cell vehicle approach, especially in the case of proteins with poor pharmacokinetic profiles, is the local delivery and release of therapeutics intratumorally. In selected instances, cellular vehicles offer advantage over other vector systems in efficacy of delivery of cancer-related therapeutics, as it has been shown in our studies where a direct comparison of therapeutic effects provided by cell-based or systemic injection of recombinant protein was undertaken. In addition to delivering anti-cancerrelated protein substances, cell vehicles can also serve as protective “coatings” for the delivery of additional payloads, such as replicating oncoselective viruses. Not only does the cell vehicle protect these viruses from the immune system, but it also allows the amplification of the initial viral load with the possibility of increasing viral spread via cell–cell contact specifically at tumor sites. Although these stem cells used for cancer gene therapy usually have some degree of tumor selectivity, one can envision strategies devised to improve tumor homing that may greatly increase the applicability and success of tumor-targeted cell carriers. Two approaches can be envisioned in this regard: to search for other promising cell populations with specific properties (such as more primitive stem cells with higher replicative or migration potential) or to use already defined and characterized cell types with engineered tumor homing properties, such as those targeting strategies applied to viral vectors. Additionally, cell homing may be manipulated by methods of specific culture due to possible differences in cell ­surface receptors expressed or by isolating “enriched” tumor homing MSC that express specific receptor profiles in response to different agents or media ­treatments.

8  Mesenchymal Stem/Stromal Cells as Cellular Vehicles for Tumor Targeting

131

The development of optimally “targeted” cell vectors may parallel advances in cell science and virology and will certainly benefit from continued innovative application of knowledge gained from basic biology. Much work remains to be done before the therapeutic potential and the full benefits of MSC-based approaches will be fully exploited The basic and application studies using MSC provides us with important and challenging research fields, emerging at the crossroads of tissue stem cell engineering, gene therapy, and cancer biology.

References Aboody KS, Brown A, Rainov NG, Bower KA, Liu S, Yang W, Small JE, Herrlinger U, Ourednik V, Black PM, Breakefield XO, Snyder EY. Neural stem cells display extensive tropism for pathology in adult brain: evidence from intracranial gliomas. Proc Natl Acad Sci U S A. 7;97(23):12846–51. Akavia UD, Veinblat O, Benayahu D. Comparing the transcriptional profile of mesenchymal cells to cardiac and skeletal muscle cells. J Cell Physiol. 2008;216(3):663–72. Alison M, Lim S, Houghton JM. Bone Marrow derived cells and epithelial tumours: Nore than just inflammatory relationships. Curr Opin Oncol. 2009;21(1):77–82. Allers C, Sierralta WD, Neubauer S, Rivera F, Minguell JJ, Conget PA. Dynamic of distribution of human bone marrow-derived mesenchymal stem cells after transplantation into adult unconditioned mice. Transplantation. 2004;78(4):503–8. Anton K, Glod J. Targeting the tumor stroma in cancer therapy. Curr Pharm Biotechnol. 2009;10(2):185–91. Review. Ayala F, Dewar R, Kieran M, Kalluri R. Contribution of bone microenvironment to leukemogenesis and leukemia progression. Leukemia. 2009;23(12):2233–41. Bagley RG, Weber W, Rouleau C, Yao M, Honma N, Kataoka S, Ishida I, Roberts BL, Teicher BA. Human mesenchymal stem cells from bone marrow express tumor endothelial and stromal markers. Int J Oncol. 2009;34(3):619–27. Bexell D, Gunnarsson S, Tormin A, Darabi A, Gisselsson D, Roybon L, Scheding S, Bengzon J. Bone marrow multipotent mesenchymal stroma cells act as pericyte-like migratory vehicles in experimental gliomas. Mol Ther. 2009;17(1):183–90. Epub 2008 Nov 4. Bierie B, Moses HL. TGF-beta and cancer. Cytokine Growth Factor Rev. 2006;17(1–2):29–40. Epub 2005 Nov 10. Review. Bissell MJ, Labarge MA. Context, tissue plasticity, and cancer: are tumor stem cells also regulated by the microenvironment? Cancer Cell. 2005;7(1):17–23. Review Biswas S, Chytil A, Washington K, Romero-Gallo J, Gorska AE, Wirth PS, Gautam S, Moses HL, Grady WM. Transforming growth factor beta receptor type II inactivation promotes the establishment and progression of colon cancer. Cancer Res. 2004;64(14):4687–92. Breau RL, Clayman GL. Gene therapy for head and neck cancer. Curr Opin Oncol. 1996;8(3): 227–31. Review. Brown LF, Dvorak AM, Dvorak HF. Leaky vessels, fibrin deposition, and fibrosis: a sequence of events common to solid tumors and to many other types of disease. Am Rev Respir Dis. 1989;140(4):1104–7. Review. Brown AB, Yang W, Schmidt NO, Carroll R, Leishear KK, Rainov NG, Black PM, Breakefield XO, Aboody KS. Intravascular delivery of neural stem cell lines to target intracranial and extracranial tumors of neural and non-neural origin. Hum Gene Ther. 2003;14(18):1777–85. Castelló-Cros R, Cukierman E. Stromagenesis during tumorigenesis: characterization of tumor-associated fibroblasts and stroma-derived 3D matrices. Methods Mol Biol. 2009; 522:275–305.

132

F. Marini et al.

Cavarretta IT, Altanerova V, Matuskova M, Kucerova L, Culig Z, Altaner C. Adipose tissue-derived mesenchymal stem cells expressing prodrug-converting enzyme inhibit human prostate tumor growth. Mol Ther. 2010;18(1):223–31. Chanda D, Isayeva T, Kumar S, Hensel JA, Sawant A, Ramaswamy G, Siegal GP, Beatty MS, Ponnazhagan S. Therapeutic potential of adult bone marrow-derived mesenchymal stem cells in prostate cancer bone metastasis. Clin Cancer Res. 2009;15(23):7175–85. Epub 2009 Nov 17. Chen, X.C.,Wang, R., Zhao, X.,Wei, Y.Q., Hu,M.,Wang, Y.S., Zhang, X.W., Zhang, R., Zhang, L., and Yao, B., et al. Prophylaxis against carcinogenesis in three kinds of unestablished tumor models via IL12-gene-engineered MSCs. Carcinogenesis. (2006) 27, 2434–41. Chen, X., Lin, X., Zhao, J., Shi, W., Zhang, H., Wang, Y., Kan, B., Du, L., Wang, B., and Wei, Y., et al. A Tumor-selective biotherapy with prolonged impact on established metastases based on cytokine gene-engineered MSCs. Mol Ther. 2008;16(4):749–56. Cheng N, Bhowmick NA, Chytil A, Gorksa AE, Brown KA, Muraoka R, Arteaga CL, Neilson EG, Hayward SW, Moses HL. Loss of TGF-beta type II receptor in fibroblasts promotes mammary carcinoma growth and invasion through upregulation of TGF-alpha-, MSP- and HGFmediated signaling networks. Oncogene. 2005;24(32):5053–68. Cho KR. Ovarian cancer update: lessons from morphology, molecules, and mice. Arch Pathol Lab Med. 2009;133(11):1775–81. Coffelt SB, Marini FC, Watson K, Zwezdaryk KJ, Dembinski JL, LaMarca HL, Tomchuck SL, Honer zu Bentrup K, Danka ES, Henkle SL, Scandurro AB. The pro-inflammatory peptide LL-37 promotes ovarian tumor progression through recruitment of multipotent mesenchymal stromal cells. Proc Natl Acad Sci U S A. 2009;106(10):3806–11. Epub 2009 Feb 20. Dalerba P, Cho RW, Clarke MF. Cancer stem cells: models and concepts. Annu Rev Med. 2007;58:267–84. Davis ID, Desai J. Clinical use of therapies targeting tumor vasculature and stroma. Curr Cancer Drug Targets. 2008;8(6):498–508. Review. De Wever O, Demetter P, Mareel M, Bracke M. Stromal myofibroblasts are drivers of invasive cancer growth. Int J Cancer. 2008;123(10):2229–38. Review. Dembinski JL, Krauss S. Characterization and functional analysis of a slow cycling stem cell-like subpopulation in pancreas adenocarcinoma. Clin Exp Metastasis. 2009;26(7):611–23. Epub 2009 May 7. Dembinski JL, Spaeth EL, Fueyo J, Gomez-Manzano C, Studeny M, Andreeff M, Marini FC. Reduction of nontarget infection and systemic toxicity by targeted delivery of conditionally replicating viruses transported in mesenchymal stem cells. Cancer Gene Ther. 2009 Oct 30. Denys H, Derycke L, Hendrix A, Westbroek W, Gheldof A, Narine K, Pauwels P, Gespach C, Bracke M, De Wever O. Differential impact of TGF-beta and EGF on fibroblast differentiation and invasion reciprocally promotes colon cancer cell invasion. Cancer Lett. 2008;266(2):263–74. Devine SM, Bartholomew AM, Mahmud N, Nelson M, Patil S, Hardy W, Sturgeon C, Hewett T, Chung T, Stock W, Sher D, Weissman S, Ferrer K, Mosca J, Deans R, Moseley A, HoffmanR. Mesenchymal stem cells are capable of homing to the bone marrow of non-human primates following systemic infusion. Exp Hematol. 2001;29(2):244–55. Ding YT, Kumar S, Yu DC. The role of endothelial progenitor cells in tumour vasculogenesis. Pathobiology. 2008;75(5):265–73. Epub 2008 Oct 15. Review. Duan X, Guan H, Cao Y, Kleinerman ES. Murine bone marrow-derived mesenchymal stem cells as vehicles for interleukin-12 gene delivery into Ewing sarcoma tumors. Cancer. 2009;115(1):13–22. Review. Duband JL, Blavet C, Jarov A, Fournier-Thibault C. Spatio-temporal control of neural epithelial cell migration and epithelium-to-mesenchyme transition during avian neural tube development. Dev Growth Differ. 2009;51(1):25–44. Dvorak HF. Tumors: wounds that do not heal. Similarities between tumor stroma generation and wound healing. N Engl J Med. 1986;315(26):1650–9. Review. Dvorak HF. Thrombosis and cancer. Hum Pathol. 1987;18(3):275–84. Review.

8  Mesenchymal Stem/Stromal Cells as Cellular Vehicles for Tumor Targeting

133

Erices AA, Allers CI, Conget PA, Rojas CV, Minguell JJ. Human cord blood-derived mesenchymal stem cells home and survive in the marrow of immunodeficient mice after systemic infusion. Cell Transplant. 2003;12(6):555–61. Erickson AC, Barcellos-Hoff MH. The not-so innocent bystander: the microenvironment as a therapeutic target in cancer. Expert Opin Ther Targets. 2003;7(1):71–88. Review. Falanga V, Iwamoto S, Chartier M, Yufit T, Butmarc J, Kouttab N, Shrayer D, Carson P. Autologous bone marrow-derived cultured mesenchymal stem cells delivered in a fibrin spray accelerate healing in murine and human cutaneous wounds. Tissue Eng. 2007;13(6):1299–312. Gao D, Mittal V. The role of bone-marrow-derived cells in tumor growth, metastasis initiation and progression rends Mol Med. 2009;15(8):333–43. Epub 2009 Aug 7. Gerber PA, Hippe A, Buhren BA, Müller A, Homey B. Chemokines in tumor-associated angiogenesis. Biol Chem. 2009;390(12):1213–23. Gilbert CA, Ross AH. Cancer stem cells: cell culture, markers, and targets for new therapies. J Cell Biochem. 2009;108(5):1031–8. Gonda TA, Varro A, Wang TC, Tycko B. Molecular biology of cancer-associated fibroblasts: Can these cells be targeted in anti-cancer therapy? Semin Cell Dev Biol. 2010;21(1):2–10. Grande MT, López-Novoa JM. Fibroblast activation and myofibroblast generation in obstructive nephropathy. Nat Rev Nephrol. 2009;5(6):319–28. Review. Gu C, Li S, Tokuyama T, Yokota N, Namba H. Therapeutic effect of genetically engineered mesenchymal stem cells in rat experimental leptomeningeal glioma model. Cancer Lett. 2010;291(2):256–62. Epub 2009 Nov 27. Guest I, Ilic Z, Ma J, Grant D, Glinsky G, Sell S. Direct and indirect contribution of bone marrow derived cells to cancer. Int J Cancer. 2010;126(10):2308–18. Guturu P, Shah V, Urrutia R. Interplay of tumor microenvironment cell types with parenchymal cells in pancreatic cancer development and therapeutic implications. J Gastrointest Cancer. 2009;40(1–2):1–9. Epub 2009 Jun 10. Hakkarainen T, Särkioja M, Lehenkari P, Miettinen S, Ylikomi T, Suuronen R, Desmond RA, Kanerva A, Hemminki A. Human mesenchymal stem cells lack tumor tropism but enhance the antitumor activity of oncolytic adenoviruses in orthotopic lung and breast tumors. Hum Gene Ther. 2007;18(7):627–41. Hall B, Andreeff M, Marini F. The participation of mesenchymal stem cells in tumor stroma formation and their application as targeted-gene delivery vehicles. Handb Exp Pharmacol. 2007;(180):263–83. Review. Hall B, Dembinski J, Sasser AK, Studeny M, Andreeff M, Marini F. Mesenchymal stem cells in cancer: tumor-associated fibroblasts and cell-based delivery vehicles. Int J Hematol. 2007;86(1):8–16. Hamada H, Kobune M, Nakamura K, et al. Mesenchymal stem cells (MSC) as therapeutic cytoreagents for gene therapy. Cancer Sci. 2005;96:149–156. Hata N, Shinojima N, Gumin J, Yong R, Marini F, Andreeff M, Lang FF. Platelet-derived growth factor BB mediates the tropism of human mesenchymal stem cells for malignant gliomas. Neurosurgery. 2010;66(1):144–56; discussion 156–7. Herrera MB, Bussolati B, Bruno S, Fonsato V, Romanazzi GM, Camussi G. Mesenchymal stem cells contribute to the renal repair of acute tubular epithelial injury. Int J Mol Med. 2004;14(6):1035–41. Hofer EL, La Russa V, Honegger AE, Bullorsky EO, Bordenave RH, Chasseing NA. Alteration on the expression of IL-1, PDGF, TGF-beta, EGF, and FGF receptors and c-Fos and c-Myc proteins in bone marrow mesenchymal stroma cells from advanced untreated lung and breast cancer patients. Stem Cells Dev. 2005;14(5):587–94. Ishii G, Sangai T, Ito T, et al. In vivo and in vitro characterization of human fibroblasts recruited selectively into human cancer stroma. Int J Cancer. 2005;117:212–220. Jayanthi NV, Rowan-Hull AM, Teague WJ, Johnson PR. The importance of pancreatic embryonic epithelium for mesenchyme-to-epithelial transition during islet development. Transplant Proc. 2005;37(8):3485–6.

134

F. Marini et al.

Jiang J, Chen W, Zhuang R, Song T, Li P. The effect of endostatin mediated by human mesenchymal stem cells on ovarian cancer cells in  vitro. J Cancer Res Clin Oncol. 2010;136(6):873–81. Epub 2009 Nov 17. Johnson B, Osada T, Clay T, Lyerly H, Morse M. Physiology and therapeutics of vascular endothelial growth factor in tumor immunosuppression. Curr Mol Med. 2009;9(6):702–7. Review. Kähler CM, Wechselberger J, Hilbe W, Gschwendtner A, Colleselli D, Niederegger H, Boneberg EM, Spizzo G, Wendel A, Gunsilius E, Patsch JR, Hamacher J. Peripheral infusion of rat bone marrow derived endothelial progenitor cells leads to homing in acute lung injury. Respir Res. 2007;8:50. Kallifatidis G, Beckermann BM, Groth A, Schubert M, Apel A, Khamidjanov A, Ryschich E, Wenger T, Wagner W, Diehlmann A, Saffrich R, Krause U, Eckstein V, Mattern J, Chai M, Schütz G, Ho AD, Gebhard MM, Büchler MW, Friess H, Büchler P, Herr I. Improved lentiviral transduction of human mesenchymal stem cells for therapeutic intervention in pancreatic cancer. Cancer Gene Ther. 2008;15(4):231–40. Epub 2008 Jan 18. Kalluri R, Zeisberg M. Fibroblasts in cancer. Nat Rev Cancer. 2006;6(5):392–401. Review. Kanehira M, Xin H, Hoshino K, Maemondo M, Mizuguchi H, Hayakawa T, Matsumoto K, Nakamura T, Nukiwa T, and Saijo Y. Targeted delivery of NK4 to multiple lung tumors by bone marrow-derived mesenchymal stem cells. Cancer Gene Ther (2007)14, 894–903. Keeley EC, Mehrad B, Strieter RM. The role of circulating mesenchymal progenitor cells (fibrocytes) in the pathogenesis of fibrotic disorders. Thromb Haemost. 2009;101(4):613–8. Review. Kenny PA, Lee GY, Bissell MJ. Targeting the tumor microenvironment. Front Biosci. 2007;12:3468–74. Review. Kidd S, Spaeth E, Klopp A, Andreeff M, Hall B, Marini FC. The (in) auspicious role of mesenchymal stromal cells in cancer: be it friend or foe. Cytotherapy. 2008;10(7):657–67. Review. Kim SK, Kim SU, Park IH, Bang JH, Aboody KS, Wang KC, Cho BK, Kim M, Menon LG, Black PM, Carroll RS. Human neural stem cells target experimental intracranial medulloblastoma and deliver a therapeutic gene leading to tumor regression. Clin Cancer Res. 2006;12(18):5550–6. Kim SM, Lim JY, Park SI, Jeong CH, Oh JH, Jeong M, Oh W, Park SH, Sung YC, Jeun SS. Gene therapy using TRAIL-secreting human umbilical cord blood-derived mesenchymal stem cells against intracranial glioma. Cancer Res. 2008;68(23):9614–23. Klopp AH, Spaeth EL, Dembinski JL, Woodward WA, Munshi A, Meyn RE, Cox JD, Andreeff M, Marini FC. Tumor irradiation increases the recruitment of circulating mesenchymal stem cells into the tumor microenvironment. Cancer Res. 2007;67(24):11687–95. Kopfstein L, Christofori G. Metastasis: cell-autonomous mechanisms versus contributions by the tumor microenvironment. Cell Mol Life Sci. 2006;63(4):449–68. Review. Korc M. Pancreatic cancer-associated stroma production. Am J Surg. 2007;194(4 Suppl):S84–6. Review. Krampera M, Pasini A, Rigo A, Scupoli MT, Tecchio C, Malpeli G, Scarpa A, Dazzi F, Pizzolo G, Vinante F. HB-EGF/HER-1 signaling in bone marrow mesenchymal stem cells: inducing cell expansion and reversibly preventing multilineage differentiation. Blood. 2005;106(1):59–66. Kucerova L, Matuskova M, Pastorakova A, Tyciakova S, Jakubikova J, Bohovic R, Altanerova V, and Altaner C. Cytosine deaminase expressing human mesenchymal stem cells mediated tumour regression in melanoma bearing mice. J Gene Med. 2008;10(10):1071–82. Kumamoto M, Nishiwaki T, Matsuo N, Kimura H, Matsushima K. Minimally cultured bone marrow mesenchymal stem cells ameliorate fibrotic lung injury. Eur Respir J. 2009;34(3):740–8. Epub 2009 Mar 26. Kyriakou CA, Yong KL, Benjamin R, Pizzey A, Dogan A, Singh N, Davidoff AM, Nathwani AC. Human mesenchymal stem cells (hMSCs) expressing truncated soluble vascular endothelial growth factor receptor (tsFlk-1) following lentiviral-mediated gene transfer inhibit growth of Burkitt’s lymphoma in a murine model. J Gene Med. 2006;8(3):253–64. Le Blanc K. Mesenchymal stromal cells: Tissue repair and immune modulation. Cytotherapy. 2006;8(6):559–61. Review.

8  Mesenchymal Stem/Stromal Cells as Cellular Vehicles for Tumor Targeting

135

Le Blanc K, Frassoni F, Ball L, Locatelli F, Roelofs H, Lewis I, Lanino E, Sundberg B, Bernardo ME, Remberger M, Dini G, Egeler RM, Bacigalupo A, Fibbe W, Ringdén O; Developmental Committee of the European Group for Blood and Marrow Transplantation. Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versus-host disease: a phase II study. Lancet. 2008;371(9624):1579–86. Liao D, Luo Y, Markowitz D, Xiang R, Reisfeld RA. Cancer associated fibroblasts promote tumor growth and metastasis by modulating the tumor immune microenvironment in a 4T1 murine breast cancer model. PLoS One. 2009;4(11):e7965. Liao W, Zhong J, Yu J, Xie J, Liu Y, Du L, Yang S, Liu P, Xu J, Wang J, Han Z, Han ZC. Therapeutic benefit of human umbilical cord derived mesenchymal stromal cells in intracerebral hemorrhage rat: implications of anti-inflammation and angiogenesis. Cell Physiol Biochem. 2009;24(3–4):307–16. Epub 2009 Aug 3. Lin EY, Li JF, Bricard G, Wang W, Deng Y, Sellers R, Porcelli SA, Pollard JW. Vascular endothelial growth factor restores delayed tumor progression in tumors depleted of macrophages. Mol Oncol. 2007;1(3):288–302. Loebinger MR, Eddaoudi A, Davies D, Janes SM. Mesenchymal stem cell delivery of TRAIL can eliminate metastatic cancer. Cancer Res. 2009;69(10):4134–42. Epub 2009 May 12. Lorusso G, Rüegg C. The tumor microenvironment and its contribution to tumor evolution toward metastasis. Histochem Cell Biol. 2008;130(6):1091–103. Epub 2008 Nov 6. Review. Luetzkendorf J, Mueller LP, Mueller T, Caysa H, Nerger K, Schmoll HJ. Growth-inhibition of colorectal carcinoma by lentiviral TRAIL-transgenic human mesenchymal stem cells requires their substantial intratumoral presence. J Cell Mol Med. 2009 Jun 5. Mahadevan D, Von Hoff DD. Tumor-stroma interactions in pancreatic ductal adenocarcinoma. Mol Cancer Ther. 2007;6(4):1186–97. Epub 2007 Apr 3. Review Maijenburg MW, Noort WA, Kleijer M, Kompier CJ, Weijer K, van Buul JD, van der Schoot CE, Voermans C. Cell cycle and tissue of origin contribute to the migratory behaviour of human fetal and adult mesenchymal stromal cells. Br J Haematol. 2010;148(3):428–40. Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, Zhou AY, Brooks M, Reinhard F, Zhang CC, Shipitsin M, Campbell LL, Polyak K, Brisken C, Yang J, Weinberg RA. The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell. 2008;133(4):704–15. Mansilla E, Marín GH, Drago H, Sturla F, Salas E, Gardiner C, Bossi S, Lamonega R, Guzmán A, Nuñez A, Gil MA, Piccinelli G, Ibar R, Soratti C. Bloodstream cells phenotypically identical to human mesenchymal bone marrow stem cells circulate in large amounts under the influence of acute large skin damage: new evidence for their use in regenerative medicine. Transplant Proc. 2006;38(3):967–9. Markowitz CE. Interferon-beta: mechanism of action and dosing issues. Neurology. 2007;68(24 Suppl 4):S8–11. Review. Matsumoto G, Sunamura M, Shimamura H, Kodama T, Hashimoto W, Kobari M, Kato K, Takeda K, Yagita H, Okumura K, Hamada H, Matsuno S. Adjuvant immunotherapy using fibroblasts genetically engineered to secrete interleukin 12 prevents recurrence after surgical resection of established tumors in a murine adenocarcinoma model. Surgery. 1999;125(3):257–64. Matuskova M, Hlubinova K, Pastorakova A, Hunakova L, Altanerova V, Altaner C, Kucerova L. HSV-tk expressing mesenchymal stem cells exert bystander effect on human glioblastoma cells. Cancer Lett. 2010;290(1):58–67. Menon LG, Kelly K, Yang HW, Kim SK, Black PM, Carroll RS. Human bone marrow-derived mesenchymal stromal cells expressing S-TRAIL as a cellular delivery vehicle for human glioma therapy. Stem Cells. 2009;27(9):2320–30. Menon LG, Kelly K, Yang HW, Kim SK, Black PM, Carroll RS. Human bone marrow-derived mesenchymal stromal cells expressing S-TRAIL as a cellular delivery vehicle for human glioma therapy. Stem Cells. 2009;27(9):2320–30. Mohr A, Lyons M, Deedigan L, Harte T, Shaw G, Howard L, Barry F, O’Brien T, Zwacka R. Mesenchymal stem cells expressing TRAIL lead to tumour growth inhibition in an experimental lung cancer model. J Cell Mol Med. 2008;12(6B):2628–43.

136

F. Marini et al.

Moserle L, Amadori A, Indraccolo S. The angiogenic switch: implications in the regulation of tumor dormancy. Curr Mol Med. 2009;9(8):935–41. Muehlberg FL, Song YH, Krohn A, Pinilla SP, Droll LH, Leng X, Seidensticker M, Ricke J, Altman AM, Devarajan E, Liu W, Arlinghaus RB, Alt EU. Tissue-resident stem cells promote breast cancer growth and metastasis. Carcinogenesis. 2009;30(4):589–97. Epub 2009 Jan 30. Nakamizo A, Marini F, Amano T, Khan A, Studeny M, Gumin J, Chen J, Hentschel S, Vecil G, Dembinski J, Andreeff M, Lang FF. Human bone marrow-derived mesenchymal stem cells in the treatment of gliomas. Cancer Res. 2005;65(8):3307–18. Nakamura K, Ito Y, Kawano Y, Kurozumi K, Kobune M, Tsuda H, Bizen A, Honmou O, Niitsu Y, and Hamada H. Antitumor effect of genetically engineered mesenchymal stem cells in a rat glioma model. Gene Ther 2004;11:1155–1164. Nelson CM, Bissell MJ. Of extracellular matrix, scaffolds, and signaling: tissue architecture regulates development, homeostasis, and cancer. Annu Rev Cell Dev Biol. 2006;22:287–309. Nyberg P, Salo T, Kalluri R. Tumor microenvironment and angiogenesis. Front Biosci. 2008;13:6537–53. Review. Okamoto S, Okamoto A, Nikaido T, Saito M, Takao M, Yanaihara N, Takakura S, Ochiai K, Tanaka T. Mesenchymal to epithelial transition in the human ovarian surface epithelium focusing on inclusion cysts. Exp Cell Res. 2009;315(11):1819–31. Epub 2009 Apr 8. Palmero EI, Achatz MI, Ashton-Prolla P, Olivier M, Hainaut P. bTumor protein 53 mutations and inherited cancer: beyond Li-Fraumeni syndrome. Curr Opin Oncol. 2010;22(1):64–9. Park CC, Bissell MJ, Barcellos-Hoff MH. The influence of the microenvironment on the malignant phenotype. Mol Med Today. 2000;6(8):324–9. Review. Pereira RF, Halford KW, O’Hara MD, Leeper DB, Sokolov BP, Pollard MD, Bagasra O, Prockop DJ. Cultured adherent cells from marrow can serve as long-lasting precursor cells for bone, cartilage, and lung in irradiated mice. Proc Natl Acad Sci U S A. 1995;92(11):4857–61. Petrulio CA, Kim-Schulze S, Kaufman HL. The tumour microenvironment and implications for cancer immunotherapy. Expert Opin Biol Ther. 2006;6(7):671–84. Review. Pittet MJ. Behavior of immune players in the tumor microenvironment. Curr Opin Oncol. 2009;21(1):53–9. Review. Pollard JW. Macrophages define the invasive microenvironment in breast cancer. J Leukoc Biol. 2008;84(3):623–30. Epub 2008 May 8. Review Ponte AL, Marais E, Gallay N, Langonné A, Delorme B, Hérault O, Charbord P, Domenech J. The in vitro migration capacity of human bone marrow mesenchymal stem cells: comparison of chemokine and growth factor chemotactic activities. Stem Cells. 2007;25(7):1737–45. Epub 2007 Mar 29. Quirici N, Scavullo C, de Girolamo L, Lopa S, Arrigoni E, Lambertenghi Deliliers G, Brini AT. Anti-L-NGFR and -CD34 Monoclonal Antibodies identify multipotent mesenchymal stem cells in human adipose tissue. Stem Cells Dev. 2009 Nov 23. Rachakatla RS, Marini F, Weiss ML, Tamura M, Troyer D. Development of human umbilical cord matrix stem cell-based gene therapy for experimental lung tumors. Cancer Gene Ther. 2007;14(10):828–35. Epub 2007 Jun 29. Rachakatla RS, Pyle MM, Ayuzawa R, Edwards SM, Marini FC, Weiss ML, Tamura M, Troyer D. Combination treatment of human umbilical cord matrix stem cell-based interferon-beta gene therapy and 5-fluorouracil significantly reduces growth of metastatic human breast cancer in SCID mouse lungs. Cancer Invest. 2008;26(7):662–70. Radisky ES, Radisky DC. Stromal induction of breast cancer: inflammation and invasion. Rev Endocr Metab Disord. 2007;8(3):279–87. Review. Reddy K, Zhou Z, Schadler K, Jia SF, Kleinerman ES. Bone marrow subsets differentiate into endothelial cells and pericytes contributing to Ewing’s tumor vessels. Mol Cancer Res. 2008;6(6):929–36. Ren C, Kumar S, Chanda D, Kallman L, Chen J, Mountz JD, Ponnazhagan S. Cancer gene therapy using mesenchymal stem cells expressing interferon-beta in a mouse prostate cancer lung metastasis model. Gene Ther. 2008;15(21):1446–53.

8  Mesenchymal Stem/Stromal Cells as Cellular Vehicles for Tumor Targeting

137

Reya T, Morrison SJ, Clarke MF, Weissman IL. Stem cells, cancer, and cancer stem cells. Nature. 2001;414(6859):105–11. Sangai T, Ishii G, Kodama K, et al. Effect of differences in cancer cells and tumor growth sites on recruiting bone marrow-derived endothelial cells and myofibroblasts in cancer-induced stroma. Int J Cancer. 2005;115:885–892. Sasportas LS, Kasmieh R, Wakimoto H, Hingtgen S, van de Water JA, Mohapatra G, Figueiredo JL, Martuza RL, Weissleder R, Shah K. Assessment of therapeutic efficacy and fate of engineered human mesenchymal stem cells for cancer therapy. Proc Natl Acad Sci U S A. 2009;106(12):4822–7. Epub 2009 Mar 5. Satoh H, Kishi K, Tanaka T, Kubota Y, Nakajima T, Akasaka Y, Ishii T. Transplanted mesenchymal stem cells are effective for skin regeneration in acute cutaneous wounds. Cell Transplant. 2004;13(4):405–12. Schatton T, Frank NY, Frank MH. Identification and targeting of cancer stem cells. Bioessays. 2009;31(10):1038–49. Schmid SA, Dietrich A, Schulte S, Gaumann A, Kunz-Schughart LA. Fibroblastic reaction and vascular maturation in human colon cancers. Int J Radiat Biol. 2009;85(11):1013–25. Secchiero P, Melloni E, Corallini F, Beltrami AP, Alviano F, Milani D, D’Aurizio F, di Iasio MG, Cesselli D, Bagnara GP, Zauli G. Tumor necrosis factor-related apoptosis-inducing ligand promotes migration of human bone marrow multipotent stromal cells. Stem Cells. 2008;26(11):2955–63. Epub 2008 Sep 4. Semedo P, Correa-Costa M, Cenedeze MA, Malheiros DM, Antonia Dos Reis M, Shimizu MH, Seguro AC, Pacheco-Silva A, Câmara NO. Mesenchymal Stem Cells Attenuate Renal Fibrosis Through Immune Modulation and Remodeling Properties in a Rat Remnant Kidney Model. Stem Cells. 2009 Sep 11. [Epub ahead of print] Shake JG, Gruber PJ, Baumgartner WA, Senechal G, Meyers J, Redmond JM, Pittenger MF, Martin BJ. Mesenchymal stem cell implantation in a swine myocardial infarct model: engraftment and functional effects. Ann Thorac Surg. 2002;73(6):1919–25; discussion 1926. Silzle T, Kreutz M, Dobler MA, Brockhoff G, Knuechel R, Kunz-Schughart LA. Tumorassociated fibroblasts recruit blood monocytes into tumor tissue. Eur J Immunol. 2003;33(5):1311–20. Spaeth E, Klopp A, Dembinski J, Andreeff M, Marini F. Inflammation and tumor microenvironments: defining the migratory itinerary of mesenchymal stem cells. Gene Ther. 2008;15(10):730–8. Epub 2008 Apr 10. Review. Spaeth EL, Dembinski JL, Sasser AK, Watson K, Klopp A, Hall B, Andreeff M, Marini F. Mesenchymal stem cell transition to tumor-associated fibroblasts contributes to fibrovascular network expansion and tumor progression. PLoS One. 2009;4(4):e4992. Stagg J, Lejeune L, Paquin A, and Galipeau J. Marrow stromal cells for interleukin-2 delivery in cancer immunotherapy. Hum Gene Ther 2004;75:597–608. Stoff-Khalili MA, Rivera AA, Mathis JM, Banerjee NS, Moon AS, Hess A, Rocconi RP, Numnum TM, Everts M, Chow LT, Douglas JT, Siegal GP, Zhu ZB, Bender HG, Dall P, Stoff A, Pereboeva L, Curiel DT. Mesenchymal stem cells as a vehicle for targeted delivery of CRAds to lung metastases of breast carcinoma. Breast Cancer Res Treat. 2007;105(2):157–67. Studeny M, Marini FC, Champlin RE, Zompetta C, Fidler IJ, Andreeff M. Bone marrow-derived mesenchymal stem cells as vehicles for interferon-beta delivery into tumors. Cancer Res. 2002;62(13):3603–8. Studeny M, Marini FC, Dembinski JL, Zompetta C, Cabreira-Hansen M, Bekele BN, Champlin RE, Andreeff M. Mesenchymal stem cells: potential precursors for tumor stroma and targeteddelivery vehicles for anticancer agents. J Natl Cancer Inst. 2004;96(21):1593–603. Sugimoto H, Mundel TM, Kieran MW, Kalluri R. Identification of fibroblast heterogeneity in the tumor microenvironment. Cancer Biol Ther. 2006;5(12):1640–6. Epub 2006 Dec 5. Sun B, Roh KH, Park JR, Lee SR, Park SB, Jung JW, Kang SK, Lee YS, Kang KS Therapeutic potential of mesenchymal stromal cells in a mouse breast cancer metastasis model. Cytotherapy. 2009;11(3):289–98.

138

F. Marini et al.

Sund M, Kalluri R. Tumor stroma derived biomarkers in cancer. Cancer Metastasis Rev. 2009;28(1–2):177–83. Review. Szegezdi E, O’Reilly A, Davy Y, Vawda R, Taylor DL, Murphy M, Samali A, Mehmet H. Stem cells are resistant to TRAIL receptor-mediated apoptosis. J Cell Mol Med. 2009;13(11–12):4409–14. Thu MS, Najbauer J, Kendall SE, Harutyunyan I, Sangalang N, Gutova M, Metz MZ, Garcia E, Frank RT, Kim SU, Moats RA, Aboody KS. Iron labeling and pre-clinical MRI visualization of therapeutic human neural stem cells in a murine glioma model. PLoS One. 2009;4(9):e7218. Tolar J, Nauta AJ, Osborn MJ, Panoskaltsis Mortari A, McElmurry RT, Bell S, Xia L, Zhou N, Riddle M, Schroeder TM, Westendorf JJ, McIvor RS, Hogendoorn PC, Szuhai K, Oseth L, Hirsch B, Yant SR, Kay MA, Peister A, Prockop DJ, Fibbe WE, Blazar BR. Sarcoma derived from cultured mesenchymal stem cells. Stem Cells. 2007;25(2):371–9. Epub 2006 Oct 12. Troyer DL, Weiss ML. Wharton’s jelly derived cells are a primitive stromal cell population. Stem Cells. 2008;26(3):591–9. Review. Uchibori R, Okada T, Ito T, Urabe M, Mizukami H, Kume A, Ozawa K. Retroviral vector-producing mesenchymal stem cells for targeted suicide cancer gene therapy. J Gene Med. 2009;11(5):373–81. van Velthoven CT, Kavelaars A, van Bel F, Heijnen CJ. Mesenchymal stem cell treatment after neonatal hypoxic-ischemic brain injury improves behavioral outcome and induces neuronal and oligodendrocyte regeneration. Brain Behav Immun. 2010;24(3):387–93. Vitale G, van Eijck CH, van Koetsveld Ing PM, Erdmann JI, Speel EJ, van der Wansem Ing K, Mooij DM, Colao A, Lombardi G, Croze E, Lamberts SW, Hofland LJ. Type I interferons in the treatment of pancreatic cancer: mechanisms of action and role of related receptors. Ann Surg. 2007;246(2):259–68. Wang JF, Wang LJ, Wu YF, Xiang Y, Xie CG, Jia BB, Harrington J, McNiece IK. Mesenchymal stem/progenitor cells in human umbilical cord blood as support for ex vivo expansion of CD34(+) hematopoietic stem cells and for chondrogenic differentiation. Haematologica. 2004;89(7):837–44. Wang M, Crisostomo PR, Herring C, Meldrum KK, Meldrum DR. Human progenitor cells from bone marrow or adipose tissue produce VEGF, HGF, and IGF-I in response to TNF by a p38 MAPK-dependent mechanism. Am J Physiol Regul Integr Comp Physiol. 2006;291(4):R880– 4. Epub 2006 May 25. Wang P, Bowl MR, Bender S, Peng J, Farber L, Chen J, Ali A, Zhang Z, Alberts AS, Thakker RV, Shilatifard A, Williams BO, Teh BT. Parafibromin, a component of the human PAF complex, regulates growth factors and is required for embryonic development and survival in adult mice. Mol Cell Biol. 2008;28(9):2930–40. Epub 2008 Jan 22. Wang Y, Crisostomo PR, Wang M, Markel TA, Novotny NM, Meldrum DR. TGF-alpha increases human mesenchymal stem cell-secreted VEGF by MEK- and PI3-K- but not JNK- or ERKdependent mechanisms. Am J Physiol Regul Integr Comp Physiol. 2008;295(4):R1115–23. Epub 2008 Aug 6. Weaver VM, Howlett AR, Langton-Webster B, Petersen OW, Bissell MJ. The development of a functionally relevant cell culture model of progressive human breast cancer. Semin Cancer Biol. 1995;6(3):175–84. Review. Xiang J, Tang J, Song C, Yang Z, Hirst DG, Zheng QJ, Li G. Mesenchymal stem cells as a gene therapy carrier for treatment of fibrosarcoma. Cytotherapy. 2009;11(5):516–26. Xin, H., Kanehira, M., Mizuguchi, H., Hayakawa, T., Kikuchi, T., Nukiwa, T., and Saijo, Y. Targeted deliveryof CX3CL1 to multiple lung tumors by mesenchymal stem cells. Stem Cells (2007) 25, 1618–1626. Xin H, Sun R, Kanehira M, Takahata T, Itoh J, Mizuguchi H, Saijo Y. Intratracheal delivery of CX3CL1-expressing mesenchymal stem cells to multiple lung tumors. Mol Med. 2009;15(9– 10):321–7. Epub 2009 Jun 18. Yang B, Wu X, Mao Y, Bao W, Gao L, Zhou P, Xie R, Zhou L, Zhu J. Dual-targeted antitumor effects against brainstem glioma by intravenous delivery of tumor necrosis factor-related, apoptosis-inducing, ligand-engineered human mesenchymal stem cells. Neurosurgery. 2009;65(3):610–24.

8  Mesenchymal Stem/Stromal Cells as Cellular Vehicles for Tumor Targeting

139

Yong RL, Shinojima N, Fueyo J, Gumin J, Vecil GG, Marini FC, Bogler O, Andreeff M, Lang FF. Human bone marrow-derived mesenchymal stem cells for intravascular delivery of oncolytic adenovirus Delta24-RGD to human gliomas. Cancer Res. 2009;69(23):8932–40. Epub 2009 Nov 17. You MH, Kim WJ, Shim W, Lee SR, Lee G, Choi S, Kim DY, Kim YM, Kim H, Han SU. Cytosine deaminase-producing human mesenchymal stem cells mediate an antitumor effect in a mouse xenograft model. J Gastroenterol Hepatol. 2009;24(8):1393–400. Epub 2009 May 28. Zeisberg EM, Potenta S, Xie L, Zeisberg M, Kalluri R. Discovery of endothelial to mesenchymal transition as a source for carcinoma-associated fibroblasts. Cancer Res. 2007;67(21):10123–8. Zipori D, Reichman N, Arcavi L, Shtalrid M, Berrebi A, Resnitzky P. In vitro functions of stromal cells from human and mouse bone marrow. Exp Hematol. 1985;13(7):603–9. Zischek C, Niess H, Ischenko I, Conrad C, Huss R, Jauch KW, Nelson PJ, Bruns C.Targeting tumor stroma using engineered mesenchymal stem cells reduces the growth of pancreatic carcinoma. Ann Surg. 2009;250(5):747–53. Erratum in: Ann Surg. 2010;251(1):187. Zumsteg A, Christofori G. Corrupt policemen: inflammatory cells promote tumor angiogenesis. Curr Opin Oncol. 2009;21(1):60–70. Review.

Chapter 9

Retargeting Adenovirus for Cancer Gene Therapy Erin E. Thacker and David T. Curiel

Abstract  Despite the rapid progress in developing clinically relevant cancer gene therapies, successful treatment options remain elusive for many tumor types, and complete remission in patients is difficult to achieve, especially for metastatic cancers. With this in mind, viral vectors have been extensively investigated as delivery vehicles for a variety of cancer gene therapy approaches. In particular, Adenovirus (Ad)based vectors have emerged as attractive vectors for clinical application due to their high capacity for gene transfer and their safety profile. Ad vectors have demonstrated great promise in the development of gene therapy and immunotherapy vaccines for cancer. In addition, the recent development of oncolytic Ad vectors has added significantly to the therapeutic potential of Ad-based cancer therapies. However, previous investigations with Ad vectors identified complex interactions with blood factors and nontarget cells, as well as anti-Ad immune responses, which diminish the therapeutic capacity and increase the potential for unintended side-effects. Therefore, efforts are now focused on retargeting Ad vectors to specifically bind target cells, while avoiding unfavorable native interactions. This chapter reviews the significant advances made toward retargeting Ad vectors to achieve the ultimate goal of developing a safe and effective therapy with application for a variety of cancers. Keywords  Adenovirus • Targeting • Cancer • Immunotherapy • Gene therapy

1 Introduction The utility of Adenoviruses (Ads) as therapeutic vectors for tumor eradication has been widely investigated in the context of cancer gene therapy virotherapy and immunotherapy. The serotype most commonly used for human therapies, Ad5, is an attractive

D.T. Curiel (*) Comprehensive Cancer Center, and Gene Therapy Center, University of Alabama at Birmingham, 901 19th Street South BMR2-508, Birmingham, AL 35294, USA e-mail: [email protected] J.A. Roth (ed.), Gene-Based Therapies for Cancer, Current Cancer Research, DOI 10.1007/978-1-4419-6102-0_9, © Springer Science+Business Media, LLC 2010

141

142

E.E. Thacker and D.T. Curiel

vector for clinical use due to its relative safety profile, capacity to transduce dividing and nondividing cells, and ease of manipulation. However, less than optimal results from clinical trials utilizing Ad-based strategies clearly indicate the need for vector improvement. It is hypothesized that efficient and specific targeting of Ad5 vectors will facilitate the realization of safe and effective cancer therapy (reviewed in Hemminki and Alvarez 2002). Many approaches for attaining this goal are currently being evaluated, and will be introduced in this chapter. Due to the breadth of information covered, review articles have been cited where possible, and the reader is highly encouraged to refer to the original articles cited within these reviews for more in-depth information.

2 Adenovirus Life Cycle and Genomic Organization A thorough understanding of the Ad genome and life cycle is critical for developing retargeted Ad vectors that maintain therapeutic utility. It is well-established that the double stranded DNA core of Ad is protected by a nonenveloped protein capsid composed predominately of the major capsid protein, hexon, in association with minor capsid proteins (Fig.  1). Protruding fiber proteins support globular knob domains that mediate vector tropism through recognition of cellular receptors (reviewed in Waehler et  al. 2007, Nemerow et  al. 2009). The Ad5 knob domain binds to the coxsackievirus and adenovirus receptor (CAR) expressed on the ­surface of many cells, but likely interacts with other receptors as well. Several Ads belonging to other serotypes also bind efficiently to CAR, while others preferentially bind alternative receptors (reviewed in Arnberg 2009). Receptor-binding brings Ad5 into proximity with cellular integrins, facilitating vector endocytosis.

pIX

DNA

Hexon Fiber Knob Fig.  1  Schematic representation of Ad capsid proteins. The Ad capsid is composed of major capsid proteins, such as hexon, and minor capsid proteins, such as pIX. The fiber and knob domains are largely responsible for determining vector tropism

9  Retargeting Adenovirus for Cancer Gene Therapy

143

Viral transcription, replication, and packaging occur in the nucleus, followed by cell lysis and virus release from the cell (reviewed in Nemerow et al. 2009). The 36  kb Ad genome is organized into early and late genes. The first region expressed after infection is the Early region 1A (E1A) transcription unit, which manipulates host cell cycle proteins necessary for virus replication and regulates expression of the remaining early genes, E1B, E2, E3, and E4. These regions also encode proteins that regulate host cell functions, including transcription and translation and immune responses to Ad (reviewed in Yang et al. 2007, Akusjarvi 1993, White 2001). Late genes primarily encode the capsid proteins. The crucial discovery that most Ad genes can be provided in trans by cell lines or helper viruses made the development of Ad vectors for gene therapy possible (Grable and Hearing 1992).

3 Strategies for Ad-Based Cancer Gene Therapy 3.1 Cancer Gene Therapy Vectors Ad-based gene therapy vectors were originally designed to treat inherited genetic disorders by delivering an encoded therapeutic transgene to mutant cells, but these vectors have also demonstrated great promise in the context of acquired diseases, including cancer (reviewed in Kaplan 2005). To prevent the Ad-mediated killing of targeted cells, “first generation” nonreplicating vectors were engineered with deletions in E1 regions essential for Ad replication (Berkner 1988). E1 deletions also allow larger therapeutic transgenes to be inserted without surpassing the total DNA packaging capacity of Ad vectors (Bett et al. 1993). Nonreplicating Ad-based vectors have been employed to deliver a variety of genetic payloads, including proapoptotic genes, inhibitory RNA sequences, or “suicide” genes to tumor cells (reviewed in Yang et al. 2007, Shirakawa 2008). Ad vectors specifically targeting the vasculature required by solid tumors for growth and survival are also being investigated (reviewed in Liu et al. 2007, Khalighinejad et al. 2008)

3.2 Immunotherapy Vectors Nonreplicating Ad vectors have also demonstrated utility for cancer immunotherapy, whereby tumor-associated antigens (TAA) are encoded as transgenes for delivery to antigen-presenting cells (APCs) of the immune system. Transduced APCs translate and process the TAA for epitope presentation via major histocompatability complex I (MHCI) and/or MHCII on the cell surface. T cells that recognize and bind the MHC/TAA are then activated to mount a systemic immune directed against tumor cells expressing the TAA (reviewed in Mossoba and Medin 2006). High antigenspecific immune responses are typically generated against Ad5-encoded transgene products, and may provide enhanced immunotherapeutic efficacy. Vectors encoding transgenes that modulate the function of tumor-infiltrating immune cells are likely to provide adjuvant benefits as well (Eruslanov et al. 2009).

144

E.E. Thacker and D.T. Curiel

3.3 Virotherapy Vectors While the above strategies have shown promise, complete tumor eradication by cancer gene therapy vectors depends on successful transduction and transgene expression in all tumor cells. Furthermore, antitumor immune responses generated by immunotherapeutic vectors are often suppressed by the tumor environment (reviewed in Alemany and Cascallo 2009), and immune responses often decline with age. Therefore, therapeutic strategies employing nonreplicating Ad vectors may not be sufficient for eradicating all tumors. In light of this, researchers have capitalized on the intrinsic lytic capacity of replication-competent Ads to develop oncolytic vectors (reviewed in Yang et  al. 2007, Alemany 2007, Jounaidi et  al. 2007). Oncolytic Ad vectors maintain the ability to replicate and lyse cells, releasing progeny to infect and kill surrounding tumor cells. Thus, it is not as critical that every tumor cell be transduced upon initial vector administration. Moreover, initial viral-induced tumor cell lysis may lead to an immune response against the tumor cells, which could aid in further tumor elimination (reviewed in Alemany and Cascallo 2009). Oncolytic vectors can also sensitize tumor cells to chemotherapy or radiation, and vectors can be modified to encode therapeutic transgenes, enhancing the potential for effective tumor cell lysis.

4 A Need for Retargeted Adenovirus-Based Vectors for Cancer Therapy Several factors limit the efficacy of gene therapy applications in general and Ad-based vaccines in particular. Systemic delivery of gene therapy vectors is necessary to reach metastatic cells, and could be useful for reaching immune cells and many solid tumors. However, vector extravasation from the vasculature is limited, preventing efficient delivery (reviewed in Waehler et  al. 2007). Unfortunately, administering higher doses of Ad vectors does not enhance delivery, and can lead to toxic or lethal side-effects (Raper et al. 2003). CAR expression by tumor cells is also decreased as a result of the hypoxic tumor environment, and APCs normally express very little, if any, CAR. Therefore, Ad5 vectors are most likely to transduce nontarget cells, reducing the therapeutic payload available for the cells of interest. In the context of oncolytic vectors or delivery of apoptotic or suicide transgenes, unintended transduction and gene expression in noncancerous cells could produce detrimental side-effects. Following systemic administration, Ad5 interacts with erythrocytes, complement and other blood factors, and is sequestered by Kupffer cells and other macrophages. These interactions lead to vector accumulation predominately in the liver and activation of innate and adaptive immune responses against Ad vectors, which can lead to toxic side-effects and further diminish vector delivery to target cells (reviewed in Parker et  al. 2008, Thacker et  al. 2009, Baker et  al. 2007). Furthermore, the presence of preexisting immunity (PEI) to Ad5 observed in the majority of humans

9  Retargeting Adenovirus for Cancer Gene Therapy

145

can significantly enhance the rate of vector clearance, again reducing vaccine efficacy. To overcome these limitations, Ad5 vectors must be redirected to specifically transduce target cells while avoiding native interactions. Strategies with demonstrated potential to retarget Ad5 and mediate tumor regression include transductional targeting, transcriptional and translational targeting, functional complementation, and the use of cell vehicles (reviewed in Yang et al. 2007, Bachtarzi et al. 2008, Alemany 2009, Noureddini and Curiel 2005).

5 Transductional Targeting Transductional targeting strategies aim to redirect Ad vectors to interact with receptors expressed mainly by the tumor or immune cells of interest. This has been accomplished through the use of targeted adapter molecules and by the genetic modification of Ad vectors.

5.1 Adapter-Based Targeting Bispecific adapter molecules are composed of a motif that binds the Ad vector, such as anti-Ad antibodies or truncated, soluble CAR (sCAR), fused to an antibody or ligand that recognizes a cell surface receptor (Fig. 2) (reviewed in Glasgow et al. 2006). Results from studies with adapter-based targeting strategies demonstrated the feasibility of retargeting Ad5 vectors to nonnative binding motifs to achieve cell-specific transduction, increase therapeutic gene delivery, and diminish toxicity in preclinical animal models (Huang et al. 2008; Li et al. 2007; Pereboev et al. 2004). a

b

c

HOST CELL NUCLEUS cellular transcription factor

Ad gene or encoded transgene

IRES Ad gene

encoded transgene

Anti-CAR antibody or sCAR Cell-specific targeting ligand or antibody

cell-specific promoter

Fig. 2  Transductional and transcriptional approaches for retargeting Ad vectors. (a) Bispecific adapters bound to the knob domain of Ad mediate transductional retargeting. (b) Vectors can also be genetically modified to express cell-specific receptor ligands or pseudotyped fibers for transductional retargeting. (c) Target cell-specific control of viral gene or encoded transgene expression can be achieved by replacing viral promoters with promoters that interact with specific cellular transcription factors

146

E.E. Thacker and D.T. Curiel

5.2 Genetic Modifications While two-component adapter-based strategies revealed the therapeutic benefits of retargeting Ad vectors, the adapter molecules cannot be replicated by the host cell, and therefore are not appropriate for targeting oncolytic vectors since released, untargeted progeny could infect surrounding noncancerous cells. This disadvantage, and issues associated with good manufacturing practices (GMP) for adapters, prompted efforts to retarget vectors by genetic modification. Strategies developed toward this goal include serotype fiber pseudotyping and genetic ligand incorporation (reviewed in Noureddini and Curiel 2005). The receptor interactions of alternate Ad serotypes have been investigated in the hope of discovering an Ad with a more restricted tropism compared to Ad5. However, vectors derived from these serotypes often do not display the clinical advantages of Ad5-based vectors. Although progress has been made toward improving the clinical utility of some Ads, fiber pseudotyping also provides a viable option (Fig. 2). In this regard, the most extensively studied vectors are chimeric Ad5 vectors engineered to display the fiber and/ or knob domain of another serotype, altering the receptor-binding affinity. Many of these vectors mediate preferential tumor cell transduction and diminish interactions with anti-Ad neutralizing antibodies (NAbs) and blood factors, leading to enhanced antitumor effects and diminished toxicity in preclinical animal models (Kanerva et al. 2003; Raki et al. 2005; Rajecki et al. 2007; Kangasniemi et al. 2006; Ulasov et al. 2007; Ni et al. 2006; Tsuruta et al. 2008; Wang et al. 2009; Liu et al. 2009; Greig et al. 2009; Jin et al. 2009). Chimeric vectors that mediate efficient transduction of APCs in vitro and generate an antigen-specific immune response in vivo have also been developed, though antitumor effects have not been specifically evaluated (Rea et al. 2001; DiPaolo et al. 2006). An alternate strategy that combines adapter targeting and genetic engineering allows targeting through candidate receptors to be evaluated quickly using a single Ad vector. This modification genetically incorporates the immunoglobulin (Ig)binding domain of Staphylococcus aureus protein A into the fiber region allowing targeting with receptor-specific antibodies even if the natural ligand for the receptor is unknown. This approach has been used to target nonreplicating vectors to tumor cells in vitro and in animal models, and APCs in vitro (reviewed in Glasgow et al. 2006). Similar to bispecific adapters, this approach cannot be applied to target oncolytic vectors, since the antibody cannot be replicated. However, Ad vectors with genetically incorporated targeting ligands or single chain antibodies (scFvs) capable of replicating along with the Ad genome have been developed (Fig. 2) (reviewed in Alemany 2009). Oncolytic vectors targeted via this strategy mediate enhanced survival in preclinical animal models, and APC targeting is being evaluated.

6 Cellular Control of Ad Vector Gene Expression While transductional targeting redirects Ad vectors to specific receptors, restricting gene expression to specific cell types is actually difficult to achieve since receptors exhibiting true cell-type specificity are rare and may not facilitate vector transduction.

9  Retargeting Adenovirus for Cancer Gene Therapy

147

Thus, transcriptional and translational targeting strategies are being investigated to further restrict the viral replication or transgene expression to target cells.

6.1 Transcriptional Targeting Transcription of the Ad genome requires host cell proteins, including RNA polymerase II, general transcription factors, enhancers, and repressors. Therefore, transgene expression or oncolytic virus replication can be controlled by exploiting transcriptional regulators specifically expressed by the target cells. Transcriptional targeting has been accomplished by substituting target cell-­ specific DNA regulatory sequences for native Ad regulatory sequences (Fig.  2). These sequences can encompass a combination of cell-specific promoters (including a transcriptional start site and recognition elements to recruit RNA polymerase), enhancer-binding regions and silencer-binding regions. However, the choice of regulatory sequences appropriate for insertion in oncolytic vectors is limited by the genomic capacity and the need for strict cell-specific control of viral gene expression, since even low (leaky) levels of E1A expression can facilitate virus replication (Hitt and Graham 1990). To meet these criteria, transcriptional targeting has also been achieved by substituting promoter fragments encompassing only the transcription start site and nearby elements or replacing viral transcription factor binding sites with cell-specific sites. Artificial promoters composed of unique combinations of regulatory sequences that respond to a variety of target cell-specific regulatory factors can further optimize transcriptional regulation. Recently developed strategies also allow the temporal control of transcription by pharmacological agents (reviewed in Yang et al. 2007, Nettelbeck 2008, Glasgow et al. 2004, Sadeghi and Hitt 2005). Placing multiple Ad proteins under cell-specific transcriptional control provides further assurance of restricted gene expression. However, the use of identical promoters to control transcription of separate viral or inserted genes may lead to genomic instability. This genomic instability, as well as genomic size restrictions, can be circumvented by fusing two genes with an internal ribosome entry site (IRES), or inserting bidirectional promoters (Fig. 2) (reviewed in Nettelbeck 2008). Careful consideration must also be given to the genes regulated, as substituting cellular promoters for viral promoters may interfere with the transcription of other genes or reduce viral replication even in target cells. Despite careful design of regulatory sequences, cryptic transcription initiation sites located upstream of E1A can facilitate the read-through transcription of downstream viral or transgenes in nontarget cells, even in the absence of a promoter. As these cryptic sites are located in the ITR and packaging sequences, which are required in cis for viral replication, they cannot be deleted. However, inserting poly A transcription termination signals or cellular insulator elements upstream of cell-selective promoters can minimize the influence of viral enhancers in some cases. Relocating the packaging sequence to the 3¢ end of the genome also attenuates nonspecific read-through, but may lead to genomic instability. In this case, the reorganization of the packaging

148

E.E. Thacker and D.T. Curiel

sequence may be a more relevant alternative. Additionally, reversing the E1A promoter region diminishes nonspecific read-through, but may also decrease the expression of the encoded gene (reviewed in Nettelbeck 2008, Sadeghi and Hitt 2005). Imposed transcriptional regulation can also be hindered by the direct interaction of cellular transcription factors with expressed Ad proteins, especially E1A. To avoid such interactions, sequences involved in E1A-transcription factor binding may be specifically mutated (Johnson et al. 2002). Helper-dependent Ad (HDAd) vectors, depleted of all viral genes except the necessary ITRs and packaging sequences, may also be employed (Shi et al. 2002). However, HDAd vectors cannot replicate without a helper virus, so the utility of these vectors for oncolytic therapy is obviously limited. Thus, overall, transcriptional targeting further restricts the expression of Ad genomes or encoded transgenes, but is likely to provide the best results when combined with other targeting strategies.

6.2 Translational Targeting The dysregulation of mRNA translation in tumor cells provides yet another opportunity to restrict Ad vector gene or transgene expression. To this end, specific sequences in the 3¢ untranslated regions (UTR) of many proteins serve to stabilize mRNA in tumor cells, increasing protein expression. Ligating E1A to such a 3¢UTR can also stabilize the Ad mRNA, increasing translation specifically in tumor cells (Ahmed et al. 2003). Similarly, ligating E1A to a complex 5¢UTR places translation under control of cellular eIF4E, which is overexpressed in tumor cells. Indeed, when compared to transcriptional targeting alone, insertion of a complex 5¢UTR further enhanced the tumor-specificity of viral replication and diminished liver toxicity in animal models (Stoff-Khalili et al. 2008). Therefore, translational targeting of Ad vectors may have critical safety benefits when combined with other targeting strategies.

6.3 Functional Complementation The signaling pathways dysregulated in tumor cells can also be exploited to allow oncolytic Ad vectors to replicate in tumor cells, but not in noncancerous cells. Key to this concept, several proteins required for Ad replication, such as E1A and E1B, must be expressed to gain control of host cell functions needed for successful virus replication, and the dysregulation of the same functions, such as cell cycle progression and apoptosis, is often observed in tumor cells. Therefore, replication of oncolytic Ad vectors engineered with mutations in essential genomic regions can be rescued in tumor cells already controlling the appropriate cell functions (reviewed in Alemany 2007, Nettelbeck 2008). However, decreased oncolytic potency has been observed with several such mutations; hence, means for optimizing these intriguing strategies are being investigated.

9  Retargeting Adenovirus for Cancer Gene Therapy

149

7 Adjunct Technologies for Delivery Harvesting autologous cells and transducing them with Ad vectors ex vivo is one way to ensure cell-specific virus or transgene expression and, presumably, ablate toxicity associated with systemic administration of Ad vectors. Vaccination with dendritic cells (DCs) transduced by nonreplicating Ad5 vectors encoding TAA avoids Ad5 PEI-mediated effects on transgene expression and prevents the production of anti-Ad5 NAbs in mice (Zabaleta et  al. 2008). Similarly, harvested stem cells and other cells that naturally home to tumor beds are being investigated as delivery vehicles for oncolytic Ad vectors (Mohr et al. 2008; Sonabend et al. 2008; Tyler et al. 2009; Stoff-Khalili et al. 2007; Nakamura et al. 2004; Lamfers et al. 2009). Viral lysis of cell vehicles in the tumor environment allows targeted viral progeny to infect and kill tumor cells. As this novel strategy is pursued, it will be important to also determine whether viral replication in the cell vehicles and the consequent presentation of viral epitopes via MHC trigger anti-Ad5 immune destruction of the vehicles prior to targeted delivery. Moreover, harvesting and ex vivo manipulation of cells present technical issues that must be addressed before vaccination with transduced cells can become standard (Proudfoot et al. 2007), but further investigation of these strategies is certainly warranted.

8 Future Directions and Conclusions Research over the past several years has resulted in significant progress toward cellspecific targeting of Ad viral replication and expression of encoded transgenes for effective cancer therapy. Yet it is becoming clear that employing more than one targeting method will be required to optimize the efficacy of Ad-based therapies while ensuring the safety of patients. Indeed, such combinatorial strategies are now being investigated (Li et al. 2009) (reviewed in Noureddini and Curiel 2005). In addition, unique ligands can be inserted in multiple Ad capsid locales, suggesting the possibility of vectors with multiple targeting motifs or imaging modalities, which could even allow the biodistribution of retargeted Ad vectors to be determined in humans prior to the clinical application of therapeutic vectors (Fig. 3) (Tang et al. 2008). Furthermore, the effects of PEI to Ad5 and vector-binding to blood cells and factors must be investigated for retargeted vectors. Substitutions in the hexon hypervariable regions (HVRs), the binding site for blood factors and many anti-Ad NAbs, may diminish toxicity and vector clearance (Fig.  3) (reviewed in Thacker et al. 2009). However, it should be noted that hexon is likely involved in the intracellular trafficking of Ad vectors required for optimal transduction in some cell types (Campos and Barry 2006). In addition, the Ad epitopes recognized by T cells are often conserved, even between human- and nonhuman-tropic Ads, and must be considered (Calcedo et al. 2009; Schirmbeck et al. 2008). Conversely, HDAd vectors invoke fewer T cell responses, and HDAds encoding cytotoxic genes have been shown to induce tumor regression in animal models even in the presence of PEI to

150

E.E. Thacker and D.T. Curiel clinical imaging motif

incorporated antigen

fluorescent imaging motif

novel targeting motif

targeting motif

Fig. 3  Combined modifications of Ad vectors. Genetic modifications have been achieved in Ad fiber/knob domains, hexon domains, and pIX domains. Ad vectors engineered with multiple targeting and imaging motifs such as these may expand clinical utility and therapeutic efficacy

Ad5 (Weaver et al. 2009; Koup et al. 2009). Thus, HDAds, which can accommodate targeting ligands or fiber pseudotyping, should be further investigated as tools for cancer gene therapy and immunotherapy. Purification of HDAds from the helper viruses required to supply viral genes in trans for vector production has proven difficult, and currently limits the clinical application of HDAds, however (reviewed in Segura et al. 2008, Brunetti-Pierri and Ng 2008). In addition, HDAds are not applicable to oncolytic virotherapy. Therefore, cell-delivery vehicles or biochemical modification with polymers such as activated polyethylene glycol (PEG) (PEGylation) or poly[N-(2-hydroxypropyl) methacrylamide] (HPMA), which can also accommodate targeting motifs, may be useful for reducing toxicity and virus clearance of both nonreplicating and oncolytic Ad vectors (reviewed in Thacker et al. 2009, Eto et al. 2008). Finally, to obtain the most stringent analysis of retargeted Ad-based vectors prior to clinical trials, caution must also be used in determining the animal models to be employed. The cell-specificity of expressed Ad receptors, in addition to other factors that contribute to liver sequestration of Ads, varies among different species, and may alter vector biodistribution. Conclusions drawn from animal studies must therefore be evaluated carefully to ensure that the most accurate safety and efficacy profiles are available for human studies (reviewed in Baker et al. 2007, Alemany 2009). In conclusion, significant progress has been made toward retargeting Ad vectors to restrict viral replication and/or expression of encoded transgenes to target cells for cancer therapy. While each strategy alone enhances specificity, complete eradication of tumors by Ad-based therapies is likely to require vectors that embody a unique combination of targeting strategies determined by the type and location of the cancer. Such optimized strategies are now within reach, and the current momentum should result in exciting progress toward the clinical application of retargeted Ad vectors for successful cancer therapy.

9  Retargeting Adenovirus for Cancer Gene Therapy

151

References Ahmed A, Thompson J, Emiliusen L et  al. A conditionally replicating adenovirus targeted to tumor cells through activated RAS/P-MAPK-selective mRNA stabilization. Nat Biotechnol 2003; 21: 771–777. Akusjarvi G. Proteins with transcription regulatory properties encoded by human adenoviruses. Trends Microbiol 1993; 1: 163-170. Alemany R. Cancer selective adenoviruses. Mol Aspects Med 2007; 28: 42–58. Alemany R. Designing adenoviral vectors for tumor-specific targeting. Methods Mol Biol 2009; 542: 57–74. Alemany R, Cascallo M. Oncolytic viruses from the perspective of the immune system. Future Microbiol 2009; 4: 527–536. Arnberg N. Adenovirus receptors: implications for tropism, treatment and targeting. Rev Med Virol 2009; 19: 165–178. Bachtarzi H, Stevenson M, Fisher K. Cancer gene therapy with targeted adenoviruses. Expert Opin Drug Deliv 2008; 5: 1231–1240. Baker AH, McVey JH, Waddington SN et al. The influence of blood on in vivo adenovirus biodistribution and transduction. Mol Ther 2007; 15: 1410–1416. Berkner KL. Development of adenovirus vectors for the expression of heterologous genes. Biotechniques 1988; 6: 616–629. Bett AJ, Prevec L, Graham FL. Packaging capacity and stability of human adenovirus type 5 vectors. J Virol 1993; 67: 5911–5921. Brunetti-Pierri N, Ng P. Progress and prospects: gene therapy for genetic diseases with helperdependent adenoviral vectors. Gene Ther 2008; 15: 553–560. Calcedo R, Vandenberghe LH, Roy S et al. Host immune responses to chronic adenovirus infections in human and nonhuman primates. J Virol 2009; 83: 2623–2631. Campos SK, Barry MA. Comparison of adenovirus fiber, protein IX, and hexon capsomeres as scaffolds for vector purification and cell targeting. Virology 2006; 349: 453–462. DiPaolo N, Ni S, Gaggar A et al. Evaluation of adenovirus vectors containing serotype 35 fibers for vaccination. Mol Ther 2006; 13: 756–765. Eruslanov E, Kaliberov S, Daurkin I et al. Altered expression of 15-hydroxyprostaglandin dehydrogenase in tumor-infiltrated CD11b myeloid cells: a mechanism for immune evasion in cancer. J Immunol 2009; 182: 7548–7557. Eto Y, Yoshioka Y, Mukai Y et al. Development of PEGylated adenovirus vector with targeting ligand. Int J Pharm 2008; 354: 3–8. Glasgow JN, Bauerschmitz GJ, Curiel DT, Hemminki A. Transductional and transcriptional targeting of adenovirus for clinical applications. Curr Gene Ther 2004; 4: 1–14. Glasgow JN, Everts M, Curiel DT. Transductional targeting of adenovirus vectors for gene therapy. Cancer Gene Ther 2006; 13: 830–844. Grable M, Hearing P. cis and trans requirements for the selective packaging of adenovirus type 5 DNA. J Virol 1992; 66: 723–731. Greig JA, Buckley SM, Waddington SN et al. Influence of coagulation factor X on in vitro and in vivo gene delivery by adenovirus (Ad) 5, Ad35, and chimeric Ad5/Ad35 vectors. Mol Ther 2009; 17: 1683–1691. Hemminki A, Alvarez RD. Adenoviruses in oncology: a viable option? Biodrugs 2002; 16: 77–87. Hitt MM, Graham FL. Adenovirus E1A under the control of heterologous promoters: wide variation in E1A expression levels has little effect on virus replication. Virology 1990; 179: 667–678. Huang D, Pereboev AV, Korokhov N et al. Significant alterations of biodistribution and immune responses in Balb/c mice administered with adenovirus targeted to CD40(+) cells. Gene Ther 2008; 15: 298–308. Jin J, Liu H, Yang C et al. Effective gene-viral therapy of leukemia by a new fiber chimeric oncolytic adenovirus expressing TRAIL: in vitro and in vivo evaluation. Mol Cancer Ther 2009; 8: 1387–1397.

152

E.E. Thacker and D.T. Curiel

Johnson L, Shen A, Boyle L et al. Selectively replicating adenoviruses targeting deregulated E2F activity are potent, systemic antitumor agents. Cancer Cell 2002; 1: 325–337. Jounaidi Y, Doloff JC, Waxman DJ. Conditionally replicating adenoviruses for cancer treatment. Curr Cancer Drug Targets 2007; 7: 285–301. Kanerva A, Zinn KR, Chaudhuri TR et al. Enhanced therapeutic efficacy for ovarian cancer with a serotype 3 receptor-targeted oncolytic adenovirus. Mol Ther 2003; 8: 449–458. Kangasniemi L, Kiviluoto T, Kanerva A et al. Infectivity-enhanced adenoviruses deliver efficacy in clinical samples and orthotopic models of disseminated gastric cancer. Clin Cancer Res 2006; 12: 3137–3144. Kaplan JM. Adenovirus-based cancer gene therapy. Curr Gene Ther 2005; 5: 595–605. Khalighinejad N, Hariri H, Behnamfar O et  al. Adenoviral gene therapy in gastric cancer: a review. World J Gastroenterol 2008; 14: 180–184. Koup RA, Lamoreaux L, Zarkowsky D et  al. Replication-defective adenovirus vectors with multiple deletions do not induce measurable vector-specific T cells in human trials. J Virol 2009; 83: 6318–6322. Lamfers M, Idema S, van Milligen F et al. Homing properties of adipose-derived stem cells to intracerebral glioma and the effects of adenovirus infection. Cancer Lett 2009; 274: 78–87. Li HJ, Everts M, Pereboeva L et  al. Adenovirus tumor targeting and hepatic untargeting by a coxsackie/adenovirus receptor ectodomain anti-carcinoembryonic antigen bispecific adapter. Cancer Res 2007; 67: 5354–5361. Li HJ, Everts M, Yamamoto M et al. Combined transductional untargeting/retargeting and transcriptional restriction enhances adenovirus gene targeting and therapy for hepatic colorectal cancer tumors. Cancer Res 2009; 69: 554–564. Liu Y, Koziol J, Deisseroth A, Borgstrom P. Methods for delivery of adenoviral vectors to tumor vasculature. Hum Gene Ther 2007; 18: 151–160. Liu Y, Wang H, Yumul R et  al. Transduction of liver metastases after intravenous injection of Ad5/35 or Ad35 vectors with and without factor X-binding protein pretreatment. Hum Gene Ther 2009; 20: 621–629. Mohr A, Lyons M, Deedigan L et al. Mesenchymal Stem Cells expressing TRAIL lead to tumour growth inhibition in an experimental lung cancer model. J Cell Mol Med 2008; 12: 2628–2643. Mossoba ME, Medin JA. Cancer immunotherapy using virally transduced dendritic cells: animal studies and human clinical trials. Expert Rev Vaccines 2006; 5: 717–732. Nakamura K, Ito Y, Kawano Y et  al. Antitumor effect of genetically engineered mesenchymal stem cells in a rat glioma model. Gene Ther 2004; 11: 1155–1164. Nemerow GR, Pache L, Reddy V, Stewart PL. Insights into adenovirus host cell interactions from structural studies. Virology 2009; 384: 380–388. Nettelbeck DM. Cellular genetic tools to control oncolytic adenoviruses for virotherapy of cancer. J Mol Med 2008; 86: 363–377. Ni S, Gaggar A, Di Paolo N et al. Evaluation of adenovirus vectors containing serotype 35 fibers for tumor targeting. Cancer Gene Ther 2006; 13: 1072–1081. Noureddini SC, Curiel DT. Genetic targeting strategies for adenovirus. Mol Pharm 2005; 2: 341–347. Parker AL, Nicklin SA, Baker AH. Interactions of adenovirus vectors with blood: implications for intravascular gene therapy applications. Curr Opin Mol Ther 2008; 10: 439–448. Pereboev AV, Nagle JM, Shakhmatov MA et al. Enhanced gene transfer to mouse dendritic cells using adenoviral vectors coated with a novel adapter molecule. Mol Ther 2004; 9: 712–720. Proudfoot O, Apostolopoulos V, Pietersz GA. Receptor-mediated delivery of antigens to dendritic cells: anticancer applications. Mol Pharm 2007; 4: 58–72. Rajecki M, Kanerva A, Stenman UH et al. Treatment of prostate cancer with Ad5/3Delta24hCG allows non-invasive detection of the magnitude and persistence of virus replication in  vivo. Mol Cancer Ther 2007; 6: 742–751. Raki M, Kanerva A, Ristimaki A et al. Combination of gemcitabine and Ad5/3-Delta24, a tropism modified conditionally replicating adenovirus, for the treatment of ovarian cancer. Gene Ther 2005; 12: 1198–1205.

9  Retargeting Adenovirus for Cancer Gene Therapy

153

Raper SE, Chirmule N, Lee FS et al. Fatal systemic inflammatory response syndrome in a ornithine transcarbamylase deficient patient following adenoviral gene transfer. Mol Genet Metab 2003; 80: 148–158. Rea D, Havenga MJ, van Den Assem M et al. Highly efficient transduction of human monocytederived dendritic cells with subgroup B fiber-modified adenovirus vectors enhances transgeneencoded antigen presentation to cytotoxic T cells. J Immunol 2001; 166: 5236–5244. Sadeghi H, Hitt MM. Transcriptionally targeted adenovirus vectors. Curr Gene Ther 2005; 5: 411–427. Schirmbeck R, Reimann J, Kochanek S, Kreppel F. The immunogenicity of adenovirus vectors limits the multispecificity of CD8 T-cell responses to vector-encoded transgenic antigens. Mol Ther 2008; 16: 1609–1616. Segura MM, Alba R, Bosch A, Chillon M. Advances in helper-dependent adenoviral vector research. Curr Gene Ther 2008; 8: 222–235. Shi CX, Hitt M, Ng P, Graham FL. Superior tissue-specific expression from tyrosinase and prostate-specific antigen promoters/enhancers in helper-dependent compared with first-generation adenoviral vectors. Hum Gene Ther 2002; 13: 211–224. Shirakawa T. The current status of adenovirus-based cancer gene therapy. Mol Cells 2008; 25: 462–466. Sonabend AM, Ulasov IV, Tyler MA et al. Mesenchymal stem cells effectively deliver an oncolytic adenovirus to intracranial glioma. Stem Cells 2008; 26: 831–841. Stoff-Khalili MA, Rivera AA, Mathis JM et al. Mesenchymal stem cells as a vehicle for targeted delivery of CRAds to lung metastases of breast carcinoma. Breast Cancer Res Treat 2007; 105: 157–167. Stoff-Khalili MA, Rivera AA, Nedeljkovic-Kurepa A et al. Cancer-specific targeting of a conditionally replicative adenovirus using mRNA translational control. Breast Cancer Res Treat 2008; 108: 43–55. Tang Y, Le LP, Matthews QL et al. Derivation of a triple mosaic adenovirus based on modification of the minor capsid protein IX. Virology 2008; 377: 391–400. Thacker EE, Timares L, Matthews QL. Strategies to overcome host immunity to adenovirus vectors in vaccine development. Expert Rev Vaccines 2009; 8: 761–777. Tsuruta Y, Pereboeva L, Breidenbach M et al. A fiber-modified mesothelin promoter-based conditionally replicating adenovirus for treatment of ovarian cancer. Clin Cancer Res 2008; 14: 3582–3588. Tyler MA, Ulasov IV, Sonabend AM et al. Neural stem cells target intracranial glioma to deliver an oncolytic adenovirus in vivo. Gene Ther 2009; 16: 262–278. Ulasov IV, Rivera AA, Han Y et al. Targeting adenovirus to CD80 and CD86 receptors increases gene transfer efficiency to malignant glioma cells. J Neurosurg 2007; 107: 617–627. Waehler R, Russell SJ, Curiel DT. Engineering targeted viral vectors for gene therapy. Nat Rev Genet 2007; 8: 573–587. Wang G, Li G, Liu H et al. E1B 55-kDa deleted, Ad5/F35 fiber chimeric adenovirus, a potential oncolytic agent for B-lymphocytic malignancies. J Gene Med 2009; 11: 477–485. Weaver EA, Nehete PN, Buchl SS et al. Comparison of replication-competent, first generation, and helper-dependent adenoviral vaccines. PLoS One 2009; 4: e5059. White E. Regulation of the cell cycle and apoptosis by the oncogenes of adenovirus. Oncogene 2001; 20: 7836–7846. Yang ZR, Wang HF, Zhao J et al. Recent developments in the use of adenoviruses and immunotoxins in cancer gene therapy. Cancer Gene Ther 2007; 14: 599–615. Zabaleta A, Llopiz D, Arribillaga L et al. Vaccination against hepatitis C virus with dendritic cells transduced with an adenovirus encoding NS3 protein. Mol Ther 2008; 16: 210–217.

Chapter 10

Lentiviruses: Vectors for Cancer Gene Therapy Yuan Lin, Amar Desai, and Stanton L. Gerson

Abstract  Lentivirus are the most efficient viral gene transfer vectors. Partitioned engineered backbones containing the essential proteins needed for reverse transcription and integration and separate elements for the transgene payload provide a 3 or 4 safety designed components that when transduced into transient producer cells yield high titre vectors. Applications of these vector systems have been designed for application for suicide gene therapy using thymidine kinase, immunotherapy and vaccine development, gene replacement and gene silencing including RNAi, anti-angiogenesis, an myelosuppression protection studies are discussed. Most of these efforts have moved from basic concept through preclinical testing and many are in early phase clinical trials. Lentiviral backbones remain a very promising approach to safe and stable gene transfer. Keywords  Lentivirus • miRNA • inducible promoters • suicide gene therapy • MGMT gene therapy

1 Introduction It was about four decades ago when researchers began to first consider the possibility that genetic diseases could potentially be reversed by correcting defective genes (Friedmann 1992). As the idea developed in the early 1980s, the practice of gene therapy began albeit with early misses and delays (Scollay 2001). The rise to prominence was due to the notion that gene therapy could replace pharmaceuticals in medical treatments. The initial goal for gene therapy was to treat monogenic diseases by supplying patients with a healthy gene to compensate for the defective gene.

S.L. Gerson (*) Center for Stem Cell and Regenerative Medicine, Case Comprehensive Cancer Center, Ireland Cancer Center, University Hospitals Case Medical Center and Case Western Reserve University, Wearn 151, 11100 Euclid Avenue, Cleveland, OH 44106, USA e-mail: [email protected] J.A. Roth (ed.), Gene-Based Therapies for Cancer, Current Cancer Research, DOI 10.1007/978-1-4419-6102-0_10, © Springer Science+Business Media, LLC 2010

155

156

Y. Lin et al.

However, early on, cancer seemed like an obvious target especially for proof of concept and assessments of toxicity (Mulligan 1993; Rosenberg et al. 1990). This is shown by the fact that about two-thirds of all gene therapy clinical trials measured in 2007 was targeting cancer (Edelstein et al. 2007). In order to generate an effective and safe vector for gene therapy, both nonviral and viral vectors have been extensively studied and used in clinical trials. There are limitations in each of the vectors, non-viral and some viral vectors efficiently transfect the cells but do not integrate their genetic payload into the genome of targeting cells, resulting in transient expression. On the other hand, gamma-retroviruses, lentiviruses, and adeno-associated viruses are able to integrate their transgenes into targeting cells to ensure long-term expression with various degrees of efficiency. Among those integrated viral vectors, lentiviral vectors have the largest payload capacity for packaging of 8–10 kb-long, compared to 8 and 5 kb size limits in gamma-retroviral vectors and adeno-associated viral vectors, respectively. While retroviral and lentiviral vectors efficiently integrate, less than 10% adeno-associated viral vectors are integrated into targeting cells (Thomas et  al. 2003). A limitation of retroviruses is that they can only transduce actively dividing cells because the integration step requires the breakdown of the nuclear membrane during infection (Miller et al. 1990; Roe et al. 1993). In the early days of gene therapy, lentiviruses, such as the human immunodeficiency virus (HIV-1), were not considered a possible gene transfer vector for clinical use because of their pathogenicity in humans. The risks appeared to be too great to consider the benefits. However, sophisticated interrogation of the lentiviral genome and identification of the essential elements for biology versus integration have yielded effective and safe vectors for clinical use. Two key observations have further enabled this effort. First, contrary to gamma-retroviruses, lentiviruses are able to transduce nondividing, quiescent cells, such as quiescent stem cells, neuronal cells, and other terminally differentiated cells (Lewis and Emerman 1994; Naldini et al. 1996). Second, clinical trials with gamma-retroviral vectors faced a major setback when an X-linked severe combined immunodeficiency (X-SCID) trial in France resulted in patients developing leukemia. This resulted in the suspension of retroviral gene therapy trials (Check 2005; HaceinBey-Abina et  al. 2003a). The mechanism of oncogenesis in these patients was shown to be the insertion and activation of LMO2 T-cell oncogene by the gammaretroviral vectors used in the trial (Hacein-Bey-Abina et al. 2003b; McCormack et  al. 2003). The explanation appears to be that gamma-retroviruses prefer the integration in open regions of DNA, typically near promoters whereas lentiviruses have a preference to integrate into active transcription without a bias for promoter regions, thus significantly reducing the risk of insertional mutagenesis (Modlich and Baum 2009). In this chapter, we discuss lentiviruses, the development of lentiviral vector system, various lentiviral applications in treating cancer, and the current progress with lentiviral vector clinical trials. We will focus more on the HIV-1 derived vector, which is currently the most widely used lentiviral vector.

10  Lentiviruses: Vectors for Cancer Gene Therapy

157

2 Lentiviruses Lentivirus belongs to the family retroviridae and has a characteristic of long clinical latency after initial infection. The genome of HIV-1 has been extensively studied in the past three decades because of its clinical relevance and its potential role as a gene therapy vector. Even though a vast amount of questions remain unanswered, the basic functions of HIV-1 viral proteins are known.

2.1 Lentiviral Genome and Structure Each HIV-1 particle has two identical copies of a single positive 9 kb RNA strand, and each RNA strain contains 9 open reading frames (ORF), which encode for 15 viral proteins (Fig.  1a). Three of the nine ORFs encode for common retroviral a

Vpr SD Vif Gag

RRE

Nef HIV-1 provirus

Env

Pol

5′ LTR

3′ LTR Tat Rev Vpu

b

Envelope (gp120 & gp41)

Capsid

Lipid membrane Matrix

RNA

Fig. 1  HIV-1 provirus and viral particle. (a) Wild-type HIV-1 provirus. (b) HIV-1 viral particle

158

Y. Lin et al.

proteins: gag (MA, CA, NC and p6), pol (protease, reverse transcriptase and integrase), and env (SU and TM). These are viral capsid proteins that are viral enzymes necessary for the reverse transcription and integration steps, and envelope glycoprotein to form new viral particles. The remaining six unique lentiviral proteins are accessory and regulatory proteins: Vif, Vpr, Vpu, Nef, Tat, and Rev (Frankel and Young 1998). The function of each of these accessory proteins has been determined, and each has been shown to affect the production of new lentiviral particles and their virulence. Viral infectivity factor (Vif) plays an important role in the production of infectious viruses. Viruses with mutated Vif are unable to produce infectious viruses because of a cell factor produced in certain cells (Cohen et al. 1996), and this factor has been identified as APOBEC3G, which can impair the infectivity of HIV viruses (Mariani et al. 2003; Sheehy et al. 2002). Viral protein R (Vpr) contains NLS (nuclear localization signal) and directs the preintegration complex (PIC) to the nucleus without the breakdown of nuclear membrane, allowing the infection of nondividing cells. Vpr is also able to induce cell cycle arrest in G2 phase to increase viral transcription and protein production. Additionally, overexpression of Vpr can eliminate T-cells through apoptosis (Emerman 1996b). Viral protein U (Vpu) also has multiple functions. It is shown to downregulate CD4 cells on host cells, interact with the cellular restriction factor tetherin, and play a critical role in the virion budding process (Nomaguchi et  al. 2008). Negative factor (Nef) also impairs the host immune response by downregulating CD4 and MHC class I expression (Piguet and Trono 1999). Two regulatory proteins, Tat and Rev, are critical in transcription elongation and viral RNA export from the nucleus. Regulator of expression (Rev) binds to the Rev responsive element (RRE) of the RNA to transport viral mRNA or unspliced viral genomic RNA out of the nucleus for translating viral proteins and packaging new viral particles (Pollard and Malim 1998). Tat binds to the 5¢ end of all nascent viral mRNA and enhances transcription, and Tat is also released to extracellular environment (Giacca 2004). Within a mature HIV-1 particle, two copies of viral genomic RNA surrounded by nucleocapsid proteins are enclosed in a conical shell formed by capsid proteins along with several viral enzymes (protease, reverse transcriptase, and integrase), p6, and three accessory proteins, Vif, Vpr, and Nef (Briggs et  al. 2004). The viral capsid is wrapped inside a lipid membrane in which viral matrix proteins cover the inner side of the lipid membrane, and viral envelope proteins localize across and outside of it (Fig. 1b).

2.2 Lentiviral Life Cycle Once the HIV-1 viral particles bind to CD4 receptors on target cells, the viral surface protein gp120 undergoes a conformational change to reveal secondary binding sites for coreceptors CCR5 or CXCR4 (Michael and Moore 1999). After the binding of gp120 to CD4 and the coreceptor, further conformational changes result in

10  Lentiviruses: Vectors for Cancer Gene Therapy

159

the fusion of the viral and cell membranes, which causes the release of the viral core into the target cells. This HIV-1 entry step has recently been contested. Miyauchi et al. (2009) suggested that the fusion of the HIV-1 membrane happens at the endosome instead of at the cell surface. After viral core gains entry into the cell, it uncoats itself and forms the reverse transcription complex. With the presence of viral reverse transcriptase in the uncoated viral core, single stranded RNA molecule is transcribed into double stranded cDNA, which can then be integrated into the host genome. Reverse transcription starts when tRNAlys3, presented in both the cell cytoplasm and viral core (Abbink and Berkhout 2008), binds to the primer binding site (PBS) on the 5¢ end of the viral genome. After the binding, a small fragment of cDNA is synthesized, termed the negative-strand strong-stop cDNA. This cDNA fragment then dissociates from the RNA–DNA complex and undergoes first strand transfer, in which the cDNA binds to the 3¢ end of the viral genome and acts as the primer for the negative strand cDNA synthesis. Once the negative strand cDNA is generated, the viral genomic RNA is degraded by RNase H, except at two polypurine tracts (PPT). One locates in the central region called the cPPT, and the other locates at the 3¢ end of viral genome. These two PPT allow for the synthesis of positive strand cDNA. The fragment of positive strand cDNA beginning from the 3¢ PPT undergoes second strand transfer, in which the cDNA fragment binds to the PBS region on the negative strand cDNA. The complete synthesis of both strands results in the final desired product of double strand cDNA. At the completion of the synthesis, there remains a 99-nucleotide overlap sequence at the center of the positive strand named the central DNA flap. This central DNA flap is critical for nuclear import of the PIC in nondividing cells, it is eventually removed by cellular endonuclease (Charneau et al. 1994). The PIC is moved to the nucleus through actions of the cellular cytoskeleton (McDonald et al. 2002). The unique feature of lentiviral vectors being able to transduce nondividing cells is due to the entry of PIC into the nucleus through formation of a nucleoprotein complex without the breakdown of the nuclear membrane. Once the PIC enters the nucleus, provirus is integrated into the cellular genome by viral integrase (Stevenson et  al. 1990). Both spliced viral mRNAs and unspliced viral genomic RNA are regulated by the Rev protein. After viral proteins are synthesized, along with the new viral RNA genome, new viral particles are generated and released from cell surface by budding.

3 HIV-1 Derived Lentiviral Vector HIV-1 is the most studied lentivirus, and it was first considered as a gene transfer vector in the 1990s. However, because of its ability to impair host immune system, lentiviral vector derived from HIV-1 required thorough examination and vigorous testing to ensure its biosafety. Compared with gamma-retroviruses, lentiviruses are unique in that they are able to transduce nondividing cells. Another important

160

Y. Lin et al.

feature is the nonbiased proviral integration site. Lentiviruses have been shown to integrate into transcriptional hot spots (Schroder et  al. 2002), which naturally increases concerns about the risk of insertional mutagenesis. However, further studies have shown that lentiviral vector has different integration patterns compared to gamma-retroviral vector (Cattoglio et al. 2007). While gamma-retroviruses like to integrate near transcription start sites and CpG islands (Wu et al. 2003), dramatically increasing the chance of insertional mutagenesis, gene activation, and enhancer-like function. Lentiviruses tend to integrate into active transcriptional units (Mitchell et  al. 2004; Schroder et  al. 2002). Thus lentiviruses have less chance to activate protooncogenes. However, this fact does not necessarily diminish the risk of inactivating a tumor suppressor gene or disrupting another important gene function. The general strategy for a safe lentiviral vector is similar to that followed for gamma-retroviral vector design, namely, separating cis-acting sequences (viral noncoding elements necessary for RNA synthesis, packaging, reverse transcription, and integration) from trans-acting sequences (viral enzymes, along with structural and accessory proteins).

3.1 Lentiviral Vector Packaging System To avoid the generation of replication-competent lentiviruses (RCL), trans-acting elements are put on separate plasmids. gag and pol sequences are put together in the packaging plasmid, and pseudotyping glycoprotein is put in envelope plasmid (Fig. 2b). Over the course of lentiviral development, there have been three generations of packaging systems evolved with increasing safety in each generation (Fig.  2a). The first generation packaging plasmid includes the entire gag and pol sequences, as well as all of the viral accessory genes and regulatory genes. To ensure that it only expresses viral proteins and enzymes for viral packaging, the packaging signal and the PBS were removed, and viral long terminal repeat (LTR) promoter was replaced with CMV promoter. Poly A tail was also added to the 3¢ end of packaging plasmid. In the secondgeneration packaging system, with more understanding of viral genes for infectivity and virulence, four accessory genes (vif, vpr, vpu and nef) were also removed without affecting viral titer and infectivity (Gasmi et al. 1999; Zufferey et al. 1997). The third generation packaging system put the regulatory gene, rev, on another separate plasmid to increase biosafety, and further remove tat by replacing the 5¢ LTR with a constitutively active promoter in the transfer vector (Dull et al. 1998). In combination with deletion in U3 region from 3¢LTR in SIN vector, viral LTR can be completely eliminated, thus further reducing the genotoxic potential of viral LTR (Montini et al. 2009).

3.2 Design and Improvement of Lentiviral Transfer Vector The transfer vector is the only genetic material to be packaged into lentivirus and integrated into targeting cell. To avoid the possible genetic recombination with

10  Lentiviruses: Vectors for Cancer Gene Therapy

a

Vpr

SD Vif CMV

161

Gag

RRE

Nef poly A

Pol Tat Rev Vpu

SD

RRE Gag

CMV

poly A

Pol Tat Rev

SD RRE CMV

Gag

REV

RSV

poly A

Pol

poly A

b CMV

c

VSVG

SD

poly A

Envelope plasmid

SA miRNAs

RSV R U5 RRE 5′ LTR

cPPT/ Promoter CTS

Transgene

∆ U3 R U5 Transfer vector plasmid

WPRE 3′ LTR

Fig.  2  Schematic representation of lentiviral vector system. (a) Three generations of lentiviral packaging systems. (b) Pseudotyping envelope plamsid. (c) Improved lentiviral gene transfer vector. RSV Rous Sarcoma Virus promoter, SD splice donor, SA splice acceptor, y packaging signal, RRE Rev response element, cPPT central polypurine tract, WPRE woodchuck hepatitis virus posttranscriptional regulatory element

wild-type lentiviruses, minimum viral genome is included in the transfer vector plasmid. Thus the lentiviral vector only consists of an expression cassette, viral cis elements, and the transgene (Fig. 2c). Several improvements have been developed over the years to make the transfer vector safer and lead to more robust transgene expression. For instance, as described above, the U3 region of the 5¢ LTR was replaced with the CMV or RSV promoter, resulting in Tat-independent transcription (Dull et  al. 1998; Kim et  al. 1998). Additionally, the self-inactivating (SIN)

162

Y. Lin et al.

lentiviral vector was a major breakthrough in vector design. It was first developed at the Salk Institute in 1998 (Miyoshi et al. 1998) and was important because it removed part of the enhancer/promoter sequence in the U3 region, thus making the LTR inactive during transgene expression. The deleted portion of the U3 region included the TATA box and the binding sites for the transcription factors Sp1 and NF-kB. The deletion does not decrease viral titer but is beneficial for safety because it even further minimizes RCL generation by reducing common viral genome with wild-type HIV-1. It also decreases the chances of host gene activation around the insertional site. Other improvements that have been made are the incorporation of the cPPT element and CTS pol gene, both of which are important in forming DNA flaps and facilitating PIC entry to the nucleus (Follenzi et al. 2000; Zennou et al. 2000), especially when transducing nondividing cells. These developments have been shown to dramatically increase transduction efficiency (Cockrell and Kafri 2007). Another important improvement in transfer vector was the addition of the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). WPRE is added to the 3¢ side of transgene, and has been shown to enhance mRNA transcript stability and increase the overall transgene expression (Popa et al. 2002; Zufferey et al. 1999). One of the ideal goals for lentiviral gene therapy is to express the transgene only in target cells while avoiding nonspecific infection. Recent developments have incorporated cellular endogenous miRNA to regulate the transgene expression from lentiviral vectors (Brown and Naldini 2009; Kelly and Russell 2009). MicroRNAs are small noncoding RNAs approximately 22 nucleotides in length that are being shown to play an important role in the natural cellular mechanism of downregulating gene expression (Ambros 2001; He and Hannon 2004). Several studies have shown effective suppression of gene expression in certain cells with the lentiviral vector containing sequences matching endogenous miRNA (Brown et  al. 2006, 2007a). When transduced cells express endogenous miRNA, transgene expression is repressed (Fig. 3b). However, careful consideration needs to be made so as to not overwhelm the endogenous microRNA system and interfere with its physiological role in target cells. Brown et al. recently published a review on how to select candidate microRNAs and design microRNA target sites (Brown and Naldini 2009). A more direct way to control transgene expression in specific tissue is through the use of a tissue-specific promoter (Endo et al. 2008; Pariente et al. 2007). Alternative methods also include using a LoxP-flanked sequence for conditional Cre-Lox regulated transgene expression (Chang and Zaiss 2003; Ventura et al. 2004) and using an inducible promoter, such as tetracycline inducible system (Kafri et  al. 2000; Shin et al. 2006; Vigna et al. 2002) (Fig. 3a). Cellular antiviral mechanisms may adversely influence transgene expression. Researchers have worked around this by adding insulator, such as chicken beta-globin locus cHS4 insulator, flanking the transgene in the transfer vector. In this way, transgene expression can be isolated from the influence of surrounding cellular genes (Arumugam et al. 2007). By the same token, this is yet another safety mechanism designed to prevent the activation of nearby genes by proviral integration (Evans-Galea et al. 2007).

10  Lentiviruses: Vectors for Cancer Gene Therapy

163

a

b

Fig.  3  Regulated transgene expression vectors. (a) Four regulated lentiviral vectors: miRNA regulated transfer vector, tissue specific promoter transfer vector, Cre-LoxP transfer vector, and inducible transfer vector. (b) miRNA regulated transfer vector can only express transgene in the cells that lack complementary endogenous miRNA. In nontargeted cells, endogenous miRNA degrades transgene mRNA, suppressing transgene expression

3.3 Non-HIV-1 Derived Lentiviral Vectors Because of the pathogenicity of HIV-1, non-HIV lentiviral vectors were suggested (Emerman 1996a). The other two primate lentiviruses, HIV-2 and SIV, have been developed as gene transfer vectors with limited success (D’Costa et al. 2003; Gilbert and Wong-Staal 2001; Salani et al. 2005). Nonprimate lentiviruses were also considered, such as FIV (Poeschla et al. 1998; Saenz and Poeschla 2004), EIAV (Olsen 1998), BIV (Molina et al. 2002), and CAEV (Mselli-Lakhal et al. 2006). Among those nonprimate lentiviral vectors, FIV and EIAV were commonly used. Their payload capacity is only around 7–8 kb (Valori et al. 2008). Non-HIV lentiviral vectors are considered to be safer for humans because they cause less or no pathogenic damage to humans in their parental forms, and these lentiviral vectors can reduce the chance of recombination

164

Y. Lin et al.

with wild-type HIV-1. As lentiviruses, they can efficiently transduce nondividing cells as well. However, because they are not pathogenic in human, their basic biology is less studied. In order to generate safe and efficient lentiviruses, similar split-genome strategy was also used to develop non-HIV-1 lentiviral vectors inspired by HIV-1 lentiviral vector development. Viral accessory genes were moved to separate packaging vectors. Improvements, such as WPRE element and SIN vector, were also applied in non-HIV lentiviral vectors (Valori et al. 2008). For safety concerns, non-HIV lentiviral vectors are also made in human 293T cells. Successful gene transfer with non-HIV lentiviral vectors has been achieved; nevertheless, for those non-HIV lentiviral vectors to be widely used in clinical setting, more extensive understanding in their basic biology and their interaction with human is warranted.

3.4 Pseudotyping Lentiviral Vector In order to broaden the tropism of lentiviral vectors outside of their native target cells, the viral envelope is replaced with different viral glycoproteins in a process called pseudotyping. This allows for the vector to extend its range of targets to various cell types in different species. The most widely used viral glycoprotein is vesicular stomatitis virus G glycoprotein (VSVG) because of its very broad tropism and stable pseudotyped viral particle (Burns et  al. 1993; Cronin et  al. 2005), which allows for further concentration of viruses by ultracentrifugation. In contrast to the traditional proposed mechanism of wild-type HIV-1 entry, VSVG pseudotyped lentiviral vectors enter the cell via an endocytic pathway (Aiken 1997). However, VSVG is cytotoxic to most mammalian cell lines, which makes it harder to generate lentiviral producer cell lines (Burns et al. 1993; Ory et al. 1996). In addition, VSVGpseudotyped HIV and FIV vectors can be targeted and eliminated by host immune responses and inactivated by human serum complement, which makes the use of VSVG pseudotyped lentiviral vectors difficult for certain in  vivo clinical applications, especially for direct intravenous injection (DePolo et al. 2000; Higashikawa and Chang 2001). This inactivation can potentially be avoided by PEGylation of VSVG pseudotyped lentiviruses and can protect the virus from inactivation in the serum and also improve the transduction efficiency (Croyle et al. 2004). Alternative viral glycoproteins derived from other viruses, such as lyssavirus (Desmaris et al. 2001), lymphocytic choriomeningitis virus (Beyer et  al. 2002), hepatitis C virus (Flint et al. 2004), and sendai virus (Kowolik and Yee 2002), can also be used for pseudotyping lentiviral vectors (Breckpot et al. 2007; Cronin et al. 2005).

3.5 Production of Lentiviral Vector In most current studies, lentiviral vectors are generated through transient cotransfection of three or four different plasmids in human embryonic kidney 293T cells, depending on the packaging system. The plasmids transfected include the packaging

10  Lentiviruses: Vectors for Cancer Gene Therapy

165

Ultracentrifugation Transient transfection

Viral protein Ultrafiltration Viral supernatant

Transfer vector plasmid Packaging plasmid Envelope plasmid

Precipitation

Fig. 4  Lentiviral production and concentration. Transfer vector plasmid, packaging plasmid, and envelope plasmid are transient transfected into 293T cells, and viral supernatant is collected and concentrated with ultracentrifugation, ultrafiltration, or precipitation

plasmid, the envelope plasmid, and the transfer vector plasmid (Fig. 4). The commonly used packaging system in both preclinical and clinical studies is the second generation packaging system with SIN lentiviral transfer vector. In order to avoid the production of empty viral particles, the plasmid ratio of packaging plasmid: transfer vector plasmid: envelope plasmid is 3:3:1. Forty-eight to seventy-two hours post transfection, lentiviral particles released into the media from 293T cells are collected and filtered through a 0.22-mm filter to remove cell debris. The titer of lentiviral vector produced by transient transfection usually has a range from 106 to 107 IU/ml. In order to obtain clinical grade lentiviruses with reproducibility and standardization, either large-scale transient production or stable packing cell lines are needed. Herein lays a difficulty because stable packaging cell lines for lentiviruses are hard to acquire. This is due to the cytotoxicity of lentiviral proteins and the pseudotype envelope protein VSVG (Li et  al. 1995; Miyazaki et  al. 1995; Patel et  al. 2000; Philippon et al. 1994). Several labs have tried to generate stable cell lines for lentiviral production (Kafri et al. 1999; Klages et  al. 2000; Kuate et  al. 2002; Strang et al. 2005; Xu et al. 2001). So far stable producer cell lines could be achieved only with an inducible expression system for the controlled expression of viral proteins and VSVG (Farson et al. 2001; Xu et al. 2001). High multiplicity of infection (MOI) studies and clinical studies requiring large quantities and high grade may need to further concentrate lentiviruses to increase the titer. Several methods are currently available for concentrating lentiviruses depending on the production levels desired. Ultracentrifugation and ultra filtration of viral supernatant are more common for making large-scale production and concentration (Coleman et al. 2003; Sena-Esteves et al. 2004), while small-scale concentration through precipitation can be done with calcium phosphate or poly-l-lysine (Pham et al. 2001; Zhang et al. 2001). Lentiviral vector supernatant can be stored in −80°C; however, the freeze-thaw cycle should be kept at minimum because it can decrease viral titer by 60% (Higashikawa and Chang 2001; Kwon et al. 2003).

166

Y. Lin et al.

4 Applications of Lentiviral Vector in Cancer Therapy There are two reasons that cancer has been a target for lentiviral gene transfer technology. First, proof-of-concept gene transfer efforts can be validated with an intention to treat advanced malignancies, and second, there are a number of targets in cancer for which there is no effective agent or treatment (Lowenstein 1997). Non-viral and viral vectors have been extensively investigated for cancer therapy with limited success. In one study, lentiviral vectors were shown to transduce human hematopoietic progenitor cells in the G1, G2/S/M phases more efficiently than those cells in the G0 phase (Sutton et al. 1999). Coincidentally, quantitative analysis of G0 and G1 phases in primary carcinomas showed that over 50% of primary cancer cells were blocked in transition in G1 and thus were not in G0 or the quiescent state (Tay et al. 1991). Lentiviral vectors seem to have a better chance at transducing cancer cells at different cell-cycle phases and thus are susceptible to lentiviral-mediated gene transfer. Below we will discuss several strategies currently employed using lentiviral vectors to treat cancer. These are suicide gene therapy, immunotherapy, gene replacement and downregulation, antiangiogenesis therapy, and myeloprotection.

4.1 Suicide Gene Therapy The concept of suicide gene therapy is to give cancer cells a suicide gene that converts a nontoxic prodrug to a cytotoxic drug, which kills cancer cells (Chang and He 2001). Commonly used suicide genes are herpes simplex virus thymidine kinase (HSV-TK) and cytosine deaminase (CD). Studies have shown that systemic administration of gancyclovir can be converted to cytotoxic gancyclovir triphosphate in the presence of HSV-TK when treating glioma cells and prostate carcinoma cells (Culver et al. 1992; Loimas et al. 2001). Small numbers of transduced tumor cells can also result in the killing of nontransduced surrounding tumor cells, a phenomenon called the “bystander effect” (Burrows et al. 2002; Hamel et al. 1996; Rubsam et al. 1999). Recent developments have shown that using suicide gene therapy in stem cells can have an impact on killing surrounding cancer cells (Huang et  al. 2005; Rath et al. 2009). Other suicide genes are also being developed. For instance, engineered human thymidylate kinase (tmpk) was introduced in cell lines with a lentiviral vector to make transduced cancer cells sensitive to 3¢-azido-3¢deoxythymidine (AZT). This led to the suppression of tumor growth in NOD/SCID mice (Sato et al. 2007).

4.2 Lentiviral Immunotherapy for Cancer One of the characteristic advantages of cancer cells is the ability to evade immune surveillance in the host (Bronte and Mocellin 2009; Foss 2002; Seliger 2005).

10  Lentiviruses: Vectors for Cancer Gene Therapy

167

APC Direct injection

LenIvirus with TAA

TAA

Presentation Infusion ex vivo transduction T cells

Dendritic cell

DC presenting TAA

Fig.  5  Lentiviral immunotherapy for cancer. Lentivirus, encoding for tumor-associated antigen (TAA), can be directly injected into patient or transduce dendritic cells ex vivo. Transduced DC can be infused back to patient to directly present to CD8 T cells, initiating targeting and the destruction of cancer cells

By identifying and introducing tumor-associated antigens (TAA) to the host immune system, a specific immune response against TAA can be initiated which could recognize and eliminate tumor cells (Boon and van der Bruggen 1996). Lentiviral vector can efficiently introduce TAA to the host immune system (Fig. 5). One approach is to inject lentiviral vector expressing TAA directly to the host to induce antitumor responses in vivo, targeting antigen-presenting cells (APC). VandenDriessche et al. (2002) showed that in vivo injection of lentiviral vectors could efficiently transduce APC in the spleen. He et al. (2006) also showed direct transfection of skin-derived dendritic cells (sDC) after the cutaneous delivery of lentiviral vectors, which resulted in potent and prolonged antigen presentation. However, the parental HIV-1 has been shown to induce both the cellular and antibody-mediated response (Humbert and Dietrich 2006; Norris and Rosenberg 2001). Recent studies have also shown that there is a host immune response toward lentiviral vectors (Brown et al. 2007b; Pichlmair et  al. 2007). It has also been shown that lentiviral vectors can efficiently transduce DC ex vivo and lead to the expression of transgenic protein (Dyall et al. 2001; Esslinger et al. 2003; Metharom et al. 2001; Schroers et al. 2000; Unutmaz et al. 1999). Transgene-derived peptides can be efficiently expressed on the cell surface of DC and lead to the activation of antigen-specific T cells (Firat et al. 2002; Sumimoto et al. 2002). In vivo studies showed that lentiviral transduced DC can efficiently inhibit established tumors and are effective against subsequent tumor challenges (Dullaers et al. 2006; He et al. 2005).

168

Y. Lin et al.

4.3 Gene Replacement and Gene Silencing Carcinogenesis occurs largely due to the loss of tumor suppressor genes and the activation of oncogenes, and great effort has been made to identify both classes of genes (Bishop 1991; Weinberg 1991). Tumor growth can contribute to the loss of a single tumor suppressor gene but by introducing a wild-type tumor suppressor gene expressed at normal levels, cellular control mechanisms can be restored and the cancer growth can be arrested. Abnormalities in the TP53 gene family have been documented in a very large number of human cancers (Benard et al. 2003; Caron de Fromentel and Soussi 1992), and studies have shown that restoring the wild-type p53 gene in human lung cancer cell lines with a gamma-retroviral vector led to significant growth arrest and apoptosis in cancer cell lines lacking p53 (Cai et al. 1993; Goyette et  al. 1992). In another study, stable lentiviral transduction of HCT116 colon cancer cells with wild-type p53 gene sensitized the transduced cells to chemotherapeutic agents (Kaeser et al. 2004). In addition to introducing tumor suppressor gene, another strategy is to use gene transfer vectors to knockdown activated oncogenes. Today, RNAi is a vastly used mechanism for posttranscriptional gene silencing in experimental system (Hammond et  al. 2000; Zamore et  al. 2000). Compared to oligonucleotide siRNA, lentiviral vectors can efficiently deliver short hairpin RNA (shRNA) to cancer cells for stable integration and consistent expression of the transgene. When shRNA is released into the cytoplasm, the linker sequence is degraded by Dicer to form siRNA, which subsequently targets the appropriate mRNA for degradation (Miyagishi et al. 2004; Sumimoto and Kawakami 2007). The ras family is among the most activated oncogenes in human cancers and thus a natural target for cancer therapeutics (Chardin 1988; Nottage and Siu 2002; Young et  al. 2009). Brummelkamp et  al. (2002) showed stable integration and expression of siRNA against the K-RASv12 allele in human tumor cells using retrovirus, and this resulted in a loss of anchorageindependent growth and tumorigenicity. Efficient RNAi silencing requires high copy numbers of siRNA. However, a drawback is that it can also lead to nonspecific gene silencing (Jackson et al. 2003). To optimize the specificity for gene silencing, multiple siRNA sequences linked in one transgene targeting one mRNA have been developed (Sumimoto et  al. 2006a, b). In addition, tissue-specific promoters can further decrease the chance of nonspecific silencing by localizing the effect (Xia et al. 2002). A detailed review of RNAi in cancer therapy can be found in chapter 1 of this book.

4.4 Antiangiogenesis Therapy For tumors to grow, angiogenesis is essential and critical. New blood vessels around tumors provide the routes for delivering nutrients and can also be the traveling vessels for metastatic cancer cells (Folkman 1990). Endogenous inhibitors of angiogenesis, including angiostatin and endostatin, have been shown to reduce

10  Lentiviruses: Vectors for Cancer Gene Therapy

169

tumor size and maintain tumor dormancy without increasing drug resistance in mice (Bergers et al. 1999; Boehm et al. 1997; O’Reilly et al. 1994, 1997). However, the requirements for long-term administration and large amounts of protein put a limitation on this potentially effective treatment (Cao 1999). Lentiviral vectors offer a solution that allows the cells to constantly express those antiangiogenic factors. Shichinohe et al. (2001) were the first to test third generation HIV-1-derived SIN lentiviral vectors expressing angiostatin or endostatin directly in endothelial cells; however, the efficiency was low, and the inhibition on cell growth was not significant. In another study, a lentiviral vector containing endostatin has been shown to decrease vascularization and inhibit tumor growth in lentiviral-transduced bladder tumor cells (Kikuchi et al. 2004).

4.5 Myeloprotection Against Chemotherapeutics Traditional treatments for cancer include radiotherapy and chemotherapy, which have severe side effects on patients. Chemotherapeutic drugs are particular toxic to bone marrow cells, causing hematopoietic cytopenia, and limit the necessary dose of chemotherapeutics needed to kill cancer cells (Gerson 2002). Instead of directly targeting and eliminating cancer cells, strategies have been developed to protect hematopoiesis in patients from cancer treatment with lentiviral-mediated gene transfer of a drug resistance gene. O6-methylguanine-DNA-methyltransferase (MGMT) is a DNA repair protein, which removes alkyl adducts on the O6 position of guanine. It has been discovered that low MGMT expression is found in bone marrow cells while MGMT over-expression is found in many types of human tumors, making tumor cells more resistant to chemotherapeutics (Erickson 1991; Kaina et al. 2007; Verbeek et al. 2008; Yarosh 1985). The mutant form of MGMT (MGMT-P140K) has been shown to be effectively resistant to O6-benzylguanine (BG), an inhibitor to wild-type MGMT, and also alkylating agents, such as Temozolomide or 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) (Chinnasamy et al. 2004; Zielske and Gerson 2002). By using a lentiviral vector to introduce MGMTP140K into hematopoietic stem cells (HSC), hematopoiesis can be restored, and transduced bone marrow cells can be protected and selected in vivo in preclinical models (Liu and Gerson 2006; Milsom and Williams 2007). MGMT-P140K provides leverage to bone marrow cells over tumor cells expressing wild-type MGMT against BG and chemotherapeutic treatments (Fig. 6).

5 Clinical Trials of Lentiviral Vectors In 2007, adenoviruses and gamma-retroviruses were equally weighted in the number of clinical trials, with each over 20% among all gene transfer clinical trials. During that time the number of studies involving lentiviral vectors were negligible

170

Y. Lin et al. Methyl adduct BG A

T

G

C wt MGMT

A

A

T

G

Cell survival

C

A

T

T

G

G

C

C

double-strand breaks

P140K-MGMT

A

T

G

C

Cell survival

Fig. 6  Myeloprotection with MGMT-P140K. Wild-type MGMT repairs DNA damage caused by alkylating agents. Treatment with benzylguanine (BG) and alkylating agents inactivates wild-type MGMT, resulting in double strand break and cell apoptosis. BG and alkylating agent treatment does not affect MGMT-P140K, which can still repair damaged DNA

(Edelstein et  al. 2007). However, with the increasing interests in research and development of lentiviral vectors, the first lentiviral clinical trial was successfully completed in 2003 with optimistic results (Levine et al. 2006). The first clinical trial was logically designed to treat HIV infection. According to the Journal of Gene Medicine website, updated on March 2009, there are a total of 21 phase I lentiviral clinical trials, and only 4 out of those 21 trials were approved for targeting cancer, and all of the lentiviral vectors are HIV-1 derived (Table  1). Three of four trials directly treat cancer target antigen receptor and T-cell receptors while the fourth clinical trial is for myeloprotection against cancer chemotherapy (see Sect.  4 for mechanism).

6 Conclusion Despite the fact that retroviruses had been shown to induce cancer through the activation of a proto-oncogene three decades ago (Hayward et al. 1981), the development of a safe retroviral vector and the benefits of a stable gene transfer has resulted in the first gamma-retroviral vector gene therapy clinical trial in treating melanoma

HIV-1 derived

HIV-1 derived

US-892

US-898

Glioma

Non-Hodgkin’s lymphoma

CD 19 antigen specific chimeric antigen receptor O6-methylguanine DNA methyltransferase

Table 1  Lentiviral clinical trials (Journal of Experiment Medicine, updated March 2009) Trial ID Vector Condition Gene inserted US-791 HIV-1 derived Malignant melanoma a and b chains of T-cell receptor specific for Mart-1 US-793 HIV-1 derived CD 19+ Leukemia and CD19 specific Zeta T cell lymphoma receptor I

T lymphocytes

I

I

T lymphocytes

CD34+ cells from peripheral blood

Phase I

Target cells T-lymphocytes

Gerson SL, Case Western Reserve University

Porter DL, University of Pennsylvania School of medicine Popplewell L, City of Hope, CA

Principal investigator Ribas A and Economou JS, UCLA

10  Lentiviruses: Vectors for Cancer Gene Therapy 171

172

Y. Lin et al.

(Rosenberg et al. 1990). After 10 years of research and hundreds of retroviral clinic trials, the onset of leukemia in the French clinical trial was a serious setback for retroviral studies and the field of gene therapy as whole. While most of the children benefited with a restoration of immune function, the potential for insertional leukemogenesis became a reality. Other similar studies in treating SCID-X1 with retroviruses have not encountered a similar fate (Gaspar et al. 2004; Ginn et al. 2005). The tremendous benefits for those successfully treated patients in these trials are truly rewarding. Because of their unique abilities to transduce both dividing and nondividing cells and to integrate into the cell genome with a lower risk of insertional mutagenesis, lentiviral vectors are receiving considerable attention in preclinical and clinical trials. Different generations of packaging systems, greatly improved transfer vectors, and pseudotyped viral envelope have made current HIV-1 derived lentiviral vectors safe but efficient. Different strategies of using lentiviral vectors in treating cancer have been developed, and the future in lentivirus-mediated gene therapy is promising. Studies on non-HIV-1 lentiviruses are being conducted in increasing numbers, and soon non-HIV-1 lentiviral vectors can be expected in clinical trials. Despite the great promise lentiviral vectors offer, constant vigilance is required in designing and applying an integrating viral vector as a gene transfer method for the treatment of either malignant or nonmalignant indications.

References Abbink TE, Berkhout B. 2008. HIV-1 reverse transcription initiation: a potential target for novel antivirals? Virus Res 134:4–18 Aiken C. 1997. Pseudotyping human immunodeficiency virus type 1 (HIV-1) by the glycoprotein of vesicular stomatitis virus targets HIV-1 entry to an endocytic pathway and suppresses both the requirement for Nef and the sensitivity to cyclosporin A. J Virol 71:5871–7 Ambros V. 2001. microRNAs: tiny regulators with great potential. Cell 107:823–6 Arumugam PI, Scholes J, Perelman N, Xia P, Yee JK, Malik P. 2007. Improved human beta-globin expression from self-inactivating lentiviral vectors carrying the chicken hypersensitive site-4 (cHS4) insulator element. Mol Ther 15:1863–71 Benard J, Douc-Rasy S, Ahomadegbe JC. 2003. TP53 family members and human cancers. Hum Mutat 21:182–91 Bergers G, Javaherian K, Lo KM, Folkman J, Hanahan D. 1999. Effects of angiogenesis inhibitors on multistage carcinogenesis in mice. Science 284:808–12 Beyer WR, Westphal M, Ostertag W, von Laer D. 2002. Oncoretrovirus and lentivirus vectors pseudotyped with lymphocytic choriomeningitis virus glycoprotein: generation, concentration, and broad host range. J Virol 76:1488–95 Bishop JM. 1991. Molecular themes in oncogenesis. Cell 64:235–48 Boehm T, Folkman J, Browder T, O’Reilly MS. 1997. Antiangiogenic therapy of experimental cancer does not induce acquired drug resistance. Nature 390:404–7 Boon T, van der Bruggen P. 1996. Human tumor antigens recognized by T lymphocytes. J Exp Med 183:725–9 Breckpot K, Aerts JL, Thielemans K. 2007. Lentiviral vectors for cancer immunotherapy: transforming infectious particles into therapeutics. Gene Ther 14:847–62 Briggs JA, Simon MN, Gross I, Krausslich HG, Fuller SD, et al. 2004. The stoichiometry of Gag protein in HIV-1. Nat Struct Mol Biol 11:672–5

10  Lentiviruses: Vectors for Cancer Gene Therapy

173

Bronte V, Mocellin S. 2009. Suppressive influences in the immune response to cancer. J Immunother 32:1–11 Brown BD, Cantore A, Annoni A, Sergi LS, Lombardo A, et al. 2007a. A microRNA-regulated lentiviral vector mediates stable correction of hemophilia B mice. Blood 110:4144–52 Brown BD, Naldini L. 2009. Exploiting and antagonizing microRNA regulation for therapeutic and experimental applications. Nat Rev Genet 10:578–85 Brown BD, Sitia G, Annoni A, Hauben E, Sergi LS, et al. 2007b. In vivo administration of lentiviral vectors triggers a type I interferon response that restricts hepatocyte gene transfer and promotes vector clearance. Blood 109:2797–805 Brown BD, Venneri MA, Zingale A, Sergi L, Naldini L. 2006. Endogenous microRNA regulation suppresses transgene expression in hematopoietic lineages and enables stable gene transfer. Nat Med 12:585–91 Brummelkamp TR, Bernards R, Agami R. 2002. Stable suppression of tumorigenicity by virusmediated RNA interference. Cancer Cell 2:243–7 Burns JC, Friedmann T, Driever W, Burrascano M, Yee JK. 1993. Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells. Proc Natl Acad Sci U S A 90:8033–7 Burrows FJ, Gore M, Smiley WR, Kanemitsu MY, Jolly DJ, et al. 2002. Purified herpes simplex virus thymidine kinase retroviral particles: III. Characterization of bystander killing mechanisms in transfected tumor cells. Cancer Gene Ther 9:87–95 Cai DW, Mukhopadhyay T, Liu Y, Fujiwara T, Roth JA. 1993. Stable expression of the wild-type p53 gene in human lung cancer cells after retrovirus-mediated gene transfer. Hum Gene Ther 4:617–24 Cao Y. 1999. Therapeutic potentials of angiostatin in the treatment of cancer. Haematologica 84:643–50 Caron de Fromentel C, Soussi T. 1992. TP53 tumor suppressor gene: a model for investigating human mutagenesis. Genes Chromosomes Cancer 4:1–15 Cattoglio C, Facchini G, Sartori D, Antonelli A, Miccio A, et  al. 2007. Hot spots of retroviral integration in human CD34+ hematopoietic cells. Blood 110:1770–8 Chang LJ, He J. 2001. Retroviral vectors for gene therapy of AIDS and cancer. Curr Opin Mol Ther 3:468–75 Chang LJ, Zaiss AK. 2003. Self-inactivating lentiviral vectors and a sensitive Cre-loxP reporter system. Methods Mol Med 76:367–82 Chardin P. 1988. The ras superfamily proteins. Biochimie 70:865–8 Charneau P, Mirambeau G, Roux P, Paulous S, Buc H, Clavel F. 1994. HIV-1 reverse transcription. A termination step at the center of the genome. J Mol Biol 241:651–62 Check E. 2005. Gene therapy put on hold as third child develops cancer. Nature 433:561 Chinnasamy D, Fairbairn LJ, Neuenfeldt J, Treisman JS, Hanson JP, Jr., et al. 2004. Lentivirusmediated expression of mutant MGMTP140K protects human CD34+ cells against the combined toxicity of O6-benzylguanine and 1,3-bis(2-chloroethyl)-nitrosourea or temozolomide. Hum Gene Ther 15:758–69 Cockrell AS, Kafri T. 2007. Gene delivery by lentivirus vectors. Mol Biotechnol 36:184–204 Cohen EA, Subbramanian RA, Gottlinger HG. 1996. Role of auxiliary proteins in retroviral morphogenesis. Curr Top Microbiol Immunol 214:219–35 Coleman JE, Huentelman MJ, Kasparov S, Metcalfe BL, Paton JF, et  al. 2003. Efficient largescale production and concentration of HIV-1-based lentiviral vectors for use in vivo. Physiol Genomics 12:221–8 Cronin J, Zhang XY, Reiser J. 2005. Altering the tropism of lentiviral vectors through pseudotyping. Curr Gene Ther 5:387–98 Croyle MA, Callahan SM, Auricchio A, Schumer G, Linse KD, et  al. 2004. PEGylation of a vesicular stomatitis virus G pseudotyped lentivirus vector prevents inactivation in serum. J Virol 78:912–21 Culver KW, Ram Z, Wallbridge S, Ishii H, Oldfield EH, Blaese RM. 1992. In vivo gene transfer with retroviral vector-producer cells for treatment of experimental brain tumors. Science 256:1550–2

174

Y. Lin et al.

D’Costa J, Harvey-White J, Qasba P, Limaye A, Kaneski CR, et al. 2003. HIV-2 derived lentiviral vectors: gene transfer in Parkinson’s and Fabry disease models in vitro. J Med Virol 71:173–82 DePolo NJ, Reed JD, Sheridan PL, Townsend K, Sauter SL, et  al. 2000. VSV-G pseudotyped lentiviral vector particles produced in human cells are inactivated by human serum. Mol Ther 2:218–22 Desmaris N, Bosch A, Salaun C, Petit C, Prevost MC, et al. 2001. Production and neurotropism of lentivirus vectors pseudotyped with lyssavirus envelope glycoproteins. Mol Ther 4:149–56 Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, et al. 1998. A third-generation lentivirus vector with a conditional packaging system. J Virol 72:8463–71 Dullaers M, Van Meirvenne S, Heirman C, Straetman L, Bonehill A, et  al. 2006. Induction of effective therapeutic antitumor immunity by direct in vivo administration of lentiviral vectors. Gene Ther 13:630–40 Dyall J, Latouche JB, Schnell S, Sadelain M. 2001. Lentivirus-transduced human monocytederived dendritic cells efficiently stimulate antigen-specific cytotoxic T lymphocytes. Blood 97:114–21 Edelstein ML, Abedi MR, Wixon J. 2007. Gene therapy clinical trials worldwide to 2007--an update. J Gene Med 9:833–42 Emerman M. 1996a. From curse to cure: HIV for gene therapy? Nat Biotechnol 14:943 Emerman M. 1996b. HIV-1, Vpr and the cell cycle. Curr Biol 6:1096–103 Endo M, Zoltick PW, Peranteau WH, Radu A, Muvarak N, et al. 2008. Efficient in vivo targeting of epidermal stem cells by early gestational intraamniotic injection of lentiviral vector driven by the keratin 5 promoter. Mol Ther 16:131–7 Erickson LC. 1991. The role of O-6 methylguanine DNA methyltransferase (MGMT) in drug resistance and strategies for its inhibition. Semin Cancer Biol 2:257–65 Esslinger C, Chapatte L, Finke D, Miconnet I, Guillaume P, et al. 2003. In vivo administration of a lentiviral vaccine targets DCs and induces efficient CD8(+) T cell responses. J Clin Invest 111:1673–81 Evans-Galea MV, Wielgosz MM, Hanawa H, Srivastava DK, Nienhuis AW. 2007. Suppression of clonal dominance in cultured human lymphoid cells by addition of the cHS4 insulator to a lentiviral vector. Mol Ther 15:801–9 Farson D, Witt R, McGuinness R, Dull T, Kelly M, et al. 2001. A new-generation stable inducible packaging cell line for lentiviral vectors. Hum Gene Ther 12:981–97 Firat H, Zennou V, Garcia-Pons F, Ginhoux F, Cochet M, et al. 2002. Use of a lentiviral flap vector for induction of CTL immunity against melanoma. Perspectives for immunotherapy. J Gene Med 4:38–45 Flint M, Logvinoff C, Rice CM, McKeating JA. 2004. Characterization of infectious retroviral pseudotype particles bearing hepatitis C virus glycoproteins. J Virol 78:6875–82 Folkman J. 1990. What is the evidence that tumors are angiogenesis dependent? J Natl Cancer Inst 82:4–6 Follenzi A, Ailles LE, Bakovic S, Geuna M, Naldini L. 2000. Gene transfer by lentiviral vectors is limited by nuclear translocation and rescued by HIV-1 pol sequences. Nat Genet 25:217–22 Foss FM. 2002. Immunologic mechanisms of antitumor activity. Semin Oncol 29:5–11 Frankel AD, Young JA. 1998. HIV-1: fifteen proteins and an RNA. Annu Rev Biochem 67:1–25 Friedmann T. 1992. A brief history of gene therapy. Nat Genet 2:93–8 Gasmi M, Glynn J, Jin MJ, Jolly DJ, Yee JK, Chen ST. 1999. Requirements for efficient production and transduction of human immunodeficiency virus type 1-based vectors. J Virol 73: 1828–34 Gaspar HB, Parsley KL, Howe S, King D, Gilmour KC, et al. 2004. Gene therapy of X-linked severe combined immunodeficiency by use of a pseudotyped gammaretroviral vector. Lancet 364:2181–7 Gerson SL. 2002. Clinical relevance of MGMT in the treatment of cancer. J Clin Oncol 20:2388–99 Giacca M. 2004. The HIV-1 Tat protein: a multifaceted target for novel therapeutic opportunities. Curr Drug Targets Immune Endocr Metabol Disord 4:277–85 Gilbert JR, Wong-Staal F. 2001. HIV-2 and SIV vector systems. Somat Cell Mol Genet 26:83–98

10  Lentiviruses: Vectors for Cancer Gene Therapy

175

Ginn SL, Curtin JA, Kramer B, Smyth CM, Wong M, et  al. 2005. Treatment of an infant with X-linked severe combined immunodeficiency (SCID-X1) by gene therapy in Australia. Med J Aust 182:458–63 Goyette MC, Cho K, Fasching CL, Levy DB, Kinzler KW, et al. 1992. Progression of colorectal cancer is associated with multiple tumor suppressor gene defects but inhibition of tumorigenicity is accomplished by correction of any single defect via chromosome transfer. Mol Cell Biol 12:1387–95 Hacein-Bey-Abina S, von Kalle C, Schmidt M, Le Deist F, Wulffraat N, et al. 2003a. A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency. N Engl J Med 348:255–6 Hacein-Bey-Abina S, Von Kalle C, Schmidt M, McCormack MP, Wulffraat N, et  al. 2003b. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 302:415–9 Hamel W, Magnelli L, Chiarugi VP, Israel MA. 1996. Herpes simplex virus thymidine kinase/ ganciclovir-mediated apoptotic death of bystander cells. Cancer Res 56:2697–702 Hammond SM, Bernstein E, Beach D, Hannon GJ. 2000. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 404:293–6 Hayward WS, Neel BG, Astrin SM. 1981. Activation of a cellular onc gene by promoter insertion in ALV-induced lymphoid leukosis. Nature 290:475–80 He L, Hannon GJ. 2004. MicroRNAs: small RNAs with a big role in gene regulation. Nat Rev Genet 5:522–31 He Y, Zhang J, Donahue C, Falo LD, Jr. 2006. Skin-derived dendritic cells induce potent CD8(+) T cell immunity in recombinant lentivector-mediated genetic immunization. Immunity 24:643–56 He Y, Zhang J, Mi Z, Robbins P, Falo LD, Jr. 2005. Immunization with lentiviral vector-transduced dendritic cells induces strong and long-lasting T cell responses and therapeutic immunity. J Immunol 174:3808–17 Higashikawa F, Chang L. 2001. Kinetic analyses of stability of simple and complex retroviral vectors. Virology 280:124–31 Huang W, Tan W, Zhong Q, Schwarzenberger P. 2005. Development of a gene therapy based bone marrow purging system for leukemias. Cancer Gene Ther 12:873–83 Humbert M, Dietrich U. 2006. The role of neutralizing antibodies in HIV infection. AIDS Rev 8:51–9 Jackson AL, Bartz SR, Schelter J, Kobayashi SV, Burchard J, et al. 2003. Expression profiling reveals off-target gene regulation by RNAi. Nat Biotechnol 21:635–7 Kaeser MD, Pebernard S, Iggo RD. 2004. Regulation of p53 stability and function in HCT116 colon cancer cells. J Biol Chem 279:7598–605 Kafri T, van Praag H, Gage FH, Verma IM. 2000. Lentiviral vectors: regulated gene expression. Mol Ther 1:516–21 Kafri T, van Praag H, Ouyang L, Gage FH, Verma IM. 1999. A packaging cell line for lentivirus vectors. J Virol 73:576–84 Kaina B, Christmann M, Naumann S, Roos WP. 2007. MGMT: key node in the battle against genotoxicity, carcinogenicity and apoptosis induced by alkylating agents. DNA Repair (Amst) 6:1079–99 Kelly EJ, Russell SJ. 2009. MicroRNAs and the regulation of vector tropism. Mol Ther 17:409–16 Kikuchi E, Menendez S, Ohori M, Cordon-Cardo C, Kasahara N, Bochner BH. 2004. Inhibition of orthotopic human bladder tumor growth by lentiviral gene transfer of endostatin. Clin Cancer Res 10:1835–42 Kim VN, Mitrophanous K, Kingsman SM, Kingsman AJ. 1998. Minimal requirement for a lentivirus vector based on human immunodeficiency virus type 1. J Virol 72:811–6 Klages N, Zufferey R, Trono D. 2000. A stable system for the high-titer production of multiply attenuated lentiviral vectors. Mol Ther 2:170–6

176

Y. Lin et al.

Kowolik CM, Yee JK. 2002. Preferential transduction of human hepatocytes with lentiviral vectors pseudotyped by Sendai virus F protein. Mol Ther 5:762–9 Kuate S, Wagner R, Uberla K. 2002. Development and characterization of a minimal inducible packaging cell line for simian immunodeficiency virus-based lentiviral vectors. J Gene Med 4:347–55 Kwon YJ, Hung G, Anderson WF, Peng CA, Yu H. 2003. Determination of infectious retrovirus concentration from colony-forming assay with quantitative analysis. J Virol 77:5712–20 Levine BL, Humeau LM, Boyer J, MacGregor RR, Rebello T, et al. 2006. Gene transfer in humans using a conditionally replicating lentiviral vector. Proc Natl Acad Sci U S A 103:17372–7 Lewis PF, Emerman M. 1994. Passage through mitosis is required for oncoretroviruses but not for the human immunodeficiency virus. J Virol 68:510–6 Li CJ, Friedman DJ, Wang C, Metelev V, Pardee AB. 1995. Induction of apoptosis in uninfected lymphocytes by HIV-1 Tat protein. Science 268:429–31 Liu L, Gerson SL. 2006. Targeted modulation of MGMT: clinical implications. Clin Cancer Res 12:328–31 Loimas S, Toppinen MR, Visakorpi T, Janne J, Wahlfors J. 2001. Human prostate carcinoma cells as targets for herpes simplex virus thymidine kinase-mediated suicide gene therapy. Cancer Gene Ther 8:137–44 Lowenstein PR. 1997. Why are we doing so much cancer gene therapy? Disentangling the scientific basis from the origins of gene therapy. Gene Ther 4:755–6 Mariani R, Chen D, Schrofelbauer B, Navarro F, Konig R, et al. 2003. Species-specific exclusion of APOBEC3G from HIV-1 virions by Vif. Cell 114:21–31 McCormack MP, Forster A, Drynan L, Pannell R, Rabbitts TH. 2003. The LMO2 T-cell oncogene is activated via chromosomal translocations or retroviral insertion during gene therapy but has no mandatory role in normal T-cell development. Mol Cell Biol 23:9003–13 McDonald D, Vodicka MA, Lucero G, Svitkina TM, Borisy GG, et al. 2002. Visualization of the intracellular behavior of HIV in living cells. J Cell Biol 159:441–52 Metharom P, Ellem KA, Schmidt C, Wei MQ. 2001. Lentiviral vector-mediated tyrosinase-related protein 2 gene transfer to dendritic cells for the therapy of melanoma. Hum Gene Ther 12:2203–13 Michael NL, Moore JP. 1999. HIV-1 entry inhibitors: evading the issue. Nat Med 5:740–2 Miller DG, Adam MA, Miller AD. 1990. Gene transfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection. Mol Cell Biol 10:4239–42 Milsom MD, Williams DA. 2007. Live and let die: in vivo selection of gene-modified hematopoietic stem cells via MGMT-mediated chemoprotection. DNA Repair (Amst) 6:1210–21 Mitchell RS, Beitzel BF, Schroder AR, Shinn P, Chen H, et al. 2004. Retroviral DNA integration: ASLV, HIV, and MLV show distinct target site preferences. PLoS Biol 2:E234 Miyagishi M, Sumimoto H, Miyoshi H, Kawakami Y, Taira K. 2004. Optimization of an siRNAexpression system with an improved hairpin and its significant suppressive effects in mammalian cells. J Gene Med 6:715–23 Miyauchi K, Kim Y, Latinovic O, Morozov V, Melikyan GB. 2009. HIV enters cells via endocytosis and dynamin-dependent fusion with endosomes. Cell 137:433–44 Miyazaki Y, Takamatsu T, Nosaka T, Fujita S, Martin TE, Hatanaka M. 1995. The cytotoxicity of human immunodeficiency virus type 1 Rev: implications for its interaction with the nucleolar protein B23. Exp Cell Res 219:93–101 Miyoshi H, Blomer U, Takahashi M, Gage FH, Verma IM. 1998. Development of a self-inactivating lentivirus vector. J Virol 72:8150–7 Modlich U, Baum C. 2009. Preventing and exploiting the oncogenic potential of integrating gene vectors. J Clin Invest 119:755–8 Molina RP, Matukonis M, Paszkiet B, Zhang J, Kaleko M, Luo T. 2002. Mapping of the bovine immunodeficiency virus packaging signal and RRE and incorporation into a minimal gene transfer vector. Virology 304:10–23 Montini E, Cesana D, Schmidt M, Sanvito F, Bartholomae CC, et al. 2009. The genotoxic potential of retroviral vectors is strongly modulated by vector design and integration site selection in a mouse model of HSC gene therapy. J Clin Invest 119:964–75

10  Lentiviruses: Vectors for Cancer Gene Therapy

177

Mselli-Lakhal L, Guiguen F, Greenland T, Mornex JF, Chebloune Y. 2006. Gene transfer system derived from the caprine arthritis-encephalitis lentivirus. J Virol Methods 136:177–84 Mulligan RC. 1993. The basic science of gene therapy. Science 260:926–32 Naldini L, Blomer U, Gallay P, Ory D, Mulligan R, et al. 1996. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272:263–7 Nomaguchi M, Fujita M, Adachi A. 2008. Role of HIV-1 Vpu protein for virus spread and pathogenesis. Microbes Infect 10:960–7 Norris PJ, Rosenberg ES. 2001. Cellular immune response to human immunodeficiency virus. AIDS 15 Suppl 2:S16–21 Nottage M, Siu LL. 2002. Rationale for Ras and raf-kinase as a target for cancer therapeutics. Curr Pharm Des 8:2231–42 O’Reilly MS, Boehm T, Shing Y, Fukai N, Vasios G, et  al. 1997. Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell 88:277–85 O’Reilly MS, Holmgren L, Shing Y, Chen C, Rosenthal RA, et  al. 1994. Angiostatin: a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma. Cell 79:315–28 Olsen JC. 1998. Gene transfer vectors derived from equine infectious anemia virus. Gene Ther 5:1481–7 Ory DS, Neugeboren BA, Mulligan RC. 1996. A stable human-derived packaging cell line for production of high titer retrovirus/vesicular stomatitis virus G pseudotypes. Proc Natl Acad Sci U S A 93:11400–6 Pariente N, Morizono K, Virk MS, Petrigliano FA, Reiter RE, et al. 2007. A novel dual-targeted lentiviral vector leads to specific transduction of prostate cancer bone metastases in vivo after systemic administration. Mol Ther 15:1973–81 Patel CA, Mukhtar M, Pomerantz RJ. 2000. Human immunodeficiency virus type 1 Vpr induces apoptosis in human neuronal cells. J Virol 74:9717–26 Pham L, Ye H, Cosset FL, Russell SJ, Peng KW. 2001. Concentration of viral vectors by coprecipitation with calcium phosphate. J Gene Med 3:188–94 Philippon V, Vellutini C, Gambarelli D, Harkiss G, Arbuthnott G, et al. 1994. The basic domain of the lentiviral Tat protein is responsible for damages in mouse brain: involvement of cytokines. Virology 205:519–29 Pichlmair A, Diebold SS, Gschmeissner S, Takeuchi Y, Ikeda Y, et  al. 2007. Tubulovesicular structures within vesicular stomatitis virus G protein-pseudotyped lentiviral vector preparations carry DNA and stimulate antiviral responses via Toll-like receptor 9. J Virol 81:539–47 Piguet V, Trono D. 1999. The Nef protein of primate lentiviruses. Rev Med Virol 9:111–20 Poeschla EM, Wong-Staal F, Looney DJ. 1998. Efficient transduction of nondividing human cells by feline immunodeficiency virus lentiviral vectors. Nat Med 4:354–7 Pollard VW, Malim MH. 1998. The HIV-1 Rev protein. Annu Rev Microbiol 52:491–532 Popa I, Harris ME, Donello JE, Hope TJ. 2002. CRM1-dependent function of a cis-acting RNA export element. Mol Cell Biol 22:2057–67 Rath P, Shi H, Maruniak JA, Litofsky NS, Maria BL, Kirk MD. 2009. Stem cells as vectors to deliver HSV/tk gene therapy for malignant gliomas. Curr Stem Cell Res Ther 4:44–9 Roe T, Reynolds TC, Yu G, Brown PO. 1993. Integration of murine leukemia virus DNA depends on mitosis. EMBO J 12:2099–108 Rosenberg SA, Aebersold P, Cornetta K, Kasid A, Morgan RA, et al. 1990. Gene transfer into humans – immunotherapy of patients with advanced melanoma, using tumor-infiltrating lymphocytes modified by retroviral gene transduction. N Engl J Med 323:570–8 Rubsam LZ, Boucher PD, Murphy PJ, KuKuruga M, Shewach DS. 1999. Cytotoxicity and accumulation of ganciclovir triphosphate in bystander cells cocultured with herpes simplex virus type 1 thymidine kinase-expressing human glioblastoma cells. Cancer Res 59:669–75 Saenz DT, Poeschla EM. 2004. FIV: from lentivirus to lentivector. J Gene Med 6 Suppl 1:S95–104 Salani B, Damonte P, Zingone A, Barbieri O, Chou JY, et al. 2005. Newborn liver gene transfer by an HIV-2-based lentiviral vector. Gene Ther 12:803–14

178

Y. Lin et al.

Sato T, Neschadim A, Konrad M, Fowler DH, Lavie A, Medin JA. 2007. Engineered human tmpk/ AZT as a novel enzyme/prodrug axis for suicide gene therapy. Mol Ther 15:962–70 Schroder AR, Shinn P, Chen H, Berry C, Ecker JR, Bushman F. 2002. HIV-1 integration in the human genome favors active genes and local hotspots. Cell 110:521–9 Schroers R, Sinha I, Segall H, Schmidt-Wolf IG, Rooney CM, et al. 2000. Transduction of human PBMC-derived dendritic cells and macrophages by an HIV-1-based lentiviral vector system. Mol Ther 1:171–9 Scollay R. 2001. Gene therapy: a brief overview of the past, present, and future. Ann N Y Acad Sci 953:26–30 Seliger B. 2005. Strategies of tumor immune evasion. BioDrugs 19:347–54 Sena-Esteves M, Tebbets JC, Steffens S, Crombleholme T, Flake AW. 2004. Optimized large-scale production of high titer lentivirus vector pseudotypes. J Virol Methods 122:131–9 Sheehy AM, Gaddis NC, Choi JD, Malim MH. 2002. Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein. Nature 418:646–50 Shichinohe T, Bochner BH, Mizutani K, Nishida M, Hegerich-Gilliam S, et al. 2001. Development of lentiviral vectors for antiangiogenic gene delivery. Cancer Gene Ther 8:879–89 Shin KJ, Wall EA, Zavzavadjian JR, Santat LA, Liu J, et al. 2006. A single lentiviral vector platform for microRNA-based conditional RNA interference and coordinated transgene expression. Proc Natl Acad Sci U S A 103:13759–64 Stevenson M, Stanwick TL, Dempsey MP, Lamonica CA. 1990. HIV-1 replication is controlled at the level of T cell activation and proviral integration. EMBO J 9:1551–60 Strang BL, Takeuchi Y, Relander T, Richter J, Bailey R, et al. 2005. Human immunodeficiency virus type 1 vectors with alphavirus envelope glycoproteins produced from stable packaging cells. J Virol 79:1765–71 Sumimoto H, Hirata K, Yamagata S, Miyoshi H, Miyagishi M, et al. 2006a. Effective inhibition of cell growth and invasion of melanoma by combined suppression of BRAF (V599E) and Skp2 with lentiviral RNAi. Int J Cancer 118:472–6 Sumimoto H, Imabayashi F, Iwata T, Kawakami Y. 2006b. The BRAF-MAPK signaling pathway is essential for cancer-immune evasion in human melanoma cells. J Exp Med 203:1651–6 Sumimoto H, Kawakami Y. 2007. Lentiviral vector-mediated RNAi and its use for cancer research. Future Oncol 3:655–64 Sumimoto H, Tsuji T, Miyoshi H, Hagihara M, Takada-Yamazaki R, et al. 2002. Rapid and efficient generation of lentivirally gene-modified dendritic cells from DC progenitors with bone marrow stromal cells. J Immunol Methods 271:153–65 Sutton RE, Reitsma MJ, Uchida N, Brown PO. 1999. Transduction of human progenitor hematopoietic stem cells by human immunodeficiency virus type 1-based vectors is cell cycle dependent. J Virol 73:3649–60 Tay DL, Bhathal PS, Fox RM. 1991. Quantitation of G0 and G1 phase cells in primary carcinomas. Antibody to M1 subunit of ribonucleotide reductase shows G1 phase restriction point block. J Clin Invest 87:519–27 Thomas CE, Ehrhardt A, Kay MA. 2003. Progress and problems with the use of viral vectors for gene therapy. Nat Rev Genet 4:346–58 Unutmaz D, KewalRamani VN, Marmon S, Littman DR. 1999. Cytokine signals are sufficient for HIV-1 infection of resting human T lymphocytes. J Exp Med 189:1735–46 Valori CF, Ning K, Wyles M, Azzouz M. 2008. Development and applications of non-HIV-based lentiviral vectors in neurological disorders. Curr Gene Ther 8:406–18 VandenDriessche T, Thorrez L, Naldini L, Follenzi A, Moons L, et al. 2002. Lentiviral vectors containing the human immunodeficiency virus type-1 central polypurine tract can efficiently transduce nondividing hepatocytes and antigen-presenting cells in vivo. Blood 100:813–22 Ventura A, Meissner A, Dillon CP, McManus M, Sharp PA, et al. 2004. Cre-lox-regulated conditional RNA interference from transgenes. Proc Natl Acad Sci U S A 101:10380–5 Verbeek B, Southgate TD, Gilham DE, Margison GP. 2008. O6-Methylguanine-DNA methyltransferase inactivation and chemotherapy. Br Med Bull 85:17–33

10  Lentiviruses: Vectors for Cancer Gene Therapy

179

Vigna E, Cavalieri S, Ailles L, Geuna M, Loew R, et al. 2002. Robust and efficient regulation of transgene expression in vivo by improved tetracycline-dependent lentiviral vectors. Mol Ther 5:252–61 Weinberg RA. 1991. Tumor suppressor genes. Science 254:1138–46 Wu X, Li Y, Crise B, Burgess SM. 2003. Transcription start regions in the human genome are favored targets for MLV integration. Science 300:1749–51 Xia H, Mao Q, Paulson HL, Davidson BL. 2002. siRNA-mediated gene silencing in  vitro and in vivo. Nat Biotechnol 20:1006–10 Xu K, Ma H, McCown TJ, Verma IM, Kafri T. 2001. Generation of a stable cell line producing high-titer self-inactivating lentiviral vectors. Mol Ther 3:97–104 Yarosh DB. 1985. The role of O6-methylguanine-DNA methyltransferase in cell survival, mutagenesis and carcinogenesis. Mutat Res 145:1–16 Young A, Lyons J, Miller AL, Phan VT, Alarcon IR, McCormick F. 2009. Ras signaling and therapies. Adv Cancer Res 102:1–17 Zamore PD, Tuschl T, Sharp PA, Bartel DP. 2000. RNAi: double-stranded RNA directs the ATPdependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101:25–33 Zennou V, Petit C, Guetard D, Nerhbass U, Montagnier L, Charneau P. 2000. HIV-1 genome nuclear import is mediated by a central DNA flap. Cell 101:173–85 Zhang B, Xia HQ, Cleghorn G, Gobe G, West M, Wei MQ. 2001. A highly efficient and consistent method for harvesting large volumes of high-titer lentiviral vectors. Gene Ther 8:1745–51 Zielske SP, Gerson SL. 2002. Lentiviral transduction of P140K MGMT into human CD34(+) hematopoietic progenitors at low multiplicity of infection confers significant resistance to BG/ BCNU and allows selection in vitro. Mol Ther 5:381–7 Zufferey R, Donello JE, Trono D, Hope TJ. 1999. Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors. J Virol 73:2886–92 Zufferey R, Nagy D, Mandel RJ, Naldini L, Trono D. 1997. Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nat Biotechnol 15:871–5

Chapter 11

Interleukin-24 Gene Therapy for Melanoma Nancy Poindexter, Rajagopal Ramesh, Suhendan Ekmekcioglu, Julie Ellerhorst, Kevin Kim, and Elizabeth A. Grimm

Abstract The interleukin (IL) -24 protein encoded by melanoma differentiation associated-7 (mda-7) gene is a novel IL-10 family cytokine with unique tumorspecific apoptotic and anti-angiogenic properties. Additional role(s) for IL-24, including the regulation of skin inflammation, as suggested by recent data, provide a teleologic role for this melanocyte- and monocyte-produced molecule. Previous studies by our group led to a Phase I trial for local adenoviral therapy delivery in advanced solid tumor patients. These clinical studies employed gene therapy with adenoviral vector–mediated delivery of mda-7/IL-24 (Ad-mda-7/IL-24) and clearly demonstrated a bystander apoptotic effect resulting from IL-24 protein production in injected tumor lesion. While these studies are useful for “proof of principle” for IL-24, we now realize that we must devise means to use this same product, IL-24, systemically to achieve therapeutic success in patients with melanoma. Therefore, gene therapy with a nanoparticle delivery vehicle is now being pursued. We propose that MDA7/IL-24 is a major skin-derived tumor suppressor/cytokine that has profound significance as a biotherapeutic for melanoma and possibly other cancers. Keywords IL-24 • MDA-7 • Cytokine • Tumor Suppressor • Melanoma • Nanoparticle

1 1.1

Biology of IL-24 a Tumor Suppressor/Cytokine Tumor Suppressor Properties

The mda-7/IL-24 gene is a novel tumor suppressor/cytokine gene that was first identified in human melanoma cell lines induced to differentiate with IFN-b and mezerein E.A. Grimm (*) Professor, Department of Experimental Therapeutics; Francis King Black Memorial Professor of Cancer Research; Deputy Head for Research Affairs, Division of Cancer Medicine; and Co-Director, Melanoma Research Program; The University of Texas MD Anderson Cancer Center, Houston, TX, USA e-mail: [email protected] J.A. Roth (ed.), Gene-Based Therapies for Cancer, Current Cancer Research, DOI 10.1007/978-1-4419-6102-0_11, © Springer Science+Business Media, LLC 2010

181

182

N. Poindexter et al.

(Jiang et al. 1995). The mda-7/IL-24 cDNA encodes a novel, evolutionarily conserved protein of 206 amino acids with a predicted size of 23.8 kDa (Mhashilkar et al. 2001). Initial studies showed mda-7/IL-24 mRNA expression in normal melanocytes and early stages of melanoma that was lost during melanoma progression (Jiang et  al. 1996). Furthermore, the ectopic expression of mda-7/IL-24 in human cancer cells, both in vitro and in vivo, resulted in the suppression of cell proliferation and induction of apoptosis with no cytotoxicity to normal cells (Jiang et al. 1996; Su et al. 1998). On the basis of these observations, it was suggested that mda-7/IL-24 is a tumor suppressor gene whose expression must be inhibited for tumor progression. Subsequent studies by us investigating IL-24 protein expression in human melanoma and non-small cell lung cancer tissues showed tumor progression was inversely correlated with IL-24 protein expression (Ellerhorst et al. 2002; Ishikawa et al. 2005). In these studies, IL-24 protein expression was detected in the early stages of the disease and was lost with disease progression. These results support mda-7/IL-24 to function as a tumor suppressor gene. On the basis of these reports, we have extensively tested and demonstrated the antitumor properties of mda-/IL-24 gene in a broad spectrum of human cancer cells in vitro that included cancer cells of the lung, ovary, prostate, breast, and melanoma (Ekmekcioglu et al. 2001; Saeki et al. 2000). These studies used plasmid-based or adenoviral vector-mediated delivery of mda-7/IL-24 (Ad-mda-7/IL-24). It is notable that in these studies, endogenous IL-24 protein was not detectable in almost all of the cancer cell lines tested by standard assays. Results from these studies clearly demonstrated mda-/IL-24 selectively killed tumor cells with minimal to no effect on normal cells. The inhibitory effect of mda-/IL-24 on tumor cells was independent of the p53, p16, ras, and Bax (Mhashilkar et al. 2001; Saeki et al. 2000). Subsequent studies by several other investigators have demonstrated similar antitumor activity of mda-7/IL-24 against numerous human cancer cell lines (Ekmekcioglu et al. 2008; Leath et  al. 2004; Lebedeva et  al. 2002, 2007; Mahasreshti et  al. 2006; Pataer et al. 2002; Sarkar et al. 2007, 2008; Su et al. 2001; Wang et al. 2007; Xie et al. 2008; Xiong et al. 2007a, 2007b; Zheng et al. 2007). All of these studies, which included the appropriate vector controls that expressed genes such as luciferase protein, provided data indicating the specificity of mda-7/IL-24-mediated tumor cell killing. The molecular mechanism of tumor cell killing was cell-type dependent. For example, in lung cancer cells, PKR (Pataer et  al. 2002) and JNK (Kawabe et al. 2002) were shown to be important for Ad-mda7/IL-24-mediated tumor cell killing. More recently, we reported that MEKK1 is upstream of the cell-signaling molecules and is required and activated by Ad-mda7/IL-24 in lung cancer cells (Oida et al. 2007). In ovarian cancer cells, we showed that Fas–FasL activation is important for mda-7/IL-24-mediated cell killing (Gopalan et al. 2005). The requirement of p38MAPK was demonstrated for mda-/IL-24-mediated killing of melanoma cells (Sarkar et al. 2002). In renal cell lines, Park et al. have demonstrated that cell death mediated by glutathione S-transferase (GST)-MDA-7/IL-24 was the result of ceramide-dependent activation of CD95 resulting in the activation of pro apoptotic pathways (Park et  al. 2009). Results from these in  vitro studies indicate that the molecular signaling death pathways activated by mda-7/IL-24 are cell-type dependent and differ upstream of the mitochondria but converge downstream of the mitochondria to activate the caspase pathway.

11  Interleukin-24 Gene Therapy for Melanoma

183

Next, we tested the antitumor properties of mda-/IL-24 in vivo against human tumor xenografts. The intratumoral administration of Ad-mda/IL-24 resulted in tumor suppression and the apoptosis of human lung and ovarian cancers (Gopalan et  al. 2007; Saeki et  al. 2002). Furthermore, the in  vivo therapeutic effect of Ad-mda/IL-24 was greater in immunocompetent mice than in immunodeficient mice suggesting Ad-mda/IL-24 can produce an enhanced therapeutic effect in the presence of a normal host immune system (Miyahara et  al. 2006). These studies demonstrated that Ad-mda/IL-24 can produce a potent anticancer activity when administered systemically in  vivo. In addition, combining Ad-mda-7/IL-24 with radiation therapy resulted in an enhanced therapeutic effect (Chada et  al. 2006; Nishikawa et  al. 2004; Yacoub et  al. 2003). Enhanced antitumor activity has also been demonstrated when Ad-mda-7/IL-24 was combined with several other therapeutic agents (Bocangel et al. 2006; Gopalan et al. 2008; Oida et al. 2005; Zheng et al. 2008). More recently, we have shown that Ad-mda-/IL-24 when combined with vitamin E succinate produced a synergistic therapeutic effect in ovarian cancer cells (Shanker et  al. 2007). All of these studies demonstrate that Ad-mda-7/IL-24, when used alone or in combination with other therapeutic strategies, functions as an effective anticancer agent both in vitro and in vivo. Molecular studies indicate that IL-24 protein is glycosylated and secreted (Mhashilkar et al. 2001; Mumm et al. 2006). Additional studies have shown the protein to undergo phosphorylation and ubiquitination (Gopalan et al. 2008). These protein modifications are similar to those observed in classic tumor suppressor genes such as p53 (Chada et al. 2008). Follow-up studies demonstrated that the IL-24 signals through two heterodimeric receptors, IL-20R1/IL-20R2 and IL-22R1/IL-20R2 complex (Dumoutier et al. 2001; Wang et al. 2002). More recent studies from our laboratory and others have shown IL-24 selectively kills human melanoma and pancreatic cancer cells that are positive for IL-24 receptors in a dose- and time-dependent manner (Chada et  al. 2004a, 2005). IL-24 had no effect on receptor-positive normal cells (Chada et al. 2004a, 2005; Su et al. 2005). The molecular mechanism for IL-24 to selectively kill receptor-positive tumor cells and not normal cells is not well understood. One possibility is that although both tumor cells and normal cells express the receptors, the intracellular signal activated upon IL-24 binding to its receptors is different. Currently, studies in our laboratory and others are ongoing to gain a better understanding of the tumor selectivity of IL-24. A more recent study by Saune et al. (Sauane et al. 2008) reports a potential autocrine regulation of IL-24-mediated tumorspecific killing. Additionally, in vivo studies showed the inhibition of contralateral tumors when injected with an oncolytic virus producing the IL-24 protein (Sarkar et al. 2007). All of these studies reveal that IL-24 can selectively kill cancer cells by a “Bystander Effect” via both autocrine and paracrine mechanisms. The only exceptions to the consensus reports (>150) by several investigators demonstrating anticancer activity for IL-24 protein are the studies by Kreis et al. (Kreis et al. 2007) who reported IL-24 does not have anti-cancer activity against cancer cells and by Sainz-Perez et al. (2006), who reported high IL-24 expression promotes the survival of chronic lymphocytic leukemia (CLL). The lack of anticancer activity reported for IL-24-mediated survival activity in CLL remains unclear. A consensus among a majority of investigators (>25) studying IL-24 is that

184

N. Poindexter et al.

the results reported by Kreis et al. (2007) are most likely do to their use of reagents from different sources of variable quality. Similarly, two recent studies testing IL-24 on hematopoietic malignancies (Qian et al. 2008) and B-lymphoblastic leukemia (Dong et al. 2008) have shown antitumor activity both in vitro and in vivo thereby negating the findings of Sainz-Perez et al. (2006). From these reports, it is evident that IL-24 exhibits antitumor activity in a majority of human cancer cell lines. Studies investigating the cytokine properties of IL-24 protein showed that it functions as a pro-Th1 cytokine unlike IL-10 which is Th-2 type cytokine (discussed in detail below) (Caudell et al. 2002; Mumm et al. 2006). Apart from the antitumor and cytokine properties of IL-24, we have previously demonstrated that IL-24 also has potent anti-angiogenic and anti-metastatic activities both in vitro and in vivo (Inoue et al. 2005, 2006; Nishikawa et al. 2004; Ramesh et al. 2004a, b). Anti-angiogenic activity was demonstrated for both the intracellular and extracellular forms of the IL-24 protein (Inoue et al. 2005, 2006, Nishikawa et al. 2004; Ramesh et al. 2004a, b). The anti-angiogenic activity exerted by the extracellular IL-24 protein was shown to be receptor-mediated (Ramesh et  al. 2003). Hence, IL-24 is a unique cytokine that undergoes protein modifications akin to classical tumor suppressor proteins and functions as a tumor suppressor/cytokine.

1.2 Cytokine Properties At least five cellular genes encoding secreted proteins with 20–30% homology to IL-10 have been found: IL-19 (Gallagher et  al. 2000), IL-20 (Blumberg et  al. 2001), IL-22 (Xie et al. 2000), IL-26 (Knappe et al. 2000), and mda-7, which was renamed IL-24 with the approval of the HUGO Gene Nomenclature Committee (Caudell et  al. 2002). Four of these genes (IL-10, IL-19, IL-20, and IL-24) are encoded within a 195-kilobase cytokine cluster on chromosome 1q31/32. All of these cytokines are secreted. The human IL-10 protein has only 23% homology to IL-24. IL-20 is the most similar to IL-24, with 33% protein homology. The relatively low amino acid sequence homology among these structurally related homodimeric IL-10 family members suggests that they have quite different biological functions. Message for IL-19, IL-22, IL-24, and IL-26 are expressed by antigen- or mitogen-stimulated human peripheral blood mononuclear cells (PBMCs) (Caudell et al. 2002; Gallagher et al. 2000; Knappe et al. 2000; Poindexter et al. 2005; Xie et  al. 2000). IL-24, as well as IL-19, expression by macrophages is strongly induced by stimulation with lipopolysaccharide (LPS) (Gallagher et  al. 2000). The stimulation of T cells with anti-CD3 causes the expression of IL-22 (Xie et  al. 2000). IL-26 has been identified in viral-transformed T cells, in normal PBMCs, and in some T cell lines (Knappe et al. 2000; Sheikh et al. 2004). All of the IL-10 family members use the class II family of receptors and signal through STAT1, STAT3, or both (Blumberg et al. 2001; Gallagher et al. 2000; Kotenko 2002; Sheikh et al. 2004; Wang et al. 2002; Xie et al. 2000). Descriptive and functional studies of the IL-24 receptor with keratinocytes or transfected cells have shown that

11  Interleukin-24 Gene Therapy for Melanoma

185

IL-24 activates STAT3 (Blumberg et  al. 2001; Kotenko 2002; Xie et  al. 2000). IL-19 stimulates human macrophages to produce both IL-6 and tumor necrosis factor (TNF)a (Liao et al. 2002). Similarly, the stimulation of PBMCs with IL-24 also leads to the production of IL-6 and TNFa (Caudell et al. 2002). More recent reports suggest that the IL-10 family of cytokines is involved in the regulation of inflammatory and immune responses (Chada et al. 2004b; Kotenko2002). IL-24 protein is endogenously expressed in normal human skin melanocytes, implying that IL-24 is involved in skin biology (Ekmekcioglu et  al. 2001). The detection of receptors for IL-24 on human keratinocytes suggests that this molecule may be involved in human wound healing as well (Wang et al. 2002). The rat homolog of IL-24, c49a, has been reported to be expressed in proliferating (Poindexter et al. 2007) fibroblasts during wound repair in a rat model (Soo et al. 1999). The relationship of IL-24 to IL-10 and its predicted cytokine features led us to study IL-24 expression in immune cells. As we describe in our publication (Poindexter et al. 2005), we found IL-24 protein to be expressed by human PBMCs as a result of mitogen or antigen stimulation. Our publications (Caudell et al. 2002; Poindexter et al. 2005) provide evidence that IL-24 is part of a cytokine network expressed during the activation of the human cellular immune response. Specifically, IL-24 is known to stimulate human PBMCs to secrete TNFa, IL-6, IL-1b, and granulocyte-macrophage colony stimulating factor (GM-CSF).

2 Expression of IL-24 in Melanocytes and Melanoma Our study (Ekmekcioglu et al. 2001) was the first to evaluate IL-24 expression in human tumors using immunohistochemical analysis. We demonstrated that IL-24 protein was expressed in normal melanocytes and early-stage melanomas; however, IL-24 expression decreased in more advanced melanomas and was completely absent in metastatic lesions, which is consistent with the tumor suppressor role of IL-24. Ellerhorst et  al. evaluated IL-24 protein expression during melanoma disease progression by using the immunohistochemical analysis of clinical tumor biopsy samples from stage III to IV melanomas (n = 82, 41 primary melanomas and 41 metastases) (Ellerhorst et  al. 2002). To determine whether IL-24 loss occurred with tumor progression (from superficial to invasive stages), the study compared IL-24 expression by tumor cells in the epidermis or superficial dermis with that of cells in the deep dermis (Fig. 1). The percentage of IL-24-positive cells and the intensity of the staining decreased significantly with tumor depth (p = 0.003 and p = 0.008, respectively). On the basis of these results, Ellerhorst et al. hypothesized that a similar pattern would be observed when comparing primary melanoma lesions with their corresponding metastases. Indeed, the examination of 24 pairs of primary tumors and their metastases showed significantly lower number (p = 0.001) and intensity (p = 0.001) scores for IL-24 staining in the metastases, relative to the primary tumors.

186

N. Poindexter et al.

Superficial

IL-24 Immunostaining

++++

++

Deep Primary Melanoma

Negative

Fig. 1  Loss of IL-24 Immunoreactivity in the invasive from of primary melanoma. The superficial portion of the tumor shows significant cytoplasmic IL-24 expression, which diminishes with invasion into the deep dermis (×40 magnification). Taken from Ellerhorst et  al. (2002) PMID 11844832

3 Re-expression of the Tumor Suppressor Preclinical studies evaluating the activity of IL-24 in tumor models have identified a complex interplay between direct tumor cell death (induced by the intracellular expression of IL-24) and complementary anti-tumor mechanisms (the so-called “bystander effect,” induced by the engagement of the IL-24 receptors). To analyze the mechanism by which IL-24 kills tumor cells, we developed mutants engineered to express IL-24 in the cytosol, nucleus, or endoplasmic reticulum, coupled with glycosylation inhibitors to alter intracellular trafficking. We observed that IL-24 appears to kill tumor cells via two distinct mechanisms. When IL-24 is expressed within a cell, a stress-response signal from the endoplasmic reticulum (ER) via caspases 7 and 12 leads to mitochondrial disruption and apoptosis (Sieger et al. 2004). Alternatively, when IL-24 protein is delivered to a cell bearing IL-24 receptors, ligand binding causes transient phosphorylation of STAT3 and initiates an apoptotic caspase cascade. Also, the mechanism of apoptosis varies depending upon the cell type studied. In a specific apoptosis pathway study that targets human ovarian tumor cell, activation of the extrinsic pathway was demonstrated by increased cellsurface Fas expression and cleavage of Bid and caspase-8, while activation of the intrinsic pathway was demonstrated by the disruption of mitochondrial potential;

11  Interleukin-24 Gene Therapy for Melanoma

187

and the activation of downstream capase-9 and caspase- 3 via cytochrome C release (Shanker et al. 2007). In cells that lack IL-24 receptors, IL-24 gene transfer causes apoptosis via the intracellular ER pathway, and exogenous IL-24 protein does not have any effect. In contrast, in cells expressing IL-24 receptors, ligand engagement activates alternate signaling pathways (Chada et  al. 2004b). In a recent study by Sauane et al., it was confirmed that IL-24 protein induces the bystander antitumor effect through an ER stress mechanism mediated by a robust activation of its own protein expression (Sauane et al. 2008). This study reported that exogenous IL-24 protein induces growth inhibition and apoptosis only in cancer cells through a mechanism identical to Ad-mda-7/IL-24 infection. They demonstrated that exogenous IL-24-mediated IL-24 receptor upregulation is essential for the IL-24-induced apoptotic effect. Blocking mda-7/IL-24 expression by RNA interference inhibits extracellular IL-24-mediated apoptosis (Sauane et al. 2008). This complex mechanism utilizing both intrinsic and extrinsic pathways of IL-24 induced cell death is summarized in Fig. 2. Therefore, having purified functional IL-24 protein provides insights into the mechanism by which this molecule exerts the bystander antitumor effects and supports alternative therapeutic approaches in addition to adenovirus delivery. We have found that preferential utilization of extrinsic versus intrinsic pathways by IL-24 is cancer-cell dependent. Treatment with Ad-mda-7/IL-24 results in

IL-20R2 Ad-mda7/IL-24

IL-24 Receptor Subunits

IL-22R1 IL-20R1

Bystander Effect via Secreted IL-24

PKR UPR stress response Caspase 12

IL-20R Type I IL-20R Type II

? NFkB STAT3

IRFs IFNb≠

iNOS Ø

Nucleus

APOPTOSIS

PERK [Ca2+] release

eIF2a Bcl family

Effector mitochondria Caspases ?

APOPTOSIS

≠BAX G1arrest

Nucleus

NFkB

STAT3

OTHER?

Nucleus

TUMOR CELLS Normal Cells NO CELL DEATH

Fig. 2  Mechanism of IL-24 induced cell death. The gene transfer of IL-24 results in direct and indirect (bystander protein) effects on normal and cancer cells

188

N. Poindexter et al.

apoptosis in melanoma tumor cells, but not in melanocytes. In melanoma, IL-24 expression induces the upregulation of growth arrest and DNA damage (GADD) and pro-apoptotic (BAX) proteins, with a concomitant downregulation of iNOS via the modulation of interferon regulatory factors (Ekmekcioglu et al. 2003; Lebedeva et al. 2002). Selective inhibition of p38 mitogen-activated protein kinase (MAPK) phosphorylation, which induces GADD, abrogates IL-24-induced apoptosis in melanoma and NSCLC cells (Mhashilkar et al. 2003; Sarkar et al. 2002). The treatment of melanoma tumor cell lines with exogenous IL-24 protein results in growth arrest and apoptotic cell death (Chada et al. 2004b). The co-administration of neutralizing antibodies against IL-24 or individual receptor subunits (anti-IL-20R1 or anti-IL-22R1) blocked tumor cell killing, thus demonstrating the specificity of the ligand-receptor interaction. IL-24 protein treatment of melanoma cells also increased the expression of BAX and p21 prior to apoptotic death. A second more novel pathway for melanoma apoptosis has been described involving the induction of endogenous IFN-b followed by IRF regulation and TRAIL/FasL system activation (Ekmekcioglu et  al. 2008). Thus, exogenous IL-24 protein can reverse the cancerous phenotype of melanoma cells and warranted its evaluation as a therapeutic approach in melanoma and other solid tumors.

4 Clinical Experience with Ad mda-7/IL-24 4.1 Metastatic Melanoma Melanoma is the most malignant of skin cancers. The incidence of melanoma in the United States is increasing at an annual rate of 3.1% and these rates have doubled in all socioeconomic status groups over the past 10-year period. Early, localized disease (radial growth phase, RGP) is effectively treated with excision, with a high cure rate (~80%). However, melanoma quickly transitions from RGP to the vertical growth phase (VGP), which leads to disseminated disease that is almost universally fatal. The median survival rate with current chemotherapy regimens for metastatic malignant melanoma has not improved significantly over the last several decades (Anderson et al. 1995). Dacarbazine (DTIC) is the only cytotoxic chemotherapeutic drug approved by the U.S. Food and Drug Administration for the treatment of metastatic melanoma. However, recent phase III studies using the Response Evaluation Criteria in Solid Tumors (RECIST) showed that the response rate of dacarbazine is less than 10% and the median progression-free survival (PFS) is only 1.5 months (Bedikian et  al. 2006, Middleton et  al. 2000). The other classes of drugs with activity between 10 and 15% are the nitrosoureas, vinca alkaloids, and cisplatin (Anderson et al. 1995). Interleukin-2 based regimens can result in long-term benefit in a small fraction of patients, but the majority of patients do not respond. Many types of combination chemotherapy regimens explored in the treatment of metastatic melanoma during the past 25 years have produced response rates of 30–40% (Chapman et al. 1999; Luikart et al. 1984). These regimens, however, were limited

11  Interleukin-24 Gene Therapy for Melanoma

189

by the short duration of responses and low rates of complete response. Although the integration of combination chemotherapy with the cytokines interleukin-2 (IL-2) and interferon-alpha (IFN-a), that is, “biochemotherapy,” has resulted in an overall response rate of more than 60%, with 10–20% complete responses, several phase III trials failed to show a meaningful survival benefit of biochemotherapy over chemotherapeutic regimens (Atkins 1997; Eton et  al. 2002; Ives et  al. 2007; Rosenberg et al. 1999). Thus, there is an urgent need to discover new ways to treat these patients with novel drugs. On the basis of all preliminary data, IL-24 therapy is likely to be more efficacious than IL-2 and IFNa and much less toxic (Fisher et al. 2003; Inoue et al. 2006; Ramesh et al. 2004b). This has proven to be the case. Ad-mda-7/IL-24 has been tested in a Phase 1 Clinical Trial in patients with advanced solid tumors.

4.2 Phase I/II intratumoral Ad-mda-7/IL-24 Gene Transfer in Patients with Advanced Solid Tumors This phase I/II clinical trial was in patients with advanced carcinoma for which intralesional injection and multiple biopsies were performed (Cunningham et  al. 2005; Tong et al. 2005). The study enrolled 28 heavily pretreated patients with a total of 15 different tumor types, including melanoma, lymphocytic lymphoma, and carcinomas of the breast, colon, head and neck, adrenal gland, renal cell, and lip. Patients were divided into eight cohorts of increasing IL-24 doses (up to 2 × 1012 viral particles (vp) per injection). Ad-mda-7/IL-24 (also called INGN-241) was administered intratumorally, and the tumors (excised at pre-established times during treatment) evaluated for vector-specific DNA and RNA, transgenic IL-24 expression, and biological effects. Successful gene transfer was demonstrated in 100% of the patients evaluated, with a parallel distribution of vector DNA, vector RNA, IL-24 protein expression, and apoptosis induction observed in all tumors that decreased with distance away from the injection site. The safety analysis reported 15 mild-to-moderate adverse events in patients completing at least one cycle of treatment (n = 22) (Cunningham et al. 2005; Tong et  al. 2005). Toxicity attributable to Ad-mda-7/IL-24 injections was self-limiting and resolved within 48 h; the most common adverse events reported were injection site pain and fever. Skin erythema, which resolved within 96 h post-injection, was reported in the cohort treated at the maximum dose. One grade 3 serious adverse event (fatigue) was reported in a patient who was subsequently removed from the study. Importantly, no maximum tolerated dose (MTD) was achieved (Cunningham et al. 2005). In the first phase of the monotherapy protocol, the injected lesions were excised 24–96  h post-treatment; thus, no conclusions could be drawn about the clinical activity of the treatment. However, minor changes in the morphology of the injected lesions from the higher-dose cohorts were observed. In a repeat-dose protocol, patients in the highest dose cohort received injections twice weekly for 3 weeks and

190

N. Poindexter et al.

had incision or core biopsies taken 30 days after the last Ad-mda-7/IL-24 injection. A clinically significant response to Ad-mda-7/IL-24 (partial regression) was observed in 40% of the patients in this cohort (n = 5) (Cunningham et al. 2005). The most dramatic clinical response occurred in a patient with a 20 × 20 mm lesion at baseline; by the 6th injection, a clear decrease in the size of the lesion was seen that was associated with erythema around the lesion. The erythema resolved and regression continued over the next 2 weeks until there was no clinical evidence of disease at that site. A second course of injections was then started on a second lesion (baseline measurement 18 × 23 mm), and an 84% reduction in size was seen by the fifth injection. After the sixth injection, the lesion was excised. An additional melanoma lesion exhibited a partial response (33% decrease as measured by RECIST). This patient was still alive more than 600  days after initiation of the Ad-mda-7/IL-24 treatment. Molecular markers of vector expression (i.e., beta-catenin redistribution/decrease, TUNEL, and in melanoma, iNOS down-regulation) were positive for all tumor types examined (Tong et al. 2005). The immunohistochemical analysis of IL-24 and TUNEL assay in tumors resected 24–96  h after injection demonstrated substantial IL-24 immunostaining (range, 20–90% positive cells) closest to the injection site and a significant correlation between apoptosis (as measured by TUNEL) and expression of IL-24 (p < 0.01). Apoptotic signals were greatest in the highest dose cohort (2 × 1012 vp) and could be detected in distal sections of the tumor in 78% of evaluable patients (n = 9). Similar to the IL-24 distribution, apoptotic activity accumulated with time, with TUNEL-positive tumor cells increasing with time post-injection. Pre-treatment samples were uniformly negative for IL-24 protein expression, and most were negative for TUNEL reactivity. Consistent with the anti-tumor effect, staining for Ki-67 (a marker of tumor proliferation) decreased after Ad-mda-7/IL-24 treatment in 67% (n = 9) evaluable tumors. Determination of the kinetics of IL-24 protein expression revealed the transient nature of gene expression and its biological effect. Taken together, the promising results from the clinical trial experience with Ad-mda-7/IL-24, combined with evidence of the loss of IL-24 protein expression during melanoma progression and invasion, support the further evaluation of IL-24 (and its signaling pathways) as a therapeutic target for melanoma.

4.3 Phase II Intratumoral Injection of Ad-mda-7/IL-24 in Patients with Advanced Melanoma At the University of Texas M. D. Anderson Cancer Center, we recently conducted a phase II study of intratumoral injection of Ad-mda-7/IL-24 in patients with advanced melanoma. In this study, patients had at least three cutaneous lesions with an injectable lesion that was 15 mm or larger in the greatest diameter. The primary objective of the study was to evaluate the biological efficacy of the injection ­treatment by measuring the induction of apoptosis in tumor cells in the noninjected cutaneous lesions. All patients underwent tumor biopsy before treatment. Patients who had

11  Interleukin-24 Gene Therapy for Melanoma

191

a small number of lesions that were amenable to curative surgery were treated with intratumoral injection of Ad-mda-7/IL-24 twice a week for 3 weeks (days 1, 4, 8, 11, 15, and 18) and underwent a surgical resection of all lesions on day 22 (Cycle 1). Those who had multiple lesions that were not amenable to curative surgery underwent a biopsy of two separate lesions, including the injected lesion, on day 22. After 1  week of rest, they received the Ad-mda-7/IL-24 treatment twice a week for 3 weeks (Cycle 2), followed by 1 week of rest, repeating every 4 weeks until their disease progressed or they could not tolerate further treatment. During the first 3 weeks, each treatment injection was performed on the same lesion. 4.3.1 Clinical Results Adverse events were mild and included primarily grade 1 toxicities such as fatigue, pain at the injection sites, and fever. No patients experienced dose-limiting toxicity. The trial was closed early because of a slow accrual rate. The requirement for multiple cutaneous lesions as well as the restriction of having to be treated twice per week for 3 weeks made accrual difficult with only five patients of the proposed 25 being treated. Lesions injected with Ad-mda-7/IL-24 regressed in all patients, and the induction of intratumoral IL-24 protein (see below) expression was observed in injected lesions. However, despite the regression of the injected lesions, the noninjected lesions continued to progress. 4.3.2 Laboratory Results The primary and secondary objectives of this study were biologic in nature. (1) To determine if intratumoral injection of Ad-mda-7/IL-24 resulted in the induction of pro-apoptotic and anti-proliferative effects in the Ad-mda-7/IL-24 injected melanoma in transit lesions. (2) To determine the effect of intratumoral Ad-mda-7/IL-24 administration on the systemic immune system. To accomplish these objectives, tumor tissue biopsies, peripheral blood for serum cytokine analysis, and peripheral blood for mononuclear cell phenotyping and ex vivo assays were collected. I nduction of Pro-apoptotic and Anti-proliferative Effects in the Ad-mda-7/IL-24 Injected Melanoma in Transit Lesions Immunohistochemical analysis was performed on tumor samples from three of the five patients who completed the trial. The sets of tissues analyzed from these patients included biopsies taken from a tumor lesion prior to treatment, the injected lesion 22 days after treatment, as well as an uninjected lesion also biopsied 22 days after the first treatment with Ad-mda-7/IL-24. Formalin-fixed, paraffin-embedded tissues were stained with a monoclonal antibody against IL-24 (Poindexter et al. 2005), Ki67, and tested for TUNEL reactivity. None of the pretreatment lesions

192

N. Poindexter et al.

were positive for IL-24. In tumor samples from two of the three patients, positive staining for IL-24 protein was found in the uninjected test lesion; while in one patient’s samples, positive staining was seen only in the injected lesion. Note that in one of the two patients, whose uninjected lesion was positive for IL-24, the injected lesion had few tumor cells remaining. We propose that this scarcity of tumor cells in the injected lesion was due to the expression of IL-24 leading to apoptosis of melanoma cells. We saw a decrease in Ki67 staining that corresponded to an increase in IL-24 staining in one of the three patients while varying degrees of TUNEL staining was seen in all tumors analyzed. On the basis of the fact that none of the pretreatment tumor biopsies were positive for MDA-7, we can conclude from these patient data that Ad-mda-7/IL-24 treatment led to IL-24 protein expression in two of the three patients’ distal tumors. Effects of Ad-mda-7/IL-24 on the Peripheral Immune System IL-24 is a positive regulator of immune reactivity causing the secretion of proinflammatory cytokines from normal peripheral blood mononuclear cells (PBMC) (Caudell et  al. 2002; Mumm et  al. 2006). Furthermore, it was observed in the completed Phase 1 Trial of Intratumoral Ad-mda-7/IL-24 gene transfer in patients with advanced solid tumors (described above) that serum IL-6, IL-10, and TNFa rose significantly in response to injection of the drug with lower increases in IFNg, GM-CSF and IL-2 also measured (Tong et  al. 2005). Given these findings, our working hypothesis was that IL-24 acts as a pro inflammatory cytokine affecting the differentiation of antigen-presenting cells resulting in an increase in the antigenspecific T cell response to tumors. We report here our analysis of samples taken from the three of five patients who completed treatment. Blood was collected during the first cycle of treatment and serum cytokine levels were analyzed using the Luminex system with antibodies for GM-CSF, IFN-g, IL-1 b, IL-2, IL-10, IL-12, and TNF-a, as previously described (Wang et al. 2008). The serum levels of IL-24 protein were measured by ELISA (R&D Systems, Minneapolis, MN). In all patients tested, a slow transient increase in serum concentration of GM-CSF, IL-1b, IFNg, and TNFa was seen during the course of intratumoral treatment with Ad-mda-7/IL-24. Because of the small number of patients tested, statistical analyses were not possible, but these results were similar to what was reported in the Phase 1 Trial of Intratumoral Ad-mda-7/IL-24 where increases in serum cytokines were noted for TNFa, IFNg, and GM-CSF (Tong et al. 2005). The serum concentration of IL-24 protein was measured in all five patients who completed treatment. In four of five patients, serum IL-24 was undetectable at the onset of treatment and three of five patients showed increases in IL-24 protein during the course of treatment reaching levels of 130 pg/ml in one patient. The further effects of IL-24 on the peripheral immune response in patients treated with intratumoral Ad-mda-7 (IL-24) were examined through phenotyping of PBMCs as well as the ex vivo stimulation of PBMC with autologous tumor cells

11  Interleukin-24 Gene Therapy for Melanoma

193

to determine if treatment with Ad-mda-7/IL-24 affected an antigen-specific T cell response as measured by intracellular cytokine production by T cells after a 6–7  day stimulation with tumor. PBMC were isolated and cryopreserved from blood samples received pretreatment, day 22 after the first treatment, and prior to the start of the second cycle of treatment. In addition, fresh tumor tissue, taken prior to the start of treatment, was enzymatically digested to prepare a single cell suspension of tumor cells (SCS). This was used as a source of autologous antigen for PBMC stimulation. Immunophenotyping of leukocytes was performed on purified PBMC with a panel of flourophore-labeled monoclonal antibodies (BD Biosciences, San Jose, CA). Complete sets of samples (blood from each of the three time points and adequate SCS) were available on only two of the five patients treated. Sufficient numbers of PBMC and tumor SCS were available to complete both phenotyping and intra­ cellular cytokine analysis. In both patients, we saw an increase in the number of CD14−/CD11b+ PBMC over the course of treatment. This phenotype is indicative of a differentiating monocyte population. Moreover, in both patients, after two cycles of treatment, we saw an increase in the number of CD83+ dendritic cells in the peripheral blood. In terms of T cell function after ex vivo stimulation, we measured increased numbers of CD69+IFNg+CD8+ cells in both patients. Patient’ PBMC were stimulated with autologous tumor SCS for 6–7 days followed by intracellular FACS analysis to measure IFNg-secreting T cells. Patient #2 showed a twofold increase in the number of CD69+/IFNg+CD8+ cells, from 4.4 to 9.9%. While patient #1 showed an increase from 0 to 6.7% over the course of treatment. These results, although quite preliminary, suggest that intratumoral injection of Ad-mda-7/IL-24 affected the peripheral immune response causing an increase in the relative number of dendritic cells that may then influence the tumor-specific T cell response as evidenced by an increase in the number of IFNg-producing T cells. The main conclusion that can be drawn from this trial is that intratumoral injection of Ad-mda-7/IL-24 resulted in regression of the injected tumors, which was accompanied by IL-24 protein expression in both injected and uninjected tumors. The accompanying positive effects on the immune response of these patients, although suggestive, must be tested in future trials.

5 Other Preclinical Studies Using Viral Delivery of IL-24 The delivery of IL-24 gene has been attempted using various vectors, including the adenoviral vector described above (Cunningham et al. 2005, Tong et al. 2005); and more recently, in the Phase II trial in metastatic melanoma patients. Other vectors also have been tested both in  vitro and in animal models of solid tumors. These include the insertion of the IL-24 gene into Ad.sp-E1A (Delta), an oncolytic ­adenovirus with a 24-bp deletion in the E1A gene, driven by a survivin promoter. Ad.sp-E1A (Delta24)-IL-24 was shown to have efficient anti-tumor effects in vitro for human nasopharyngeal, liver, lung, and cervical cancer cell lines without

194

N. Poindexter et al.

d­ amaging normal cell lines and, in  vivo, it caused the inhibition of lung tumor growth in nude mice (Zhang et al. 2009). The recombinant adeno-associated virus (rAAV) vector has been used to deliver the IL-24 gene in a recurrent and metastatic hepatocarcinoma model in nude mice. This construct employs an rAAV vector carrying alpha-fetoprotein (AFP) promoter for expressing the IL-24 gene. It was successfully tested in this HCC model, resulting in the prevention of postoperative recurrence and metastasis through the induction of tumor cell apoptosis (Yang et al. 2009). The most recent preclinical testing of IL-24 gene delivery was in combination with temazolamide (TMZ) for the treatment of experimental glioma. Kaliberova et al. constructed a VEGF-1/flt-1 conditional replicating adenoviral vector to deliver the IL-24 gene (CRAdRGDflt-IL-24). They reported that in combination with TMZ, CRAdRGDflt-IL-24 inhibited tumor growth and prolonged the survival of mice implanted with intracranial human glioma zenographs ((Kaliberova et al. 2009).

6 Systemic Delivery of Non-viral Nanoparticle-Based Gene Delivery Systems These preclinical and clinical experiences demonstrate that IL-24 is able to induce tumor regression which may be explained in terms of its pro apoptotic and anti proliferative effect on tumor cells as well as its pro inflammatory effect on the peripheral immune system. Therefore, IL-24 is functioning as both a tumor suppressor and a cytokine. However, they also emphasize the need for a systemic method of delivering IL-24 since patients with metastatic cancers have disease throughout the body. This need is most apparent in melanoma where the 10-year survival rate in patients with systemic disease is less that 10% because of the scarcity of effective treatments. We therefore have begun to investigate the effectiveness of nonviral delivery of IL-24.

6.1 Preclinical Studies Using DOTAP:chol mda7/IL-24 Several gene therapy-based clinical trials have been conducted for the treatment of numerous human diseases including cancer (Mhashilkar et  al. 2001). The most widely tested gene delivery vehicle has been the adenovirus. However, the drawback of adenovirus-based gene therapy is the ability of the virus to induce a host immune response, and potentially produce infectious virus (Kafri et  al. 1998; Yang et  al. 1994, 1996). Nonviral nanoparticle-based gene delivery systems provide an alternative gene therapy vehicle that avoids many of problems often encountered with adenovirus (Mhashilkar et al. 2001; Nabel et al. 1993; Niidome and Huang 2002). Numerous studies have documented the potential use of lipid-based nonviral vectors for gene transfer (Li et al. 2007; Liu et al. 1997; Pirollo et al. 2007; Xu et al. 2002).

11  Interleukin-24 Gene Therapy for Melanoma

195

Lipid-based nanoparticles as vehicles for gene delivery can be produced from various lipids or lipid mixtures that vary in charge, fluidity, and packing geometry. Cationic lipids are a preferred component for nucleic acid delivery, owing to the high efficiency of nucleic acid transfer associated with these lipid formulations. However, some cationic lipids are toxic when administered in vivo. The DOTAP: cholesterol (DOTAP:Chol.) lipid-based nanoparticles achieves a balance between toxicity and in vivo efficiencies. Early studies in murine models using DOTAP:Chol. nanoparticles at an equimolar ratio have demonstrated that these nanoparticles are efficient in vivo gene delivery vehicles (Gaensler et al. 1999; Lu et al. 2002; Shi et al. 2002; Templeton et al. 1997; Yotnda et al. 2004). Furthermore, the intravenous administration of DOTAP:Chol. nanoparticles carrying a reporter gene resulted in high levels of transgene expression in the mouse lung (Templeton et al. 1997). Crook et al. (1998) reported an increased in vitro delivery of nucleic acids in the presence of serum when DOTAP was mixed with the cholesterol. The presence of cholesterol in the formulation stabilizes the nanoparticles with regard to serum protein disruption and supports its use as an in vivo nucleic acid delivery vehicle. On the basis of these studies, we tested the use of DOTAP:Chol. nanoparticles as a systemic gene delivery vector in experimental mouse models. Preclinical studies from our laboratory demonstrated that tumor suppressor genes (p53, Fhit, Fus1, mda-7) encapsulated in DOTAP:Chol. nanoparticles were delivered effectively to lung tumor xenografts, which resulted in a therapeutic effect (Deng et al. 2008; It et  al. 2004a; Ramesh et  al. 2001, 2004b). Furthermore, we showed that these nanoparticles were selectively taken up by the tumors compared to surrounding normal lung tissues resulting in increased transgene expression in the tumors and an enhanced therapeutic effect (Ito et  al. 2003, 2004b). We also showed that these DNA-containing nanoparticles induced no significant toxicity in mice. On the basis of our preclinical findings demonstrating the systemic use of DOTAP:Chol. nanoparticles-based gene delivery for cancer therapy, we at M. D. Anderson Cancer Center have recently initiated a Phase I clinical trial for the systemic treatment of non-small cell lung cancer (Lu et al. 2007). In this trial, lung cancer patients, who previously failed chemotherapy, are intravenously administered DOTAP:Chol. nanoparticles carrying the Fus1 tumor suppressor gene. Initial results from this Phase I study demonstrated clinical responses in a few patients where doses were well tolerated and no treatment-associated toxicities were reported (Lu et al. 2007). One the basis of these findings, we propose to develop and test the mda-7/IL-24 gene encapsulated in DOTAP:Chol. nanoparticles as a systemic anticancer drug for metastatic melanoma.

6.2 Current Status of Non-viral Nanoparticles-Based Cancer Gene Therapy Clinical Trials Preclinical studies using targeted and nontargeted nonviral nanoparticles of different formulations administered systemically, have shown that nanoparticles are effective

196

N. Poindexter et al.

delivery systems for therapeutic genes resulting in anticancer effects (Li et al. 2007; Lu et al. 2002; Parekh 2007; Pirollo et al. 2007; Shi et al. 2002; Xu et al. 2002; Yotnda et al. 2004). However, very few of these studies have progressed from the laboratory to the clinic. One obstacle has been the inability to produce the formulation in a large scale for clinical utility. Additionally, despite the development of methods such as pegylation of the vector to reduce toxicity and increase in  vivo stability, complete elimination of toxicity has not been achieved and has hampered the testing of these non-viral vectors in the clinic. As of July 2007, only 12 nonviral vector-based clinical trials have been started and completed or are open for patient accrual (source: www.wiley.co.uk/genetherapy/clinical). Of the 12 trials, nine trials based on the intraperitoneal administration of the vector delivering E1A tumor suppressor gene (TSG) for breast and ovarian cancers were completed. These trials did not show any significant clinical benefit. In addition, treatment-related toxicity was reported in the ovarian and breast cancer trials (Hortobagyi et al. 1998, 2001). Only 3 of the 12 trials are currently focused on systemic gene delivery. The first one is our own Phase-I clinical trial delivering DOTAP:Chol.FUS1 nanoparticles systemically for lung cancer (Lu et al. 2007). The remaining two systemic trials, not yet started, aim to deliver p53 and Rb TSG using different lipid formulation for solid tumors. So, it is evident there are no any current systemic nanoparticles-based treatments for melanoma. Therefore, to successfully translate nanoparticles-based systemic treatment for melanoma in the clinic, we have begun to test the systemic delivery of IL-24-nanoparticles using human melanoma xenograft models. We believe that restoring expression of IL-24 protein, via DOTAP:Chol nanoparticles, in melanoma tumors will induce tumor-specific apoptosis, inhibit tumor cell growth, and delay time to progression in patients with metastatic melanoma.

7 Conclusion The multifunctional tumor-specific cytotoxic effects of IL-24, including tumor cell killing via intracellular pathways and bystander activities, immunomodulation, anti-angiogenesis, and direct receptor-mediated cytotoxicity make this molecule an excellent candidate for therapeutic use in melanoma. From the published report of the first Phase I/II Trial using Ad-mda-7/IL-24 as well as this report of the second Phase II Trial resulting in tumor regression at the injection site, it is apparent that IL-24 is a promising therapeutic. The development of systemic delivery of this gene is required for it to be effective against metastatic melanoma. DOTAP:Chol mda-7/ IL-24 nanoparticles are a possible solution to this problem. Melanoma often progresses rapidly from radial growth phase to vertical growth phase and in-transit metastasis, which leads to disseminated disease that is almost universally fatal. Given the limitations of current therapies for the treatment of metastatic melanoma, there is an urgent need for effective systemic treatments for this disease. Based on our preclinical data, we propose showing that treatment with IL-24 leads to tumor cell death as well as immune cell activation, that re-expression

11  Interleukin-24 Gene Therapy for Melanoma

197

of this tumor suppressor/cytokine into metastatic tumors will result in successful tumor regression. Although Ad-mda-7/IL-24 gene therapy has shown promise in preclinical and clinical studies, it is currently limited to treating localized tumors. A gene delivery system that is efficient, nonimmunogenic, and applicable for systemic therapy is the DOTAP:cholesterol nanoparticle. Ongoing preclinical studies from our group suggest that this may be a useful approach for the successful treatment of metastatic melanoma as well as other solid tumors.

References Anderson CM, Buzaid AC, Legha SS (1995) Systemic treatments for advanced cutaneous melanoma. Oncology. 9:1149–1158. Atkins MB (1997) The treatment of metastatic melanoma with chemotherapy and biologics [see comments]. Curr Opin Oncol. 9:205–213. Bedikian AY, Millward M, Pehamberger H et al. (2006) Bcl-2 antisense (oblimersen sodium) plus dacarbazine in patients with advanced melanoma: the Oblimersen Melanoma Study Group. J Clin Oncol. 24:4738–4745. Blumberg H, Conklin D, Xu WF et al. (2001) Interleukin 20: discovery, receptor identification, and role in epidermal function. Cell. 104:9–19. Bocangel D, Zheng M, Mhashilkar A et al. (2006) Combinatorial synergy induced by adenoviralmediated mda-7 and Herceptin in Her-2+ breast cancer cells. Cancer Gene Ther. 13:958–968. Caudell EG, Mumm JB, Poindexter N et al. (2002) The protein product of the tumor suppressor gene, melanoma differentiation-associated gene 7, exhibits immunostimulatory activity and is designated IL-24. J Immunol. 168:6041–6046. Chada S, Bocangel D, Ramesh R et al. (2005) mda-7/IL24 kills pancreatic cancer cells by inhibition of the Wnt/PI3K signaling pathways: identification of IL-20 receptor-mediated bystander activity against pancreatic cancer. Mol Ther. 11:724–733. Chada S, Menander KB, Bocangel D et al. (2008) Cancer targeting using tumor suppressor genes. Front Biosci. 13:1959–1967. Chada S, Mhashilkar AM, Liu Y et  al. (2006) mda-7 gene transfer sensitizes breast carcinoma cells to chemotherapy, biologic therapies and radiotherapy: correlation with expression of bcl-2 family members. Cancer Gene Ther. 13:490–502. Chada S, Mhashilkar AM, Ramesh R et  al. (2004a) Bystander activity of Ad-mda7: human MDA-7 protein kills melanoma cells via an IL-20 receptor-dependent but STAT3-independent mechanism. Mol Ther. 10:1085–1095. Chada S, Sutton RB, Ekmekcioglu S et  al. (2004b) MDA-7/IL-24 is a unique cytokine-tumor suppressor in the IL-10 Family. Int Immunopharmacol. 4:649–667. Chapman PB, Einhorn LH, Meyers ML et al. (1999) Phase III multicenter randomized trial of the Dartmouth regimen versus dacarbazine in patients with metastatic melanoma. J Clin Oncol. 17:2745–2751. Crook K, Stevenson BJ, Dubouchet M et al. (1998) Inclusion of cholesterol in DOTAP transfection complexes increases the delivery of DNA to cells in vitro in the presence of serum. Gene Ther. 5:137–143. Cunningham CC, Chada S, Merritt JA et  al. (2005) Clinical and local biological effects of an intratumoral injection of mda-7 (IL24; INGN 241) in patients with advanced carcinoma: a phase I study. Mol Ther. 11:149–159. Deng WG, Wu G, Ueda K et al. (2008) Enhancement of antitumor activity of cisplatin in human lung cancer cells by tumor suppressor FUS1. Cancer Gene Ther. 15:29–39. Dong CY, Zhang F, Duan YJ et al. (2008) mda-7/IL-24 inhibits the proliferation of hematopoietic malignancies in vitro and in vivo. Exp Hematol. 36:938–946.

198

N. Poindexter et al.

Dumoutier L, Leemans C, Lejeune D et al. (2001) Cutting edge: STAT activation by IL-19, IL-20 and mda-7 through IL-20 receptor complexes of two types. J Immunol. 167:3545–3549. Ekmekcioglu S, Ellerhorst J, Mhashilkar AM et al. (2001) Down-regulated melanoma differentiation associated gene (mda-7) expression in human melanomas. Int J Cancer. 94:54–59. Ekmekcioglu S, Ellerhorst JA, Mumm JB et al. (2003) Negative association of melanoma differentiation-associated gene (mda-7) and inducible nitric oxide synthase (iNOS) in human melanoma: MDA-7 regulates iNOS expression in melanoma cells. Mol Cancer Ther. 2:9–17. Ekmekcioglu S, Mumm JB, Udtha M et al. (2008) Killing of human melanoma cells induced by activation of class I interferon-regulated signaling pathways via MDA-7/IL-24. Cytokine. 43:34–44. Ellerhorst JA, Prieto VG, Ekmekcioglu S et al. (2002) Loss of MDA-7 expression with progression of melanoma. J Clin Oncol. 20:1069–1074. Eton O, Legha SS, Bedikian AY et al. (2002) Sequential biochemotherapy versus chemotherapy for metastatic melanoma: results from a phase III randomized trial. J Clin Oncol. 20: 2045–2052. Fisher PB, Gopalkrishnan RV, Chada S et  al. (2003) mda-7/IL-24, a novel cancer selective apoptosis inducing cytokine gene: from the laboratory into the clinic. Cancer Biol Ther. 2:S23-S37. Gaensler KM, Tu G, Bruch S et al. (1999) Fetal gene transfer by transuterine injection of cationic liposome-DNA complexes. Nat Biotechnol. 17:1188–1192. Gallagher G, Dickensheets H, Eskdale J et al. (2000) Cloning, expression and initial characterization of interleukin-19 (IL-19), a novel homologue of human interleukin-10 (IL-10). Genes Immun. 1:442–450. Gopalan B, Litvak A, Sharma S et al. (2005) Activation of the Fas-FasL signaling pathway by MDA-7/IL-24 kills human ovarian cancer cells. Cancer Res. 65:3017–3024. Gopalan B, Shanker M, Chada S et al. (2007) MDA-7/IL-24 suppresses human ovarian carcinoma growth in vitro and in vivo. Mol Cancer. 6:11–20. Gopalan B, Shanker M, Scott A et al. (2008) MDA-7/IL-24, a novel tumor suppressor/cytokine is ubiquitinated and regulated by the ubiquitin-proteasome system, and inhibition of MDA-7/ IL-24 degradation enhances the antitumor activity. Cancer Gene Ther. 15:1–8. Hortobagyi GN, Hung MC, Lopez-Berestein G (1998) A Phase I multicenter study of E1A gene therapy for patients with metastatic breast cancer and epithelial ovarian cancer that overexpresses HER-2/neu or epithelial ovarian cancer. Hum Gene Ther. 9:1775–1798. Hortobagyi GN, Ueno NT, Xia W et al. (2001) Cationic liposome-mediated E1A gene transfer to human breast and ovarian cancer cells and its biologic effects: a phase I clinical trial. J Clin Oncol. 19:3422–3433. Inoue S, Branch CD, Gallick GE et  al. (2005) Inhibition of Src kinase activity by Ad-mda7 suppresses vascular endothelial growth factor expression in prostate carcinoma cells. Mol Ther. 12:707–715. Inoue S, Shanker M, Miyahara R et al. (2006) MDA-7/IL-24-based cancer gene therapy: translation from the laboratory to the clinic. Curr Gene Ther. 6:73–91. Ishikawa S, Nakagawa T, Miyahara R et al. (2005) Expression of MDA-7/IL-24 and its clinical significance in resected non-small cell lung cancer. Clin Cancer Res. 11:1198–1202. Ito I, Began G, Mohiuddin I et al. (2003) Increased uptake of liposomal-DNA complexes by lung metastases following intravenous administration. Mol Ther. 7:409–418. Ito I, Ji L, Tanaka F et  al. (2004a) Liposomal vector mediated delivery of the 3p FUS1 gene demonstrates potent antitumor activity against human lung cancer in vivo. Cancer Gene Ther. 11:733–739. Ito I, Saeki T, Mohuiddin I et al. (2004b) Persistent transgene expression following intravenous administration of a liposomal complex: role of interleukin-10-mediated immune suppression. Mol Ther. 9:318–327. Ives NJ, Stowe RL, Lorigan P et al. (2007) Chemotherapy compared with biochemotherapy for the treatment of metastatic melanoma: a meta-analysis of 18 trials involving 2,621 patients. J Clin Oncol. 25:5426–5434.

11  Interleukin-24 Gene Therapy for Melanoma

199

Jiang H, Lin JJ, Su ZZ et  al. (1995) Subtraction hybridization identifies a novel melanoma differentiation associated gene, mda-7, modulated during human melanoma differentiation, growth and progression. Oncogene. 11:2477–2486. Jiang H, Su ZZ, Lin JJ et  al. (1996) The melanoma differentiation associated gene mda-7 suppresses cancer cell growth. Proc Natl Acad Sci U S A. 93:9160–9165. Kafri T, Morgan D, Krahl T et al. (1998) Cellular immune response to adenoviral vector infected cells does not require de novo viral gene expression: implications for gene therapy. Proc Natl Acad Sci U S A. 95:11377–11382. Kaliberova LN, Krendelchtchikova V, Harmon DK et al. (2009) CRAdRGDflt-IL24 virotherapy in combination with chemotherapy of experimental glioma. Cancer Gene Ther. Kawabe S, Nishikawa T, Munshi A et  al. (2002) Adenovirus-mediated mda-7 gene expression radiosensitizes non-small cell lung cancer cells via TP53-independent mechanisms. Mol Ther. 6:637–644. Knappe A, Hor S, Wittmann S et al. (2000) Induction of a novel cellular homolog of interleukin-10, AK155, by transformation of T lymphocytes with herpesvirus saimiri. J Virol. 74:3881–3887. Kotenko SV (2002) The family of IL-10-related cytokines and their receptors: related, but to what extent? Cytokine Growth Factor Rev. 13:223–240. Kreis S, Philippidou D, Margue C et  al. (2007) Recombinant interleukin-24 lacks apoptosisinducing properties in melanoma cells. PLoS ONE. 2:e1300 Leath CA, III, Kataram M, Bhagavatula P et al. (2004) Infectivity enhanced adenoviral-mediated mda-7/IL-24 gene therapy for ovarian carcinoma. Gynecol Oncol. 94:352–362. Lebedeva IV, Su ZZ, Chang Y et al. (2002) The cancer growth suppressing gene mda-7 induces apoptosis selectively in human melanoma cells. Oncogene. 21:708–718. Lebedeva IV, Washington I, Sarkar D et al. (2007) Strategy for reversing resistance to a single anticancer agent in human prostate and pancreatic carcinomas. Proc Natl Acad Sci U S A. 104:3484–3489. Li D, Tang GP, Li JZ et  al. (2007) Dual-targeting non-viral vector based on polyethylenimine improves gene transfer efficiency. J Biomater Sci Polym Ed. 18:545–560. Liao YC, Liang WG, Chen FW et al. (2002) IL-19 induces production of IL-6 and TNF-alpha and results in cell apoptosis through TNF-alpha. J Immunol. 169:4288–4297. Liu Y, Mounkes LC, Liggitt HD et  al. (1997) Factors influencing the efficiency of cationic liposome-mediated intravenous gene delivery. Nat Biotechnol. 15:167–173. Lu C, Sepulveda C, Ji L et al. (2007) Systemic nanoparticle FUS1 tumor suppressor gene therapy for stage IV lung cancer. American Association of Cancer Research. Lu H, Zhang Y, Roberts DD et al. (2002) Enhanced gene expression in breast cancer cells in vitro and tumors in vivo. Mol Ther. 6:783–792. Luikart SD, Kennealey GT, Kirkwood JM (1984) Randomized phase III trial of vinblastine, bleomycin, and cis-dichlorodiammine-platinum versus dacarbazine in malignant melanoma. J Clin Oncol. 2:164–168. Mahasreshti PJ, Kataram M, Wu H et  al. (2006) Ovarian cancer targeted adenoviral-mediated mda-7/IL-24 gene therapy. Gynecol Oncol. 100:521–532. Mhashilkar AM, Schrock RD, Hindi M et al. (2001) Melanoma differentiation associated gene-7 (mda-7): a novel anti-tumor gene for cancer gene therapy. Mol Med. 7:271–282. Mhashilkar AM, Stewart AL, Sieger K et al. (2003) MDA-7 negatively regulates the beta-catenin and PI3K signaling pathways in breast and lung tumor cells. Mol Ther. 8:207–219. Middleton MR, Grob JJ, Aaronson N et al. (2000) Randomized phase III study of temozolomide versus dacarbazine in the treatment of patients with advanced metastatic malignant melanoma. J Clin Oncol. 18:158–166. Miyahara R, Banerjee S, Kawano K et  al. (2006) Melanoma differentiation-associated gene-7 (mda-7)/interleukin (IL)-24 induces anticancer immunity in a syngeneic murine model. Cancer Gene Ther. 13:753–761. Mumm JB, Ekmekcioglu S, Poindexter NJ et al. (2006) Soluble human MDA-7/IL-24: characterization of the molecular form(s) inhibiting tumor growth and stimulating monocytes. J Interferon Cytokine Res. 26:877–886.

200

N. Poindexter et al.

Nabel GJ, Nabel EG, Yang ZY et al. (1993) Direct gene transfer with DNA-liposome complexes in melanoma: expression, biologic activity, and lack of toxicity in humans. Proc Natl Acad Sci U S A. 90:11307–11311. Niidome T, Huang L (2002) Gene therapy progress and prospects: nonviral vectors. Gene Ther. 9:1647–1652. Nishikawa T, Ramesh R, Munshi A et al. (2004) Adenovirus-mediated mda-7 (IL24) gene therapy suppresses angiogenesis and sensitizes NSCLC xenograft tumors to radiation. Mol Ther. 9:818–828. Oida Y, Gopalan B, Miyahara R et  al. (2007) Inhibition of nuclear factor-kappaB augments antitumor activity of adenovirus-mediated melanoma differentiation-associated gene-7 against lung cancer cells via mitogen-activated protein kinase kinase kinase 1 activation. Mol Cancer Ther. 6:1440–1449. Oida Y, Gopalan B, Miyahara R et  al. (2005) Sulindac enhances adenoviral vector expressing mda-7/IL-24-mediated apoptosis in human lung cancer. Mol Cancer Ther. 4:291–304. Parekh HS (2007) The advance of dendrimers--a versatile targeting platform for gene/drug delivery. Curr Pharm Des. 13:2837–2850. Park MA, Walker T, Martin AP et al. (2009) MDA-7/IL-24-induced cell killing in malignant renal carcinoma cells occurs by a ceramide/CD95/PERK-dependent mechanism. Mol Cancer Ther. Pataer A, Vorburger SA, Barber GN et al. (2002) Adenoviral transfer of the melanoma differentiation-associated gene 7 (mda7) induces apoptosis of lung cancer cells via up-regulation of the double-stranded RNA-dependent protein kinase (PKR). Cancer Res. 62:2239–2243. Pirollo KF, Rait A, Zhou Q et al. (2007) Materializing the potential of small interfering RNA via a tumor-targeting nanodelivery system. Cancer Res. 67:2938–2943. Poindexter N, Williams R, Powis G et  al. (2007) IL-24 and Its Role in Wound Healing. J Immunother. 30:877–877. Poindexter NJ, Walch ET, Chada S et al. (2005) Cytokine induction of interleukin-24 in human peripheral blood mononuclear cells. J Leukoc Biol. 78:745–752. Qian W, Liu J, Tong Y et  al. (2008) Enhanced antitumor activity by a selective conditionally replicating adenovirus combining with MDA-7/interleukin-24 for B-lymphoblastic leukemia via induction of apoptosis. Leukemia. 22:361–369. Ramesh R, Ito I, Gopalan B et al. (2004a) Ectopic production of MDA-7/IL-24 inhibits invasion and migration of human lung cancer cells. Mol Ther. 9:510–518. Ramesh R, Ito I, Saito Y et al. (2004b) Local and systemic inhibition of lung tumor growth after nanoparticle-mediated mda-7/IL-24 gene delivery. DNA Cell Biol. 23:850–857. Ramesh R, Mhashilkar AM, Tanaka F et al. (2003) Melanoma differentiation-associated gene 7/ interleukin (IL)-24 is a novel ligand that regulates angiogenesis via the IL-22 receptor. Cancer Res. 63:5105–5113. Ramesh R, Saeki T, Templeton NS et al. (2001) Successful treatment of primary and disseminated human lung cancers by systemic delivery of tumor suppressor genes using an improved liposome vector. Mol Ther. 3:337–350. Rosenberg SA, Yang JC, Schwartzentruber DJ et al. (1999) Prospective randomized trial of the treatment of patients with metastatic melanoma using chemotherapy with cisplatin, dacarbazine, and tamoxifen alone or in combination with interleukin-2 and interferon alfa-2b. J Clin Oncol. 17:968–975. Saeki T, Mhashilkar A, Chada S et  al. (2000) Tumor-suppressive effects by adenovirusmediated mda-7 gene transfer in non-small cell lung cancer cell in  vitro. Gene Ther. 7: 2051–2057. Saeki T, Mhashilkar A, Swanson X et al. (2002) Inhibition of human lung cancer growth following adenovirus-mediated mda-7 gene expression in vivo. Oncogene. 21:4558–4566. Sainz-Perez A, Gary-Gouy H, Portier A et al. (2006) High Mda-7 expression promotes malignant cell survival and p38 MAP kinase activation in chronic lymphocytic leukemia. Leukemia. Sarkar D, Lebedeva IV, Su ZZ et al. (2007) Eradication of therapy-resistant human prostate tumors using a cancer terminator virus. Cancer Res. 67:5434–5442.

11  Interleukin-24 Gene Therapy for Melanoma

201

Sarkar D, Su ZZ, Lebedeva IV et al. (2002) mda-7 (IL-24) Mediates selective apoptosis in human melanoma cells by inducing the coordinated overexpression of the GADD family of genes by means of p38 MAPK. Proc Natl Acad Sci U S A. 99:10054–10059. Sarkar D, Su ZZ, Park ES et  al. (2008) A cancer terminator virus eradicates both primary and distant human melanomas. Cancer Gene Ther. 15:293–302. Sauane M, Su ZZ, Gupta P et al. (2008) Autocrine regulation of mda-7/IL-24 mediates cancerspecific apoptosis. Proc Natl Acad Sci U S A. 105:9763–9768. Shanker M, Gopalan B, Patel S et  al. (2007) Vitamin E succinate in combination with mda-7 results in enhanced human ovarian tumor cell killing through modulation of extrinsic and intrinsic apoptotic pathways. Cancer Lett. 254:217–226. Sheikh F, Baurin VV, Lewis-Antes A et  al. (2004) Cutting edge: IL-26 signals through a novel receptor complex composed of IL-20 receptor 1 and IL-10 receptor 2. J Immunol. 172:2006–2010. Shi HY, Liang R, Templeton NS et al. (2002) Inhibition of breast tumor progression by systemic delivery of the maspin gene in a syngeneic tumor model. Mol Ther. 5:755–761. Sieger KA, Mhashilkar AM, Stewart A et al. (2004) The tumor suppressor activity of MDA-7/ IL-24 is mediated by intracellular protein expression in NSCLC cells. Mol Ther. 9:355–367. Soo C, Shaw WW, Freymiller E et al. (1999) Cutaneous rat wounds express c49a, a novel gene with homology to the human melanoma differentiation associated gene, mda-7. Journal of Cellular Biochemistry 74:1–10. Su Z, Lebedeva IV, Gopalkrishnan RV et  al. (2001) A combinatorial approach for selectively inducing programmed cell death in human pancreatic cancer cells. Proc Natl Acad Sci U S A. 98:10332–10337. Su Z, Emdad L, Sauane M et  al. (2005) Unique aspects of mda-7/IL-24 antitumor bystander activity: establishing a role for secretion of MDA-7/IL-24 protein by normal cells. Oncogene. 24:7552–7566. Su ZZ, Madireddi MT, Lin JJ et al. (1998) The cancer growth suppressor gene mda-7 selectively induces apoptosis in human breast cancer cells and inhibits tumor growth in nude mice. Proc Natl Acad Sci U S A. 95:14400–14405. Templeton NS, Lasic DD, Frederik PM et  al. (1997) Improved DNA: liposome complexes for increased systemic delivery and gene expression. Nat Biotechnol. 15:647–652. Tong AW, Nemunaitis J, Su D et al. (2005) Intratumoral injection of INGN 241, a nonreplicating adenovector expressing the melanoma-differentiation associated gene-7 (mda-7/IL24): biologic outcome in advanced cancer patients. Mol Ther. 11:160–172. Wang M, Tan Z, Zhang R et al. (2002) Interleukin 24 (MDA-7/MOB-5) signals through two heterodimeric receptors, IL-22R1/IL-20R2 and IL-20R1/IL-20R2. J Biol Chem. 277:7341–7347. Wang X, Ye Z, Zhong J et al. (2007) Adenovirus-mediated Il-24 expression suppresses hepatocellular carcinoma growth via induction of cell apoptosis and cycling arrest and reduction of angiogenesis. Cancer Biother Radiopharm. 22:56–63. Wang XS, Shi Q, Williams LA et  al. (2008) Serum interleukin-6 predicts the development of multiple symptoms at nadir of allogeneic hematopoietic stem cell transplantation. Cancer. 113:2102–2109. Xie MH, Aggarwal S, Ho WH et  al. (2000) Interleukin (IL)-22, a novel human cytokine that signals through the interferon receptor-related proteins CRF2-4 and IL-22R. J Biol Chem. 275:31335–31339. Xie Y, Sheng W, Xiang J et al. (2008) Recombinant human IL-24 suppresses lung carcinoma cell growth via induction of cell apoptosis and inhibition of tumor angiogenesis. Cancer Biother Radiopharm. 23:310–320. Xiong J, Peng ZL, Tan X (2007a) [Effects of adenoviral-mediated mda-7/IL-24 gene infection on the growth and drug-resistance of drug-resistant]. Sichuan Da Xue Xue Bao Yi Xue Ban. 38:433-436. Xiong J, Peng ZL, Tan X et  al. (2007b) [Effect of adenovirus-mediated mda-7/IL-24 gene infection on apoptosis of drug-resistant human ovarian cancer cell lines OVCAR-3 and OVCAR-8/TR]. Ai Zheng. 26:371–376.

202

N. Poindexter et al.

Xu L, Huang CC, Huang W et al. (2002) Systemic tumor-targeted gene delivery by anti-transferrin receptor scFv-immunoliposomes. Mol Cancer Ther. 1:337–346. Yacoub A, Mitchell C, Lebedeva IV et  al. (2003) mda-7 (IL-24) Inhibits growth and enhances radiosensitivity of glioma cells in vitro via JNK signaling. Cancer Biol Ther. 2:347–353. Yang Y, Ertl HC, Wilson JM (1994) MHC class I-restricted cytotoxic T lymphocytes to viral antigens destroy hepatocytes in mice infected with E1-deleted recombinant adenoviruses. Immunity. 1:433–442. Yang Y, Jooss KU, Su Q et al. (1996) Immune responses to viral antigens versus transgene product in the elimination of recombinant adenovirus-infected hepatocytes in  vivo. Gene Ther. 3:137–144. Yang YJ, Chen DZ, Li LX et al. (2009) Targeted IL-24 gene therapy inhibits cancer recurrence after liver tumor resection by inducing tumor cell apoptosis in nude mice. Hepatobiliary Pancreat Dis Int. 8:174–178. Yotnda P, Davis AR, Hicks MJ et al. (2004) Liposomal enhancement of the antitumor activity of conditionally replication-competent adenoviral plasmids. Mol Ther. 9:489–495. Zhang KJ, Wang YG, Cao X et al. (2009) Potent Antitumor Effect of Interleukin-24 Gene in the Survivin Promoter and Retinoblastoma Double-Regulated Oncolytic Adenovirus. Hum Gene Ther. Zheng M, Bocangel D, Doneske B et  al. (2007) Human interleukin 24 (MDA-7/IL-24) protein kills breast cancer cells via the IL-20 receptor and is antagonized by IL-10. Cancer Immunol Immunother. 56:205–215. Zheng M, Bocangel D, Ramesh R et al. (2008) Interleukin-24 overcomes temozolomide resistance and enhances cell death by down-regulation of O6-methylguanine-DNA methyltransferase in human melanoma cells. Mol Cancer Ther. 7:3842–3851.

Chapter 12

Herpes Simplex Virus 1 for Cancer Therapy Richard L. Price, Balveen Kaur, and E. Antonio Chiocca

Abstract  The obvious inadequacy of conventional therapies to combat cancer has spawned the development of a variety of nonconventional treatment modalities such as cancer-specific replication competent viruses. Herpes simplex virus 1 (HSV1) is a traditionally pathologic lytic virus. It forms latent infection in sensory ganglion and can recur in the form of herpetic skin lesions or even deadly encephalitis. A little under 20 years ago, scientists discovered that certain mutant HSV1 can selectively replicate and lyse neoplastic cells while sparing normal cells. Intense research has uncovered a variety of genetic alterations in the HSV1 genome that render it to have cancer cell-specific replication. Modifications include deleting nonessential regulatory proteins, proteins involved with nucleic acid metabolism, and host immunomodulating proteins. Five oncolytic HSV1 viruses are currently in clinical trials. So far, 200 patients with various types of cancer have been safely treated and ongoing large Phase III clinical trials will determine the potential for therapeutic efficacy. In this chapter, we will discuss the development of each of these HSV1derived viruses and the results of their clinical testing in human patients. Keywords  Herpes simplex virus 1 • Oncolytic virus • Glioblastoma • Biological cancer therapy

1 Herpes Simplex Virus 1 for Cancer Therapy Cancer is a disease that will affect one in three people during their lifetime. Since Richard Nixon declared War against Cancer in 1971, many advances have been made in understanding the molecular genetic basis of tumor growth and

E.A. Chiocca (*) Dardinger Laboratory for Neuro-oncology and Neurosciences, Department of Neurological Surgery, James Comprehensive Cancer Center and The Ohio State University Medical Center, Suite 385B Wiseman Hall/CCC, 400 West 12th Avenue, Columbus, OH, USA e-mail: [email protected] J.A. Roth (ed.), Gene-Based Therapies for Cancer, Current Cancer Research, DOI 10.1007/978-1-4419-6102-0_12, © Springer Science+Business Media, LLC 2010

203

204

R.L. Price et al.

p­ rogression as well as toward engineering new therapeutics to control the disease. Despite some advances in diagnosis and therapy, cancer remains a devastating disease for most patients and their families. Unconventional therapies such as viral, cellular, and gene therapies hold tremendous promise as possible treatments and cures. Oncolytic viral therapy is one such “unconventional” treatment modality that relies on cancer cell-specific replication of a virus leading to cancer cell lytic destruction. Less than 20 years ago, an attenuated Herpes Simplex Virus 1 (HSV1) was demonstrated to have oncolytic ability against glioblastomas in vitro and in mice (Martuza et  al. 1991). Since that crucial discovery, several HSV1derived viruses have been developed and are currently being tested in clinical trials. In this chapter, we will focus on the different oncolytic HSV1-based therapies being investigated in clinical trials.

2 Herpes Simplex Virus 1 HSV1 is a double-stranded enveloped DNA virus that can infect many types of human cells. The properties of HSV1 that make it a good candidate to be reprogrammed for oncolytic viral therapy include the following: (1) It is a well-characterized genome, (2) it has inherent cytolytic ability, (3) up to 30 kb of DNA can be replaced by transgenes, (4) it does not incorporate in the host genome, (5) it infects most types of tumor cells, (6) low multiplicity of infection (MOI) is needed for total cytotoxicity, (7) host immune reactions may enhance tumor cell death, (8) several antiherpetic drugs are available to control infection, and (9) there are well-established animal models which mimic human infection. The genome of HSV1 encodes for 74 proteins in 152 Kbp of genomic material (Fig. 1a). On the basis of their function, genes are divided into alpha, beta, or gamma genes. Alpha genes are the first to be transcribed and help regulate gene transcription. The function of beta genes is to promote viral DNA synthesis. And finally, gamma genes help to modulate the environment to promote favorable viral protein translation. Of these genes, 40 “core” genes are required for replication and are conserved in all viruses in the herpes family. The remaining genes are known as accessory genes and are not considered to be essential for viral replication (Roizman 1996). The very well-characterized genome of HSV1 permits genetic manipulations essential to reprogram this normally pathogenic virus to selectively infect and destroy cancer cells (Fig. 1a). To achieve this end, the deletion of some accessory gene functions, including regulatory proteins, proteins involved with nucleic acid meta­bolism, and host immunomodulating proteins, have been tested. Some of these mutant viruses demonstrate severely compromised viral replication in normal cells, but replicate efficiently in cancer cells. In this first section, we will discuss some of the genes that have been deleted to generate oncolytic viruses and will then review the safety and efficacy results from their use in clinics. Later, we will describe oncolytic HSV1 clinical trials to date.

12  Herpes Simplex Virus 1 for Cancer Therapy

205

Fig.  1  (a) Schematic representation of the HSV1 genome. It is composed of a long and short region. The terminal ends of each region are repeated on the respective end of each region. This creates a duplication of the end of each region (note gamma34.5). Highlighted are genes that are modified in oncolytic HSV1 currently in clinical trials. (b) Method of action of ICP34.5. PKR in infected cells phosphorylate eIF2a to halt protein synthesis as a way to control viral replication. HSV1 circumvents this defense mechanism via ICP34.5. This protein binds PP1 to dephosphorylates eIF2a, which allows protein translation to resume. (c) HSV1 deficient in ribonucleotide reductase deficiency replicates preferentially in cells lacking p16. Cells lacking p16 allow the constitutive activation of E2F1 and transcription of mammalian ribonucleotide reductase that compensates for inherent viral deficiency. (d) Bystander effect. Treating HSV1 tumor cells with gancyclovir leads to a spread of toxic metabolites to neighboring cells leading to enhanced tumor death. (e) ICP47 mechanism. In an infected cell, viral peptides are transported from the cytoplasm to the endoplasmic reticulum (ER) via TAP1/2. In the ER, the peptides are loaded onto MHC class I molecules and are translocated to the cell surface to present to CD8+ cytotoxicity cells. ICP47 inhibits TAP1/2 resulting in a decrease of MHC class I expression and thus a decreased immune response

3 Thymidine Kinase (UL23/ICP36) Thymidine kinase (TK) is a beta protein that is found in the UL23 region of the genome (Fig. 1). It is active during DNA synthesis, functioning to phosphorylate deoxythymidine to form deoxythymidine 5¢-phosphate. Despite its role in DNA

206

R.L. Price et al.

synthesis, it has been found to be dispensable for viral replication in all cells (Field and Wildy 1978; Jamieson et al. 1974). Coen and colleagues (1989) found that TK was not required for HSV1 replication in mouse corneas and that tk-deficient virus could form latent infections in mouse ganglia. Cellular TK expressed in dividing cells can compensate for the lack of virus-encoded TK, and thus tk-deficient virions could replicate effectively in dividing cells. In 1991, Dr. Robert Martuza tested the intrinsic ability of dlsptk, a TK-defective HSV1, to selectively replicate in dividing cancer cells (Martuza et al. 1991). In this study, dlsptk effectively killed short-term and long-term glioma cell populations in culture. Furthermore, tests in athymic mice revealed that the oncolytic virus could inhibit U87 glioma cells implanted subcutaneously and that the virus prolonged the life of mice injected intracranially with U87 glioma cells when administered intratumorally (Martuza et  al. 1991). Importantly, it was the first bit of evidence that HSV1 viruses could be exploited to be oncolytic. In a subsequent study, another variant of tk-deficient HSV1, dl18.36tk, was shown to cause regression or substantially reduce brain tumors in immunocompetent rats (Kaplitt et  al. 1994). This oncolytic virus strain contains a 360  bp deletion of the tk gene. Importantly, this virus did not cause pathology upon viral injection and the modified virus produced as much progeny as wild type virus in growing cells. Unlike its mammalian counterpart, TK from HSV1 has the ability to phosphorylate and convert some nucleotide analogs into toxic metabolites. This property has been exploited in the creation of several antiherpetic drugs, such as acyclovir and gancyclovir. Thus tk-defective viruses are insensitive to these powerful drugs designed to control HSV1 infection. Hence, despite promising preclinical activity, concerns about their resistance to commonly used antiherpetic drugs have limited further clinical development of tk-deficient HSV1 as oncolytic agents.

4 ICP 34.5 (RL1) RL1 was identified to encode for ICP34.5 by Chou et al. (1990). Since it is present within the “ab” inverted repeat sequences, which flank the unique long (UL) sequence, two copies of the gene are present in each HSV1 genome (Ackermann et al. 1986; Chou and Roizman 1986, 1990). The gene encodes for a 263 amino acid protein with a 159 amino acid N-terminal domain and a 74 amino acid carboxy-terminal domain (Chou and Roizman 1990). Upon infection, cells activate an antiviral response initiated by the activation of RNA-dependent protein kinase (PKR), a known tumor suppressor (Koromilas et  al. 1992; Meurs et  al. 1993). Activated PKR kinase complexes with p90 phosphoprotein phosphorylates cellular eukaryotic translation initiation factor 2-alpha (eIF2a) which leading to the shutoff of host protein synthesis (Fig.  1b) (Chou et  al. 1995; Clemens and Elia 1997; Coffey et al. 1998). HSV1 encoded ICP34.5 binds and activates PP1 which dephosphorylates eIF2a, thus alleviating the host shutoff of protein synthesis and

12  Herpes Simplex Virus 1 for Cancer Therapy

207

permitting viral replication (Chou and Roizman 1992; He et al. 1998). The role of ICP34.5 in countering cellular PKR is bolstered by the observation that the deletion of cellular PKR is sufficient to restore the neurovirulence of ICP34.5-deficient HSV1 (Leib et al. 2000). The PKR-mediated translation shutoff occurs after the expression of alpha genes but before the gamma genes, suggesting that the signal is linked to viral DNA synthesis (He et  al. 1996). Interestingly, restoration of protein translation is not affected by mutations in the N-terminal portion of the ICP34.5 protein but is ablated by carboxy terminal mutations (Chou and Roizman 1994). The carboxy-terminal is homologous to mammalian GADD34, a protein that is induced by conditions that favor growth arrest (Fornace et al. 1989). ICP34.5 has also been shown to form a complex with proliferating cell nuclear antigen (PCNA), a protein involved in DNA replication and repair. The formation of this complex allows for cellular DNA replication to proceed, creating an environment conducive for viral replication (Brown et al. 1997). ICP34.5 is not necessary for viral replication in cells that have active PCNA (i.e. dividing cells). In a study by Detta et al. (2003) they showed that metastatic tumors that expressed PCNA supported viral replication. This suggests that for oncolytic viruses deficient in ICP34.5 to replicate and be effective, a tumor must express PCNA. The ability of ICP34.5 to disarm cellular antiviral responses allows virus replication even in terminally differentiated nerve terminals, and is thus considered to be the neurovirulence gene (Chou et  al. 1990; Thompson et  al. 1983). Accordingly, ICP34.5 mutants replicate about 100,000 times less in neurons compared to wildtype virus. Despite the attenuated replication in normal neurons, ICP34.5-deleted HSV1 was found to infect and replicate in neoplastic cells leading to eventual lysis (Kesari et al. 1995; Kucharczuk 1997; Randazzo et al. 1995). Such observations led to the development of ICP34.5-deficient HSV1 as therapeutic agents for oncolytic viral therapy for various cancers (Mineta et al. 1995). Several HSV1 mutants were tested in a study by Markert and colleagues (1993) to determine an effective glioma killing virus with a safe profile. They tested two viruses with point mutations in the viral DNA polymerase, one recombinant virus with at least two attenuating lesions, and R3616 that has mutations in both copies of gamma34.5. R3616 was effective at killing both long-term glioma lines and early passage human gliomas and showed antitumor activity in a subcutaneous glioma model. Also, the virus enhanced the life of athymic mice with intracranial gliomas compared with controls with no residual tumor found in four out of six surviving mice (Markert et al. 1993). Many HSV1 vectors used for cancer gene therapy harbor mutations in one or both copies of gamma34.5. The attenuated replication of ICP34.5 mutants results in lower viral yields, which translates to reduced efficacy in  vivo (McKie et  al. 1996; Todo et al. 2001). Newer transcriptionally targeted oncolytic viruses being tested in preclinical models include viruses expressing ICP34.5, albeit under tumorspecific promoters (see review by (Hardcastle et al. 2007)). Such transcriptionally targeted viruses retain the ICP34.5-deficient safety profile in normal cells but allow for ICP34.5 expression and increased viral replication in tumor cells.

208

R.L. Price et al.

5 Ribonucleotide Reductase (ICP 6) Mammalian Ribonucleotide Reductase (RR) is a heterodimeric enzyme composed of one large subunit (R1) and one small subunit (R2) that catalyzes the reduction of ribonucleotides to their corresponding deoxyribonucleotides, essential for DNA synthesis (Bacchetti et al. 1986; Thelander and Reichard 1979). In mammalian cells, expression is tightly regulated with cell cycling, and is undetectable in quiescent cells (Thelander and Reichard 1979). HSV1 UL39 encodes for ICP6, the viral counterpart of mammalian large subunit of RR, which facilitates DNA synthesis and viral replication in quiescent cells (Honess and Roizman 1973, 1974). The virulence of ICP6-deleted HSV1 is reduced by 106-fold compared to wild-type virus indicating that it contributes towards for pathogenicity in adult mice (Cameron et al. 1988; Yamada et  al. 1991). Whereas, an RR-deficient HSV1 with RR restored is within 100-fold as virulent as wild-type HSV1; however, viral RR expression is not required for HSV1 propagation in cells in culture, indicating that mammalian RR could compensate for the lack of ICP6 (Goldstein and Weller 1988b). The expression of cellular RR in neoplastic cells compensates for the loss of viral RR in HSV1 deficient for UL39, hence allowing for virus replication in cancer-specific cells (Goldstein and Weller 1988a; Preston et al. 1984). This suggests that HSV1 lacking RR can replicate in dividing cells, which express high levels of mammalian RR. More recently, RR expression was found to depend on p16 status of cells. Deletions in the p16 tumor suppressor were found to increase endogenous levels of RR even in quiescent embryonic fibroblasts (Fig. 1c) (Aghi et al. 2008). p16 is a tumor suppressor gene that prevents E2F1 from transcribing downstream genes, such as RR (Elledge et  al. 1992; Kamiryo et  al. 2002). Thus p16 loss facilitates high levels of cellular RR expression, which supports the replication of RR deleted HSV1. These observations led to investigations testing the ability of UL39 deleted viruses as oncolytic viruses. In order to better study the function and impact of this protein on viral replication, Goldstein and Weller created a mutant HSV1 (hrR3) with an in frame gene disrupting insertion of beta-galactosidase gene within the ICP6 locus, of a KOS strain HSV. The LacZ cassette ablates ICP6 gene activity while producing LacZ instead, which allows viral activity to be visualized with X-Gal staining (Goldstein and Weller 1988a). Combination with TK targeting gancyclovir in conjunction with hrR3 has also shown efficacy in animal models of tumor (Boviatsis et  al. 1994; Carroll et  al. 1997). The administration of gancyclovir led to the destruction of infected tumor cells as well as uninfected tumor cells by a bystander effect (Fig. 1d). More recently, the combination of RR-deleted viruses with antiangiogenic agents has also shown promise for therapy (Kurozumi et al. 2007).

6 ICP 47 Throughout the course of evolution, viruses have devised methods for escaping host immunity in order to survive and spawn progeny. HSV1 is no exception. Several viruses, including HSV1, have devised ways to downregulate MHC class I ­molecules

12  Herpes Simplex Virus 1 for Cancer Therapy

209

(Gooding 1992; McFadden and Kane 1994). MHC class I molecules on the cell surface are involved in antigen presentation to cytotoxic T cells to activate killing of the infected host cell. HSV1 has been shown to downregulate the cell surface expression of MHC class I molecules on the surface of infected cells (Hill et  al. 1994; Jennings et al. 1985). The reduced expression of MHC class I upon HSV1 infection hence results in a reduced cytotoxic CD8+ T cell host immune response, which is corroborated in studies that show humans infected with HSV1 show a greater CD4+ than CD8+ cell response to the infection (Schmid and Rouse 1992). This phenomenon is mediated by ICP 47, a nonessential gene that is encoded by an immediate early (alpha) HSV1 gene product (Mavromara-Nazos et al. 1986; Umene 1986). ICP47 blocks the function of the transporter associated with antigen presentation (TAP) 1 and 2, which translocate peptides from the cytoplasm to the endoplasmic reticulum for loading onto MHC class I molecules (Früh et al. 1995; Hill et al. 1995; York et al. 1994). Blockade of TAP function results in the accumulation of unloaded MHC class I molecules in the endoplasmic reticulum which are then targeted for degradation. This effectively reduces viral antigenic expression on the cell, hence limiting the recognition and elimination of virally infected cells by CD8+ cytotoxic T cells (Fig. 1e). The loss of ICP47 in the HSV1 genome also results in the early activation of viral gene US11 (He et al. 1997; Mohr and Gluzman 1996). US11 expressed early in the viral replication cycle can also bind to and inactivate antiviral cellular PKR. Thus, while the loss of ICP47 permits for MHC class I processing, which can be exploited to increase antitumor immune responses, the consequent early expression of US11 also supports efficient viral replication in infected cells. This leads to a more potent antitumor response against the tumor cell. G47D is an ICP47 and ICP34.5-deleted oncolytic HSV1 is currently being investigated in preclinical studies for safety and efficacy (Todo et al. 2001). This virus has shown promise as an anticancer therapy alone and in combination with chemotherapy when tested in animal models of brain tumors and prostate adenocarcinomas (Todo et al. 2001) (Fukuhara et al. 2005a, b). As discussed later in this chapter ongoing clinical trials using OncoVEX are starting to show that mutations in ICP47 are safe and well tolerated in the host (Hu et al. 2006).

7 HSV1 Viruses in Clinical Trials for Cancer 7.1 G207 G207 was the first HSV1 oncolytic virus to be cleared for human trials in North America. It is derived from the HSV1 wild type F strain with attenuating deletions in both copies of ICP34.5 gene as well as a gene disrupting beta-galactosidase (bGal) insertion within the viral RR gene (Fig. 2, Dix et al. 1983; Mineta et al. 1995). The ablation of ICP34.5 protein made the virus sensitive to PKR neutralization and the disruption of RR further decreased virus replication (Chou and Roizman 1992;

210

R.L. Price et al.

Honess and Roizman 1973). The beta galactosidase insertion also permits for a simple method to track virally infected cells in the tissue upon subsequent replications. Furthermore, retaining the tk gene allows for gancyclovir hypersensitivity, thus allowing for a way of controlling the virus as well as enhanced tumor bystander killing (Boviatsis et  al. 1994). Hence, the resulting oncolytic virus is severely attenuated in its replication and represents the second generation of HSV1 oncolytic viruses that were tested in clinics. For these reasons, G207 has many favorable properties to be used for cancer therapy. Before being tested in human patients, G207 was investigated for antitumor efficacy in rodents. In initial experiments, the use of G207 showed the killing of glioma cells in culture as well as killing subcutaneous gliomas placed in the flanks of mice (Mineta et al. 1995). Later experiments showed further efficacy in combating other types of cancer including lung, meningioma and neuroblastoma, breast, hepato­ cellular carcinoma, squamous cell carcinoma, and prostate (Chahlavi et al. 1999; Kanai et al. 2006; Song et al. 2006; Toda et al. 1998; Yazaki et al. 1995). Apart from tumor cells, G207 has also been shown to be able to effectively infect, replicate, and lyse endothelial cells. Thus, along with the ability to kill cancer, it can also function as an antiangiogenic agent (Cinatl et  al. 2004). While it may have a direct antiangiogenic effect, more recent studies by Dr. Martuza’s and our laboratory have also revealed that infection with G207 can also switch the homeostatic balance maintained between angiogenic and angiostatic molecules to increase angiogenesis after viral clearance (Aghi et al. 2007; Kurozumi et al. 2007). Also, G207 has been shown to replicate more efficiently in hypoxic environments, which is common in tumor cores (Aghi et al. 2009; Thomlinson and Gray 1955). Preclinical safety for G207 was initially tested in rodents. Mice with intracerebral and intraventricular inoculation of G207 (up to 107 p.f.u.) did not demonstrate any deleterious symptoms for over 20 weeks (Sundaresan et al. 2000). And upon sacrifice, their brain did not show any abnormal pathology. After demonstrating safety in mice, G207 was inoculated into Aotus monkeys, a model organism to test HSV1 safety (Hunter et al. 1999; Todo et al. 2000). These nonhuman primates are very sensitive to HSV1; an inoculation of only 103 p.f.u. of F strain has proven to be fatal. Titers up to 109 p.f.u. were injected intracranially with no evidence of complications, viral shedding, or histological changes. Also, intraprostatic inoculations up to 107 were tested in Aotus for use of G207 for prostate cancer in humans (Varghese et  al. 2001). More recently, the safety of G207 administration in the developing mammalian brain was tested by intracerebral inoculation of G207 in 4-day-old mice (Radbill et al. 2007). No significant difference in long-term physical development, cognitive performance, or exploratory behaviors was found in mice treated with G207 compared to control mice. However, unilateral ventriculomegaly ipsilateral to the site of injection was observed frequently in mice treated with G207, indicating that an initial study of G207 in children should exclude those patients with tumors in or near the ventricular system as well as patients less than 2 years of age. Between 1998 and 2000, 21 patients with anaplastic astrocytomas or glioblastoma multiformes (GBMs) were enrolled in the first trial (Markert et  al. 2000).

12  Herpes Simplex Virus 1 for Cancer Therapy

211

Patients that failed traditional treatment strategies were assigned to escalating doses between 106 and 109 p.f.u. of G207 to test safety of the virus in humans (Table 1). Inoculations were performed intracranially immediately following tumor resection. During this safety and dose escalation trial, no patient showed evidence of toxic effects or adverse events that could be unequivocally ascribed to G207 administration. Significantly, no patients showed the symptoms of viral encephalitis. In some patients, there was evidence of antitumor activity and viral DNA present in tumor remnants months after inoculation. In a subsequent Phase Ib trial, six patients were enrolled to test the safety of two separate inoculations of G207 (Markert et al. 2009). In this trial, patients were subjected to 1.15 × 109 p.f.u. of G207 in two separate doses. The first dose, 13% of the total dose, was administered 2 or 5 days via a stereotactic injected catheter before resection of the tumor. Postresection, the remaining dose was injected into the resection cavity (Table 1). This trial was designed to answer several questions including the safety of multiple injections and viral replication in the tumor. Though one patient experienced decreased mental status, increased temperature, and left hemiparesis that was successfully treated with dexamethasone; nobody opted out of the treatment. Immunohistochemistry for viral glycoprotein C was observed in tumor tissues of two out six patients. All but one patient’s tumor samples displayed cytoplasmic viral staining, with more intense staining occurring at the 5-day cohort. Furthermore, all patients demonstrated the presence of HSV DNA via PCR. This trial demonstrates the safety of administering G207 into the adjoining brain postresection. These findings bolster the potential of this powerful therapeutic modality and support its further development.

7.2 HSV1716 A serendipitous discovery in the early 1990s led to the identification of HSV1716, another HSV1 variant for oncolytic viral therapy. MacLean and colleagues discovered that HSV1714, a mutant HSV1, lacked neurovirulence, yet still grew in high titers in vitro and was not host cell type restricted (MacLean et al. 1991). Along with a tk deletion, a 759 base pair deletion within the RL1 gene (encoding for ICP34.5) was identified (Fig. 2). Furthermore, this strain had an LD50 of 7 × 106 p.f.u. when injected intracranially in 3-week old mice, approximately 106 greater than the parental wild type strain (which was lethal at less than 10 p.f.u.). Subsequently, this deletion was introduced into the parental Glasgow strain 17+, to restore XbaI sites and the tk gene, to generate the HSV1716 oncolytic virus. HSV1716 replicates as effectively as the parental virus in cell lines, but display reduced neurovirulence in mice. Neurovirulence and oncolytic potential of HSV1716, was studied in human embryonal carcinoma cells (NT2) in  vitro (Kesari et  al. 1995). In addition to forming tumors, these cells resemble neural progenitor cells and can be differentiated into neurons (NT2N) by administering retinoic acid. HSV1716 was unable to replicate in NT2N cells while replicating in and killing the NT2 cells, suggesting reduced neurovirulence while retaining

2 doses totalling 1.15 × 109 p.f.u.

21

GBM

stereotactic inoculation

106, 107, 3 × 107, 108, 3 × 108, 109, 3 × 109 p.f.u.

none unequivocally related

mean survival of 12.8 months (4 alive at publication of paper)

Number of Pts

Cancer Treated

Route of administration

Dose

Adverse Events

Outcomes

phase II trial underway

{Markert, 2009 #471}

References

23 months from initial diagnosis and 6.6 months from virus administration

hemiparesis, increased mental status, and high temperature

GBM

6

Additional Comments

{Markert, 2000 #460}

sterotactic catheter before resection and

Phase I

Type of Trial

Phase Ib

Deletion of both gamma34.5 copies, inactivatied UL39

Virus Modifications

G207

{Rampling, 2000 #419}

4 alive at publication of paper (14, 17, 19, and 22 months)

none unequivocally related

103, 104, 105 p.f.u.

stereotactic inoculation

Recurrent Malignant Glioma

9

Dose Escalation

105 p.f.u.

Resection followed by intratumoral injection in 8-10 adjacent sites

High Grade Glioma

12

Safety in Normal Brain

See Figure 3

2 patients are free of tumor progression

{Papanastassiou, 2002 #6} {Harrow, 2004 #29}

infectious virus was recovered from tumor samples

infectious virus was recovered from tumor samples

none unequivocally related none unequivocally related

105 p.f.u.

stereotactic inoculation followed by resection 4-9 days later

High Grade Glioma (11 GBM, 1 AA)

12

Proof of Prinicple

deletions of both RL1 (gamma 34.5) copies

HSV1716

Table 12.1  Summary of Oncolytic HSV1 Clinical Trials for Cancer Therapy

{MacKie, 2001 #399}

Dose

one patient with flattening of nodule, evidence of virus replication confined to tumor cells

no adverse effects

1, 2, or 4 injections of 103 p.f.u.

Intratumoral injection into subcutaneous nodules

Metastatic Melanoma

5

Dose Escalation

{Mace, 2008 #121}

little evidence of viral evidence or efficacy

no adverse effects

105 or 5 × 105

intratumoral injection 1, 3, or 14 days prior to resection

Oral Squamous Cell Carcinoma

20

Proof of Principle

24% average devrease of CEA, 30 –100% cancer 2 patients with reduction in death based on tumor size histopathologic data

patients also received chemotherapy pump after oncolytic therapy

{Kemeny, 2006 #425}

Outcomes

Additional Comments

References

Pilot Study

N/A

N/A

3 daily doses of 105 p.f.u. or 5 × 105 p.f.u.

catheter in tumor mass

unresectable pancreatic cancer

2

pyrexia and inflammation at injection site

no dose limiting toxicities

single dose or three same as previous trial, dose regimen of 106, additional doses on 107, or 108 p.f.u./ml days 22, 43, and 64 (up to 4 ml)

intratumoral into lesion

{Fujimoto, 2006 #474}

{Hu, 2006 #402}

{K. Harrington, 2009 #253}

patients received radiotherapy with concomitant cisplatin

replication and tumor necrosis in at 23 month followup spread of virus, cell most biopsies, some 65% of patients in death of tumor cells disease inhibition complete remission

no adverse effects

2.5 × 105 p.f.u. or 5 × 105 p.f.u. daily for 3 days

Phase I/II 17

subcutanoues lesions HNSCC from metastatic cancer

26

Dose Escalation

intratumoral into intratumoral into subcutaneous lesion nodule, followed by excision at day 13 or 15 post injection

metastatic HNSCC

2

Pilot Study

{N. Senzer, 2009 #254}

phase III trial underway

26% respnose rate

flu-like symptoms, not dose limiting

106 p.f.u./ml split between injectable tumors followed by 108 p.f.u./ml every two weeks afterwards

intratumoral into lesion

Metastatic Melanoma

50

Dose Escalation

p.f.u. - plaque forming units; UL - Unique Long; CEA - carcinoembryonic antigen; tk - thymidine kinase; GBM - glioblastoma multiforme; AA - Anaplastic Astrocytoma; HNSCC - head and neck squamous cell carcinoma

{Nakao, 2004 #376} {Nakao, 2007 #36}

no adverse effects

rise in gamma-glutamyl transferase, diarrhea, and leukocytosis

Adverse Events

5 × 103 up to 2.5 × 105 for three days

3 × 106, 107, 3 × 107, 108

Dose

Route of intra-hepatic artery administration infusion

intratumoral injection into nodules followed by resection 14 days later

recurrent breast cancer with metastatic nodules

Cancer Treated

Colorectal cancer metastisized to liver

6

Number of Pts 12

Dose Escalation

Phase I

OncoVEXGMCSF

Type of Trial

HF10

deletion of endogenous tk and 2 Kbp and 4 Kbp deletion, followed by insertion of part of 4 Kbp deletion of gamma34.5 and alpha47 from clinically isolated strain, large region containing one deletion in an inverted manner (results in 2 copies of UL53, 54, and 55; addition of GM-CSF copy of gamm34.5, insertion of one copy of UL52 and non-functional UL56) exogenous tk and HSV2 DNA

Virus Modifications

NV1020

Table 12.1  (continued)

214

R.L. Price et al.

Fig. 2  Graphical representations of each HSV1 in clinical trials. G207 features deletions in both copies of gamma34.5 and an inactivating insertion of LacZ into the ICP6 gene. HSV1716 is characterized by deletions in both copies of gamma34.5. Endogenous tk is deleted from NV1020. It also contains a sizeable deletion in the IRL/S junction that results in deletion of one copy of gamma34.5. HSV2 DNA and an exogenous copy of tk under control of the ICP4 promoter is inserted into this deletion. A deletion in the IRL region and an insertion in the TRL region are the modifications of HF10. The attenuating mutation is believed to be the loss of UL56. OncoVEXGM-CSF lacks both copies of gamma34.5 and ICP47. GM-CSF transgenes are inserted into each gamma34.5 locus in order to stimulate the immune response against the tumor

tumor cell-killing ability. Electron microscopy and DNA fragmentation studies revealed that infected tumor cells were killed via a lytic virus burst as opposed to an apoptotic death. Apart from in  vitro cell killing HSV1716 treatment of NT2 tumor-bearing mice showed significant antitumor efficacy in vivo. Further studies have shown that it can replicate in an array of tumor models and can eliminate metastatic brain tumors in mice while failing to replicate in normal tissue (Kucharczuk et al. 1997; Randazzo et al. 1995). Similar to G207, HSV1716 has also been shown to infect and lyse proliferating tumor endothelium while sparing normal vasculature resulting in a potent antitumorigenic effect (Benencia et  al. 2005). The oncolytic potential of HSV1716 was also evaluated in multiple different preclinical models of melanoma, medulloblastoma, human embryonic carcinoma, mesothelioma, head and neck squamous cell carcinoma, and nonsmall cell lung carcinoma (Kesari et al. 1995; Kucharczuk et al. 1997; Lasner et al. 1996; Randazzo et al. 1995; Toyoizumi et al. 1999).

12  Herpes Simplex Virus 1 for Cancer Therapy

215

Pathological and immune responses following HSV1716 treatment were studied by intracerebral inoculation of the ICP34.5 null mutant in mice (McKie et al. 1996). Initial low grade meningoencephalitis, with a limited inflammatory response was cleared by day 28 postinoculation with no evidence of pathology (McKie et al. 1998). Limited pathological response in mice after intracerebral inoculation combined with antitumor efficacy observed in animal models underscores the therapeutic potential of this virus. Like G207, HSV1716 was first tested in patients suffering from highgrade glioma. Nine patients with recurrent malignant gliomas, all of which had failed radical treatment, were included in the first study (Rampling et al. 2000). The goal of this study was to determine if an effective dose could safely be given to a human. Accordingly, three patients each were given one dose of 103, 104, or 105 p.f.u. injected stereotactically into the tumor (Table 1). No patients showed any adverse effects related directly to virus administration. Follow-up studies revealed neither viral shedding nor encephalitis. At the time of the initial publication of the study, four patients were still alive. One patient was alive at 24 months after HSV1716 injection without any other antitumor treatment. After proving that HSV1716 is safe at 105 p.f.u., physicians proceeded to test the efficacy potential by demonstrating intratumoral replication. In the next study, patients with high-grade gliomas received an intratumoral injection of 105 p.f.u. of HSV1716 followed by resection of the tumor. The resected tumor was analyzed an assayed for evidence of viral replication (Papanastassiou et al. 2002). As seen in the previous study, none of the patients experience any adverse effects related virus administration. In two of the patients, infectious virus was recovered from the tumor samples. In one of these patients, a titer yielded 4 × 105 p.f.u., an amount greater than the titer of the initial dose thus indicating viral replication in the tumor. Furthermore, HSV1 DNA was found via PCR in 10 of 12 patient samples and 4 of 12 patient samples at a distal tumor site. Immunohistochemical staining revealed the UL42 protein in tumor samples from two patients. This is evidence of de novo protein synthesis of the virus in the tumor. A subsequent study investigated the safety of injecting the virus in adjacent “tumor cell infiltrated normal” brain tumor postresection (Harrow et al. 2004). Using HSV1716 to destroy these cells may be an effective way to eliminate residual tumor cells, which could prevent recurrence and cure GBM. In this study, 12 patients with high-grade gliomas underwent tumor resection followed by HSV1716 inoculation. Aliquots of 0.1–0.2 ml were injected into eight to ten sites adjacent to the tumor totaling 1 ml of 105 p.f.u. of HSV1716 (Table 1). In concurrence with the other studies, HSV1716 inoculation caused no evidence of toxicity as well as no adverse events directly associated with the virus. At the time of publication of this particular study, three treated patients were alive (15, 18, and 22 months (Fig. 3 for imaging) after HSV1716 administration). The results of this study demonstrate the safety as well as suggest the potential efficacy of HSV1716 injected into the tumor periphery at the time of resection. Taken together, these studies suggest that HSV1716 is a potential treatment for high-grade gliomas. The use of HSV1716 in gliomas has been expanded to more patients in recent years. Besides gliomas, the safety and efficacy of HSV1716 administration in human patients suffering from metastatic melanoma and head and neck squamous cell carcinoma has also been studied. After HSV1716 was shown to cause cell death in

216

R.L. Price et al.

Fig.  3  MRI images of a glioma patient treated with HSV1716 showing a complete response. From left column to right: T2-weighted, T1-weighted, and Thallium-201 SPECT axial sections of tumor. (a) Pre-resection and viral injection. (b) 48 h postresection. (c) 6-week follow-up. (d) 22-month follow-up. Reprinted with permission from Macmillan Publishers Ltd: Harrow et al. (2004). © 2004 Nature Publishing Group

12  Herpes Simplex Virus 1 for Cancer Therapy

217

human melanoma cell lines and selectively replicate in melanoma tissue in nude mice, a clinical trial was approved (Randazzo et al. 1997). Five patients with metastatic melanoma were enrolled in the first study (MacKie et al. 2001). The patients received either one, two, or four injections of 103 p.f.u. of HSV1716 directly into the subcutaneous nodules of the cancer (Table 1). After injection, biopsies of all the nodules revealed viral replication and all patients that received two or more injections revealed evidence of tumor necrosis including the flattening of a previously palpable nodule observed in one patient. The effect of HSV1716 administration in 20 patients with a variety of resectable head and neck tumors was published in 2008 (Mace et al. 2008). Each patient received an intratumoral injection of 105 or 5 × 105 p.f.u. at 1 day, 3 days, or 14 days prior to resection injected directly into the tumors as well as into the contralateral buccal mucosa (Table 1). All patients tolerated the virus well with no signs of adverse events. Though there was little evidence of significant antitumor efficacy, two patients tested positive for HSV1 DNA at the tumor site. Currently, adolescent patients with non-CNS solid tumors are being recruited to test HSV1716 in pediatric malignancies (ClinicalTrials.gov 2009). The goal of the study is to test the safety of administering oncolytic virus to children and young adults. The safety observed in patients treated with HSV1716 encourages testing for efficacy in a wide variety of cancers in the future. To date, all patients treated with HSV1716 tolerated the virus well with no signs of adverse events. HSV1716 has proven to be safe at a dose up to 5 × 105 p.f.u. without any adverse effects related to virus administration. However, the lack of a robust antitumor response in these studies has been attributed to the limited dose that was administered and underscores the need for additional preclinical research to evaluate ways to enhance this therapeutic modality.

7.3 NV1020 (RV7020) Unlike other oncolytic viruses, NV1020 was not initially developed for cancer therapy, but is a nonselected clone of R7020 originally developed by Bernard Roizman’s lab as a potential vaccine for HSV1 and HSV2 infections (Meignier et al. 1988). R7020 was constructed by using HSV1 strain F as a backbone. This backbone was attenuated by deleting a 700 base pair portion of the UL region eliminating viral tk gene and a portion of the UL24 promoter in addition to a 14,500 base pair deletion within the internal repeated region (Fig. 2). The deletion of the IRL region resulted in the removal of one copy of ICP34.5, which attenuated the neurovirulence of the hybrid virus, and permitted for the insertion of HSV2 DNA and an exogenous tk gene. The HSV2 DNA consists of genes that encode glycoproteins D, G, I, and a portion of E. The addition of the HSV2 glycoproteins genes was intended to confer HSV2 immunogenicity to the putative vaccine. Also, the addition of TK allowed for restored viral replicative ability and sensitivity to antiviral medication, though outside of traditional endogenous regulation and under the control of the ICP4 promoter. This potential herpes vaccine construct was proven

218

R.L. Price et al.

safe in mice, rabbits, and guinea pigs as well as Aotus monkeys (Meignier et al. 1988, 1990). In Aotus monkeys, doses up to 106 p.f.u of R7020 resulted in local lesions and viral shedding but no disseminated disease (Meignier et al. 1990). This is considerably greater than the lethal dose of 100–1,000 p.f.u. of the wild type virus. After safety in other animals was confirmed, the potential vaccine was cleared for clinical trials. Though RV7020 showed preclinical promise, clinical trials showed that the virus was ineffective as a vaccine. R7020 was first tested as an oncolytic virus in chemotherapy/radiation-resistant epidermoid carcinoma and androgen-independent prostate adenocarcinoma cell lines in vitro and in vivo (Advani et al. 1999). Mice bearing subcutaneous tumors treated with 2 × 106 p.f.u. of R7020 showed tumor mass regression. In the same study, R7020 was also found to have increased antitumor efficacy when combined with ionizing radiation in vivo. In addition to these tumor types, antitumor efficacy of NV1020 has been demonstrated against a wide variety of tumors including bladder, mesothelioma, gastric, prostate, and hepatoma carcinomas (Adusumilli et  al. 2006; Bennett et  al. 2001, 2002; Chung et  al. 2002; Cozzi et  al. 2001, 2002). Interestingly, NV1020 retained effectiveness even in animals with pre existing immunity against HSV1 (Delman et al. 2000). Due to the preexisting safety studies of R7020 in animals and humans (Cadoz et al. 1992), NV1020 was quickly approved for testing safety and efficacy in human patients for colorectal cancer metastatic to the liver. Twelve patients with colorectal adenocarcinoma metastatic to the liver that were refractory to conventional chemotherapy were divided into four cohorts with three patients in each cohort receiving doses of 3 × 106, 107, 3 × 107, or 108 p.f.u. infused directly into the hepatic artery (Table 1; Delman et al. 2000). This was the first study to test an intravascular delivery approach for an oncolytic virus. It was chosen in order to minimize toxicities to other tissues and reduce antibody exposure to the virus (Fong et  al. 2009). Only three patients experienced adverse side effects (rise in gamma-glutamyl transferase, diarrhea, and leukocytosis) that could possibly be related to NV1020 administration. No patients experienced disseminated herpes infection and only one saliva sample and two serum samples from the same patient tested positive for the virus. Tumor reduction of 20% and 39% was observed in two patients who received 3×107 and 108 virus particles, respectively. Seven patients remained stable while the cancer in the other three patients progressed (Kemeny et al. 2006). A follow-up study of these patients revealed the evidence of HSV replication in liver biopsies, along with an average reduction in carcinoembryonic antigen (a common colorectal cancer marker) of 24% along with no wild-type viral reactivation in treated patients (Fong et al. 2009). Taken together, the clinical evidence to date indicates that NV1020 is safe to deliver intra-arterially and suggests that it may be efficacious in treating colorectal cancer that has metastasized to the liver.

7.4 HF10 HF10 is another replication competent oncolytic HSV1 derivative currently being tested in clinical trials. Unlike many other oncolytic viruses tested in the clinic, HF10

12  Herpes Simplex Virus 1 for Cancer Therapy

219

is not genetically engineered but is a nonselective clone from the non-neuroinvasive HSV1 strain HF (Nishiyama et al. 1991). Though the virus typically replicates more efficiently in cultured cells as compared to wild type HSV1, peripheral routes of infection show an attenuated phenotype. HF10 contains a 3,832 bp deletion at the right end of the UL and the UL/IRL junction region of HSV1 genome, (Nishiyama et  al. 1991; Teshigahara et  al. 2004; Umene et  al. 1984) and additionally has 6  Kbp of its DNA (110,488  bp to 116,514  bp) inserted in reverse orientation (Fig. 2). Collectively, these mutations result in a mutant virus (HF10) containing two copies of UL 53, 54, and 55, and incomplete copies of UL52 and UL56 (Kimata et al. 2006). UL56 has been localized to the golgi apparatus and endosomes, suggesting that it is involved in vesicular trafficking and may be involved in anterograde axonal transport of the virus (Kimata et al. 2006; Teshigahara et al. 2004). Sequencing of HF10 revealed an overall 99.1% similarity to HSV1 strain 17 in amino acid identity; and also has amino acid changes in genes involved in the regulation of synctia formation including UL1, UL20, UL22, UL24, UL27, and UL53 (Ushijima et al. 2007). However, most scientists agree that the loss of UL56 accounts for its attenuated phenotype. Significant antitumor efficacy of HF10 has been observed in a variety of tumor models including breast, peritoneal, and bladder cancers and malignant melanoma tumors in mice (Kimata et  al. 2003; Kohno et  al. 2005; Takakuwa et  al. 2003; Teshigahara et  al. 2004; Watanabe et  al. 2008). Other recent studies have shown that paclitaxel, an established chemotherapeutic, enhances the effect of HF10 in mice with peritoneally disseminated colon cancer (Shimoyama et  al. 2007). It is currently being tested in Japan for safety and efficacy in patients with breast and pancreatic cancer as well as head and neck squamous cell carcinoma. A pilot study containing six patients with recurrent breast cancer with metastatic nodules were enrolled in the first study (Table 1). Each patient received 0.5 ml of varying doses of HF10 directly injected into the nodule as well as 0.5  ml of saline in another control nodule (Kimata et  al. 2006). The doses ranged from 1 dose of 104 p.f.u./0.5  ml to 3 daily doses of 5 × 105 p.f.u./0.5  ml. After 14  days, the nodules were excised to study histopathologic changes. All patients tolerated the virus well. There were neither adverse effects nor evidence of shedding or activation. Tumor cell death was confirmed by nuclear inclusion bodies and viral replication was readily seen by immunoflourescence in biopsy samples. It is important to note that all patients were seropositive to HSV1 before the trial, suggesting that seropositivity does not affect the replication potential of the virus. This small study suggests that humans can tolerate the virus well. HF10 was also tested in three patients with nonresectable pancreatic cancer. Once the patient was deemed unresectable during laparotomy, a catheter was placed in the tumor mass (Nakao et  al. 2007). Two patients received three doses of 10 5 p.f.u. the day of the surgery and the two following days. The other patient received 5 × 105 p.f.u. on the same dosing schedule. Unfortunately, published data regarding patient outcomes have not been released. A third small trial was conducted for two patients with skin nodules from metastatic squamous cell carcinoma (Table 1; Fujimoto et al. 2006). Each patient was injected intratumorally once a day for 3 days with either 105 p.f.u./1 ml/3 days (one patient) or 105 p.f.u./0.5 ml/3 days (two patients). Besides a low-grade fever,

220

R.L. Price et al.

both patients tolerated the virus well without any other adverse effects. After the resection of nodules, evidence of viral replication was only found in tumor cells. These trials show that the virus is well tolerated with minimal adverse effects. Further investigation of the efficacy of HF10 is warranted.

7.5 OncoVex GM-CSF OncoVEXGM-CSF represents the first “armed” oncolytic HSV1 to be tested in human patients. It was created from JS1 strain of HSV1 obtained from reactivating cold sores in human patients (Liu et al. 2003). This strain killed tumor cells much more effectively than the laboratory strain 17+, and so was selected as the viral backbone to create a novel oncolytic virus. To control viral replication both copies of ICP34.5 were deleted in this oncolytic virus. An additional deletion of ICP47 was done such that the adjoining US11 gene was now expressed under the IE promoter facilitating the early expression of US11. Since the early expression of US11 blocks intracellular PKR phosphorylation, and the ensuing antiviral host responses, this engineering facilitated efficient virus replication in infected cells (Cassady et  al. 1998; Poppers et al. 2000). Furthermore, the deletion of ICP47 disinhibits MHC class I expression on infected cells (see Fig. 12.1e). Greater MHC class I expression on infected tumor cells stimulate a greater immune response against the tumor cell. The resultant mutated virus is armed with GM-CSF gene driven by the CMV promoter to activate a systemic antitumor immune response after initial virus infection and tumor cell lysis. Preclinical studies with OncoVEXGM-CSF revealed efficient tumor killing in vitro and in vivo. Interestingly, mice that were cured of their tumor by OncoVEX treatment were resistant to subsequent tumor cell implantation demonstrating induction of a protective antitumor immunity in these mice. From these results, the clinically isolated virus is sufficiently attenuated with enhanced antitumor immunity (Liu et al. 2003). OncoVEXGM-CSF was tested for safety in 26 patients with cutaneous or subcutaneous lesions from malignant melanoma, head and neck, breast, or gastrointestinal, cancers refractory to prior treatments (Hu et al. 2006). In this dose escalation study, the virus was injected directly into the lesion as a single dose of 106, 107, or 108 p.f.u./ml or as a three-dose regimen with each patient receiving a combination of the three doses (Table 1). The dose amount was based on tumor size with lesions greater than 2.5 cm in diameter receiving up to 4 ml of the target dose. Thirteen patients received a single dose and the remaining patients received an escalating combination of the three doses. The timing of multiple doses varied between 1 and 3 weeks and was determined by injection site healing, adverse events, and seroconversion. Overall, the virus was well tolerated and no patients dropped from the study. The most common side effects were pyrexia and local inflammation at the injection site. After analysis of all the patients, the optimal dosing schedule was determined to be an initial dose of 106 p.f.u./ml followed by two doses of 108 p.f.u./ ml every 2–3 weeks. Although, complete or partial responses were not observed;

12  Herpes Simplex Virus 1 for Cancer Therapy

221

stable disease was observed in several patients. Of interest, most tumor biopsies showed tumor necrosis, and were positive for HSV1 (Hu et al. 2006). As a result of proving safety of the virus, trials of OncoVEXGM-CSF have been expanded. At the 2009 ASCO meeting, the preliminary results of a Phase I/II study in head and neck cancer was presented (Harrington et al. 2009; Coffin et al. 2007). The virus was being studied in lymph node positive stage III/IV head and neck cancer. Patients were assigned to one of four dosing cohorts based on the previous Phase I trial. Each patient received four doses on days 1, 22, 43 and 64. Additionally, each patient received radiotherapy with concomitant cisplatin. At 6–10  weeks postradiation, neck dissection was performed. At the time of the 2009 meeting, 17 patients had undergone treatment (Table 1). The neck dissection showed pathological complete response in 94% of the patients. Levels of HSV1 were detected in injected as well as non-injected tumors, with some patients showing higher titers than originally injected. At the 23-month follow-up, 65% of the patients remain in complete remission. During the same time, OncoVEXGM-CSF trials involving metastatic melanoma were also being expanded (Senzer et al. 2009). Fifty patients with stage III/IV metastatic melanoma received a primary intratumoral dose of 106 p.f.u./ml split between injectable tumors. Three weeks later up to 24 sequential injections of 108 p.f.u./ml were administered every 2 weeks or until significant disease progression or overall complete response was observed (Table 1). Consistent with the head neck cancer trials, both injected and non-injected lesions showed a response, indicating the activation of a systemic antitumor immune response. Similar to previous data from the virus, side effects were limited and involved flu-like symptoms. Overall, the response rate in this study was observed to be 26% with the 1-year survival rate being 61% and median overall survival of 16+ months. On the basis of safety profile established by this study, OncoVEXGM-CSF is currently being studied in a Phase III trial for malignant melanoma (ClinicalTrials.gov 2009).

8 The Future of Oncolytic HSV1 Cancer Therapy Since the initial discovery by Martuza et  al. demonstrating cancer cell killing properties of oncolytic HSV1, several of these viruses have been tested in clinical trials. While evidence of significant antitumor efficacy of these viruses is being evaluated in ongoing Phase III randomized clinical trials, many investigators around the world are investigating ways to enhance efficacy of this promising therapeutic modality. While the large size of HSV1 genome has proved to be cumbersome for genetic manipulations in  vitro, the recent creation of two rapid methods which facilitate this process has greatly expanded the ability of making new armed vectors (Fukuhara et al. 2005a, b; Terada et al. 2006). This technology has been utilized to arm oncolytic viruses with prodrug activating enzymes to enhance tumor specific killing of these viruses. rRp450 is one such virus that has a deletion in ICP6 to attenuate the virus and an insertion of the rat CYP2B1 gene, which allows for the activation of cyclophosphamide into its active form

222

R.L. Price et al.

(Chase et al. 1998; Clarke and Waxman 1989). Cyclophosphamide has been shown in experiments to augment oncolytic viral activity by suppressing innate immune responses (Ikeda et al. 1999, 2000). This virus has been proven to be more effective than the parental ICP6-deleted virus in subcutaneous glioma tumor xenograft model in athymic mice and liver metastasis of colon cancer model in mice (Chase et  al. 1998). Subsequently, MGH2 a dually armed oncolytic virus expressing both CYP2B1 gene and human intestinal carboxylesterase was created in our laboratory. This virus has shown effective and increased glioma cell killing in the presence of cyclophosphamide and irinotecan (Tyminski et  al. 2005). Similarly, HSV1 armed with cytosine deaminase (CD) also show increased cytotoxicity in the presence of 5-fluorocytosine (Nakamura et  al. 2001). Prodrug arming of second generation OncoVEXGM-CSF with CD has also shown promise in preclinical studies (Simpson et al. 2006). Apart from arming oncolytic viruses, the reintroduction of originally deleted viral genes under the governance of cancer cell specific promoters to increase the replication potential in a cancer cell specific manner has also been investigated. Several tumor or tissue specific promoters such as b-myb, Nestin, Calponin Albumin and CEA have been used to drive the potency of attenuated oncolytic HSV1 in a cancer cell restricted manner (Chung et al. 1999; Kambara et al. 2005; Mullen et  al. 2002; Yamamura et  al. 2001). Interested readers are referred to a recent review by Hardcastle et al. (2007) for details about this approach. The combination of oncolytic viruses with pharmacologic adjuvants is also currently being investigated as ways to enhance antitumor efficacy. For example, the synergistic augmentation of oncolytic virus therapy with cyclophosphamide has been investigated in several preclinical studies (Currier et  al. 2008; Fulci et  al. 2006). On the basis of these observations, cyclophosphamide combined with an oncolytic measles virus encoding thyroidal sodium iodide symporter is currently being tested in a ClinicalTrials.gov (2009). Several recent reports have investigated the synergistic effects of combining antiangiogenic and other antineoplastic agents to increase oncolytic efficacy and dissemination of these viruses. Interested readers are referred to a recent review by (Alvarez-Breckenridge et  al. 2009) for more details on this subject. The tumor microenvironment is increasingly being recognized as a very significant contributor to disease progression and response to therapy. Oncolytic viruses armed with extracellular matrix (ECM) modulating genes that can suppress angiogenesis or modulate the ECM have been generated to increase viral dissemination through the tumor matrix and augment its efficacy. For example, the incorporation of antiangiogenic genes such as Vasculostatin, TIMP3, and Platelet Factor 4 within HSV1 backbone has shown promising results in preclinical models (Hardcastle et al. 2009; Liu et al. 2006; Mahller et al 2008). Like the OncoVEXGM-CSF, second generation oncolytic HSV1 expressing immunomodulatory genes such as IL-4, IL-12, and B lymphocyte activation antigen-1 are being tested in preclinical studies (Kaur and Chiocca 2009). The limitation of tumor ECM on viral efficacy is currently being recognized. A recent report studying the cellular effects of G207 treatment on glioblastoma microenvironment concluded that the transduction of a

12  Herpes Simplex Virus 1 for Cancer Therapy

223

significant volume of tumor was necessary to achieve significant efficacy with oncolytic viruses (Huszthy et  al. 2009). Combining oncolytic HSV1 with matrix modulating enzymes has shown efficacy in several preclinical studies; future generations of HSV1 derived viruses armed with such genes will evaluate if such an approach improves therapy.

9 Conclusion The last couple of decades has seen HSV1, a traditionally pathogenic and sometimes deadly virus, become attenuated and targeted for the use of oncolytic therapy. Using such a pathogenic virus is an exotic idea and required exhaustive studies in preclinical and Phase I trials before it was accepted as a safe therapy. Now that several viruses have been shown to be safe in humans, several viruses are proceeding to further clinical trials to test efficacy. Accordingly, the field has erupted with a plethora of different viruses with all sorts of modifications to make them more oncolytic. Unfortunately, early data has shown that oncolytic viruses are not as effective as hoped. The reason for this is unknown but could stem from the virus being too attenuated, the seropositivity of patients, or early immune clearance. Before the HSV1 therapy becomes the standard of care for certain types of cancer, efficacy needs to be enhanced. Instead of focusing on the creation of new modifications of the virus, efforts should be focused on how to make the virus more effective in humans. On the horizon, there are many new clinical trials planned. Hopefully, an oncolytic virus with the right combination of safety, efficacy, and tolerability will emerge in these trials.

References Ackermann M, Chou J, Sarmiento M et al (1986) Identification by antibody to a synthetic peptide of a protein specified by a diploid gene located in the terminal repeats of the L component of herpes simplex virus genome. J Virol, 58: 843–850 Adusumilli PS, Stiles BM, Chan MK et al (2006) Imaging and therapy of malignant pleural mesothelioma using replication-competent herpes simplex viruses. J Gene Med, 8: 603–615 Advani SJ, Chung SM, Yan SY et al (1999) Replication-competent, nonneuroinvasive genetically engineered herpes virus is highly effective in the treatment of therapy-resistant experimental human tumors. Cancer Res, 59: 2055–2058 Aghi M, Rabkin SD, Martuza RL (2007) Angiogenic response caused by oncolytic herpes simplex virus-induced reduced thrombospondin expression can be prevented by specific viral mutations or by administering a thrombospondin-derived peptide. Cancer Res, 67: 440–444 Aghi M, Visted T, Depinho RA et  al (2008) Oncolytic herpes virus with defective ICP6 specifically replicates in quiescent cells with homozygous genetic mutations in p16. Oncogene, 27: 4249–4254 Aghi MK, Liu TC, Rabkin S et al (2009) Hypoxia enhances the replication of oncolytic herpes simplex virus. Mol Ther, 17: 51–56

224

R.L. Price et al.

Alvarez-Breckenridge C, Kaur B, Chiocca EA (2009) Pharmacologic and chemical adjuvants in tumor virotherapy. Chem Rev, 109: 3125–3140 Bacchetti S, Evelegh MJ, Muirhead B (1986) Identification and separation of the two subunits of the herpes simplex virus ribonucleotide reductase. J Virol, 57: 1177–1181 Benencia F, Courreges MC, Conejo-García JR et al (2005) Oncolytic HSV exerts direct antiangiogenic activity in ovarian carcinoma. Hum Gene Ther, 16: 765–778 Bennett JJ, Malhotra S, Wong RJ et al (2001) Interleukin 12 secretion enhances antitumor efficacy of oncolytic herpes simplex viral therapy for colorectal cancer. Ann Surg, 233: 819–826 Bennett JJ, Delman KA, Burt BM et al (2002) Comparison of safety, delivery, and efficacy of two oncolytic herpes viruses (G207 and NV1020) for peritoneal cancer. Cancer Gene Ther, 9: 935–945 Boviatsis EJ, Park JS, Sena-Esteves M et  al (1994) Long-term survival of rats harboring brain neoplasms treated with ganciclovir and a herpes simplex virus vector that retains an intact thymidine kinase gene. Cancer Res, 54: 5745–5751 Brown SM, MacLean AR, McKie EA et  al (1997) The herpes simplex virus virulence factor ICP34.5 and the cellular protein MyD116 complex with proliferating cell nuclear antigen through the 63-amino-acid domain conserved in ICP34.5, MyD116, and GADD34. J Virol, 71: 9442–9449 Cadoz M, Seigneurin JM, Mallaret MR, Baccard C, and Morand P (1992). Phase I trial of R7020: A live attenuated recombinant herpes simplex virus (HSV) candidate vaccine. Paper presented at the Program and Abstracts of the 32nd Interscience Con- ference on Antimicrobial Agents and Chemotherapy. (American So- ciety of Microbiology, Washington, D.C.). Cameron JM, McDougall I, Marsden HS et al (1988) Ribonucleotide reductase encoded by herpes simplex virus is a determinant of the pathogenicity of the virus in mice and a valid antiviral target. J Gen Virol, 69 ( Pt 10): 2607–2612 Carroll NM, Chase M, Chiocca EA et  al (1997) The effect of ganciclovir on herpes simplex virus-mediated oncolysis. J Surg Res, 69: 413–417 Cassady KA, Gross M, Roizman B (1998) The herpes simplex virus US11 protein effectively compensates for the gamma1(34.5) gene if present before activation of protein kinase R by precluding its phosphorylation and that of the alpha subunit of eukaryotic translation initiation factor 2. J Virol, 72: 8620–8626 Chahlavi A, Todo T, Martuza RL et al (1999) Replication-competent herpes simplex virus vector G207 and cisplatin combination therapy for head and neck squamous cell carcinoma. Neoplasia, 1: 162–169 Chase M, Chung RY, Chiocca EA (1998) An oncolytic viral mutant that delivers the CYP2B1 transgene and augments cyclophosphamide chemotherapy. Nat Biotechnol, 16: 444–448 Chou J, Roizman B (1986) The terminal a sequence of the herpes simplex virus genome contains the promoter of a gene located in the repeat sequences of the L component. J Virol, 57: 629–637 Chou J, Roizman B (1990) The herpes simplex virus 1 gene for ICP34.5, which maps in inverted repeats, is conserved in several limited-passage isolates but not in strain 17syn+. J Virol, 64: 1014–1020 Chou J, Roizman B (1992) The gamma 1(34.5) gene of herpes simplex virus 1 precludes neuroblastoma cells from triggering total shutoff of protein synthesis characteristic of programed cell death in neuronal cells. Proc Natl Acad Sci U S A, 89: 3266–3270 Chou J, Roizman B (1994) Herpes simplex virus 1 gamma(1)34.5 gene function, which blocks the host response to infection, maps in the homologous domain of the genes expressed during growth arrest and DNA damage. Proc Natl Acad Sci U S A, 91: 5247–5251 Chou J, Kern ER, Whitley RJ et al (1990) Mapping of herpes simplex virus-1 neurovirulence to gamma 134.5, a gene nonessential for growth in culture. Science, 250: 1262–1266 Chou J, Chen JJ, Gross M et al (1995) Association of a M(r) 90,000 phosphoprotein with protein kinase PKR in cells exhibiting enhanced phosphorylation of translation initiation factor eIF-2 alpha and premature shutoff of protein synthesis after infection with gamma 134.5- mutants of herpes simplex virus 1. Proc Natl Acad Sci U S A, 92: 10516–10520 Chung RY, Saeki Y, Chiocca EA (1999) B-myb promoter retargeting of herpes simplex virus gamma34.5 gene-mediated virulence toward tumor and cycling cells. J Virol, 73: 7556–7564

12  Herpes Simplex Virus 1 for Cancer Therapy

225

Chung SM, Advani SJ, Bradley JD et al (2002) The use of a genetically engineered herpes simplex virus (R7020) with ionizing radiation for experimental hepatoma. Gene Ther, 9: 75–80 Cinatl J, Michaelis M, Driever PH et al (2004) Multimutated herpes simplex virus g207 is a potent inhibitor of angiogenesis. Neoplasia, 6: 725–735 Clarke L, Waxman DJ (1989) Oxidative metabolism of cyclophosphamide: identification of the hepatic monooxygenase catalysts of drug activation. Cancer Res, 49: 2344–2350 Clemens MJ, Elia A (1997) The double-stranded RNA-dependent protein kinase PKR: structure and function. J Interferon Cytokine Res, 17: 503–524 ClinicalTrials.gov. (2009). Retrieved October 25, 2009, from http://clinicaltrials.gov/ct2/show/ NCT00769704 Coen DM, Kosz-Vnenchak M, Jacobson JG et al (1989) Thymidine kinase-negative herpes simplex virus mutants establish latency in mouse trigeminal ganglia but do not reactivate. Proc Natl Acad Sci U S A, 86: 4736–4740 Coffey MC, Strong JE, Forsyth PA et al (1998) Reovirus therapy of tumors with activated Ras pathway. Science, 282: 1332–1334 Coffin RS, Hingorani M, McNeish I, Sibtain A, Hamilton B, Love C, Nutting C, Harrington K (2007) Phase I/II trial of OncoVEXGM-CSF combined with radical chemoradiation (CRT) in patients with newly diagnosed node-positive stage III/IV head and neck cancer (HNC). Paper presented at the 2007 ASCO Annual Meeting. J Clin Oncol, 25: 14095 Cozzi PJ, Malhotra S, McAuliffe P et al (2001) Intravesical oncolytic viral therapy using attenuated, replication-competent herpes simplex viruses G207 and Nv1020 is effective in the treatment of bladder cancer in an orthotopic syngeneic model. FASEB J, 15: 1306–1308 Cozzi PJ, Burke PB, Bhargav A et al (2002) Oncolytic viral gene therapy for prostate cancer using two attenuated, replication-competent, genetically engineered herpes simplex viruses. Prostate, 53: 95–100 Currier MA, Gillespie RA, Sawtell NM et al (2008) Efficacy and safety of the oncolytic herpes simplex virus rRp450 alone and combined with cyclophosphamide. Mol Ther, 16: 879–885 Delman KA, Bennett JJ, Zager JS et al (2000) Effects of preexisting immunity on the response to herpes simplex-based oncolytic viral therapy. Hum Gene Ther, 11: 2465–2472 Detta A, Harland J, Hanif I et al (2003) Proliferative activity and in vitro replication of HSV1716 in human metastatic brain tumours. J Gene Med, 5: 681–689 Dix RD, McKendall RR, Baringer JR (1983) Comparative neurovirulence of herpes simplex virus type 1 strains after peripheral or intracerebral inoculation of BALB/c mice. Infect Immun, 40: 103–112 Elledge SJ, Zhou Z, Allen JB (1992) Ribonucleotide reductase: regulation, regulation, regulation. Trends Biochem Sci, 17: 119–123 Field HJ, Wildy P (1978) The pathogenicity of thymidine kinase-deficient mutants of herpes simplex virus in mice. J Hyg (Lond), 81: 267–277 Fong Y, Kim T, Bhargava A et  al (2009) A herpes oncolytic virus can be delivered via the vasculature to produce biologic changes in human colorectal cancer. Mol Ther, 17: 389–394 Fornace AJ, Nebert DW, Hollander MC et al (1989) Mammalian genes coordinately regulated by growth arrest signals and DNA-damaging agents. Mol Cell Biol, 9: 4196–4203 Früh K, Ahn K, Djaballah H et  al (1995) A viral inhibitor of peptide transporters for antigen presentation. Nature, 375: 415–418 Fujimoto Y, Mizuno T, Sugiura S et  al (2006) Intratumoral injection of herpes simplex virus HF10 in recurrent head and neck squamous cell carcinoma. Acta Otolaryngol, 126: 1115–1117 Fukuhara H, Ino Y, Kuroda T et  al (2005) Triple gene-deleted oncolytic herpes simplex virus vector double-armed with interleukin 18 and soluble B7-1 constructed by bacterial artificial chromosome-mediated system. Cancer Res, 65: 10663–10668 Fukuhara H, Martuza RL, Rabkin SD et al (2005) Oncolytic herpes simplex virus vector g47delta in combination with androgen ablation for the treatment of human prostate adenocarcinoma. Clin Cancer Res, 11: 7886–7890

226

R.L. Price et al.

Fulci G, Breymann L, Gianni D et al (2006) Cyclophosphamide enhances glioma virotherapy by inhibiting innate immune responses. Proc Natl Acad Sci U S A, 103: 12873–12878 Goldstein DJ, Weller SK (1988a) Factor(s) present in herpes simplex virus type 1-infected cells can compensate for the loss of the large subunit of the viral ribonucleotide reductase: characterization of an ICP6 deletion mutant. Virology, 166: 41–51 Goldstein DJ, Weller SK (1988b) Herpes simplex virus type 1-induced ribonucleotide reductase activity is dispensable for virus growth and DNA synthesis: isolation and characterization of an ICP6 lacZ insertion mutant. J Virol, 62: 196–205 Gooding LR (1992) Virus proteins that counteract host immune defenses. Cell, 71: 5–7 Hardcastle J, Kurozumi K, Chiocca EA et al (2007) Oncolytic viruses driven by tumor-specific promoters. Curr Cancer Drug Targets, 7: 181–189 Hardcastle J, Kurozumi K, Dmitrieva N et al (2009) Enhanced antitumor efficacy of vasculostatin (Vstat120) expressing oncolytic HSV-1. Mol Ther, 18(2): 285–294 Harrington K, Hingorani M, Tanay M, Matthews J, Newbold K, Renouf L, Coffin RS, McNeish I, Nutting C (2009) Phase I/II dose escalation study of OncoVexGM-CSF and chemoradiotherapy (CRT) in untreated stage III/IV squamous cell cancer of the head and neck (SCCHN). Paper presented at the 2009 ASCO Annual Meeting Harrow S, Papanastassiou V, Harland J et al (2004) HSV1716 injection into the brain adjacent to tumour following surgical resection of high-grade glioma: safety data and long-term survival. Gene Ther, 11: 1648–1658 He B, Chou J, Liebermann DA et al (1996) The carboxyl terminus of the murine MyD116 gene substitutes for the corresponding domain of the gamma(1)34.5 gene of herpes simplex virus to preclude the premature shutoff of total protein synthesis in infected human cells. J Virol, 70: 84–90 He B, Chou J, Brandimarti R et al (1997) Suppression of the phenotype of gamma(1)34.5- herpes simplex virus 1: failure of activated RNA-dependent protein kinase to shut off protein synthesis is associated with a deletion in the domain of the alpha47 gene. J Virol, 71: 6049–6054 He B, Gross M, Roizman B (1998) The gamma134.5 protein of herpes simplex virus 1 has the structural and functional attributes of a protein phosphatase 1 regulatory subunit and is present in a high molecular weight complex with the enzyme in infected cells. J Biol Chem, 273: 20737–20743 Hill AB, Barnett BC, McMichael AJ et al (1994) HLA class I molecules are not transported to the cell surface in cells infected with herpes simplex virus types 1 and 2. J Immunol, 152: 2736–2741 Hill A, Jugovic P, York I et  al (1995) Herpes simplex virus turns off the TAP to evade host immunity. Nature, 375: 411–415 Honess RW, Roizman B (1973) Proteins specified by herpes simplex virus. XI. Identification and relative molar rates of synthesis of structural and nonstructural herpes virus polypeptides in the infected cell. J Virol, 12: 1347–1365 Honess RW, Roizman B (1974) Regulation of herpesvirus macromolecular synthesis. I. Cascade regulation of the synthesis of three groups of viral proteins. J Virol, 14: 8–19 Hu JC, Coffin RS, Davis CJ et  al (2006) A phase I study of OncoVEXGM-CSF, a secondgeneration oncolytic herpes simplex virus expressing granulocyte macrophage colonystimulating factor. Clin Cancer Res, 12: 6737–6747 Hunter WD, Martuza RL, Feigenbaum F et al (1999) Attenuated, replication-competent herpes simplex virus type 1 mutant G207: safety evaluation of intracerebral injection in nonhuman primates. J Virol, 73: 6319–6326 Huszthy PC, Immervoll H, Wang J et al (2009) Cellular effects of oncolytic viral therapy on the glioblastoma microenvironment. Gene Ther, 17: 202–216 Ikeda K, Ichikawa T, Wakimoto H et al (1999) Oncolytic virus therapy of multiple tumors in the brain requires suppression of innate and elicited antiviral responses. Nat Med, 5: 881–887 Ikeda K, Wakimoto H, Ichikawa T et al (2000) Complement depletion facilitates the infection of multiple brain tumors by an intravascular, replication-conditional herpes simplex virus mutant. J Virol, 74: 4765–4775

12  Herpes Simplex Virus 1 for Cancer Therapy

227

Jamieson AT, Gentry GA, Subak-Sharpe JH (1974) Induction of both thymidine and deoxycytidine kinase activity by herpes viruses. J Gen Virol, 24: 465–480 Jennings SR, Rice PL, Kloszewski ED et al (1985) Effect of herpes simplex virus types 1 and 2 on surface expression of class I major histocompatibility complex antigens on infected cells. J Virol, 56: 757–766 Kambara H, Okano H, Chiocca EA et al (2005) An oncolytic HSV-1 mutant expressing ICP34.5 under control of a nestin promoter increases survival of animals even when symptomatic from a brain tumor. Cancer Res, 65: 2832–2839 Kamiryo T, Tada K, Shiraishi S et al (2002) Analysis of homozygous deletion of the p16 gene and correlation with survival in patients with glioblastoma multiforme. J Neurosurg, 96: 815–822 Kanai R, Eguchi K, Takahashi M et al (2006) Enhanced therapeutic efficacy of oncolytic herpes vector G207 against human non-small cell lung cancer--expression of an RNA-binding protein, Musashi1, as a marker for the tailored gene therapy. J Gene Med, 8: 1329–1340 Kaplitt MG, Tjuvajev JG, Leib DA et al (1994) Mutant herpes simplex virus induced regression of tumors growing in immunocompetent rats. J Neurooncol, 19: 137–147 Kaur BCT, Chiocca EA (2009) “Buy one get one free”: armed viruses for the treatment of cancer cells and their microenvironment. Curr Gene Ther, 9: 341–355 Kemeny N, Brown K, Covey A et al (2006) Phase I, open-label, dose-escalating study of a genetically engineered herpes simplex virus, NV1020, in subjects with metastatic colorectal carcinoma to the liver. Hum Gene Ther, 17: 1214–1224 Kesari S, Randazzo BP, Valyi-Nagy T et al (1995) Therapy of experimental human brain tumors using a neuroattenuated herpes simplex virus mutant. Lab Invest, 73: 636–648 Kimata H, Takakuwa H, Goshima F et al (2003) Effective treatment of disseminated peritoneal colon cancer with new replication-competent herpes simplex viruses. Hepatogastroenterology, 50: 961–966 Kimata H, Imai T, Kikumori T et al (2006) Pilot study of oncolytic viral therapy using mutant herpes simplex virus (HF10) against recurrent metastatic breast cancer. Ann Surg Oncol, 13: 1078–1084 Kohno S, Luo C, Goshima F et al (2005) Herpes simplex virus type 1 mutant HF10 oncolytic viral therapy for bladder cancer. Urology, 66: 1116–1121 Koromilas AE, Roy S, Barber GN et  al (1992) Malignant transformation by a mutant of the IFN-inducible dsRNA-dependent protein kinase. Science, 257: 1685–1689 Kucharczuk JC, Randazzo B, Chang MY et al (1997) Use of a “replication-restricted” herpes virus to treat experimental human malignant mesothelioma. Cancer Res, 57: 466–471 Kurozumi K, Hardcastle J, Thakur R et al (2007) Effect of tumor microenvironment modulation on the efficacy of oncolytic virus therapy. J Natl Cancer Inst, 99: 1768–1781 Lasner TM, Kesari S, Brown SM et al (1996) Therapy of a murine model of pediatric brain tumors using a herpes simplex virus type-1 ICP34.5 mutant and demonstration of viral replication within the CNS. J Neuropathol Exp Neurol, 55: 1259–1269 Leib DA, Machalek MA, Williams BR et al (2000) Specific phenotypic restoration of an attenuated virus by knockout of a host resistance gene. Proc Natl Acad Sci U S A, 97: 6097–6101 Liu BL, Robinson M, Han ZQ et al (2003) ICP34.5 deleted herpes simplex virus with enhanced oncolytic, immune stimulating, and anti-tumour properties. Gene Ther, 10: 292–303 Liu TC, Zhang T, Fukuhara H et  al (2006) Oncolytic HSV armed with platelet factor 4, an antiangiogenic agent, shows enhanced efficacy. Mol Ther, 14: 789–797 Mace AT, Ganly I, Soutar DS et al (2008) Potential for efficacy of the oncolytic Herpes simplex virus 1716 in patients with oral squamous cell carcinoma. Head Neck, 30: 1045–1051 MacKie RM, Stewart B, Brown SM (2001) Intralesional injection of herpes simplex virus 1716 in metastatic melanoma. Lancet, 357: 525–526 MacLean AR, ul-Fareed M, Robertson L et al (1991) Herpes simplex virus type 1 deletion variants 1714 and 1716 pinpoint neurovirulence-related sequences in Glasgow strain 17+ between immediate early gene 1 and the “a” sequence. J Gen Virol, 72 (Pt 3): 631–639 Mahller YY, Vaikunth SS, Ripberger MC et al (2008) Tissue inhibitor of metalloproteinase-3 via oncolytic herpesvirus inhibits tumor growth and vascular progenitors. Cancer Res, 68: 1170–1179

228

R.L. Price et al.

Markert JM, Malick A, Coen DM et al (1993) Reduction and elimination of encephalitis in an experimental glioma therapy model with attenuated herpes simplex mutants that retain susceptibility to acyclovir. Neurosurgery, 32: 597–603 Markert JM, Medlock MD, Rabkin SD et  al (2000) Conditionally replicating herpes simplex virus mutant, G207 for the treatment of malignant glioma: results of a phase I trial. Gene Ther, 7: 867–874 Markert JM, Liechty PG, Wang W et al (2009) Phase Ib trial of mutant herpes simplex virus G207 inoculated pre-and post-tumor resection for recurrent GBM. Mol Ther, 17: 199–207 Martuza RL, Malick A, Markert JM et al (1991) Experimental therapy of human glioma by means of a genetically engineered virus mutant. Science, 252: 854–856 Mavromara-Nazos P, Ackermann M, Roizman B (1986) Construction and properties of a viable herpes simplex virus 1 recombinant lacking coding sequences of the alpha 47 gene. J Virol, 60: 807–812 McFadden G, Kane K (1994) How DNA viruses perturb functional MHC expression to alter immune recognition. Adv Cancer Res, 63: 117–209 McKie EA, MacLean AR, Lewis AD et al (1996) Selective in vitro replication of herpes simplex virus type 1 (HSV-1) ICP34.5 null mutants in primary human CNS tumours – evaluation of a potentially effective clinical therapy. Br J Cancer, 74: 745–752 McKie EA, Brown SM, MacLean AR et  al (1998) Histopathological responses in the CNS following inoculation with a non-neurovirulent mutant (1716) of herpes simplex virus type 1 (HSV 1): relevance for gene and cancer therapy. Neuropathol Appl Neurobiol, 24: 367–372 Meignier B, Longnecker R, Roizman B (1988) In vivo behavior of genetically engineered herpes simplex viruses R7017 and R7020: construction and evaluation in rodents. J Infect Dis, 158: 602–614 Meignier B, Martin B, Whitley RJ et al (1990) In vivo behavior of genetically engineered herpes simplex viruses R7017 and R7020. II. Studies in immunocompetent and immunosuppressed owl monkeys (Aotus trivirgatus). J Infect Dis, 162: 313–321 Meurs EF, Galabru J, Barber GN et al (1993) Tumor suppressor function of the interferon-induced double-stranded RNA-activated protein kinase. Proc Natl Acad Sci U S A, 90: 232–236 Mineta T, Rabkin SD, Yazaki T et al (1995) Attenuated multi-mutated herpes simplex virus-1 for the treatment of malignant gliomas. Nat Med, 1: 938–943 Mohr I, Gluzman Y (1996) A herpesvirus genetic element which affects translation in the absence of the viral GADD34 function. EMBO J, 15: 4759–4766 Mullen JT, Kasuya H, Yoon SS et al (2002) Regulation of herpes simplex virus 1 replication using tumor-associated promoters. Ann Surg, 236: 502–512; discussion 512–503 Nakamura H, Mullen JT, Chandrasekhar S et al (2001) Multimodality therapy with a replicationconditional herpes simplex virus 1 mutant that expresses yeast cytosine deaminase for intratumoral conversion of 5-fluorocytosine to 5-fluorouracil. Cancer Res, 61: 5447–5452 Nakao A, Takeda S, Shimoyama S et al (2007) Clinical experiment of mutant herpes simplex virus HF10 therapy for cancer. Curr Cancer Drug Targets, 7: 169–174 Nishiyama Y, Kimura H, Daikoku T (1991) Complementary lethal invasion of the central nervous system by nonneuroinvasive herpes simplex virus types 1 and 2. J Virol, 65: 4520–4524 Papanastassiou V, Rampling R, Fraser M et al (2002) The potential for efficacy of the modified (ICP 34.5(-)) herpes simplex virus HSV1716 following intratumoural injection into human malignant glioma: a proof of principle study. Gene Ther, 9: 398–406 Poppers J, Mulvey M, Khoo D et  al (2000) Inhibition of PKR activation by the proline-rich RNA binding domain of the herpes simplex virus type 1 Us11 protein. J Virol, 74: 11215–11221 Preston VG, Palfreyman JW, Dutia BM (1984) Identification of a herpes simplex virus type 1 polypeptide which is a component of the virus-induced ribonucleotide reductase. J Gen Virol, 65 (Pt 9): 1457–1466 Radbill AE, Reddy AT, Markert JM et  al (2007) Effects of G207, a conditionally replicationcompetent oncolytic herpes simplex virus, on the developing mammalian brain. J Neurovirol, 13: 118-129

12  Herpes Simplex Virus 1 for Cancer Therapy

229

Rampling R, Cruickshank G, Papanastassiou V et  al (2000) Toxicity evaluation of replicationcompetent herpes simplex virus (ICP 34.5 null mutant 1716) in patients with recurrent malignant glioma. Gene Ther, 7: 859–866 Randazzo BP, Kesari S, Gesser RM et al (1995) Treatment of experimental intracranial murine melanoma with a neuroattenuated herpes simplex virus 1 mutant. Virology, 211: 94–101 Randazzo BP, Bhat MG, Kesari S et al (1997) Treatment of experimental subcutaneous human melanoma with a replication-restricted herpes simplex virus mutant. J Invest Dermatol, 108: 933–937 Thomlinson RH, Gray LH (1955) The histological structure of some human lung cancers and the possible implications for radiotherapy. Br J Cancer, 9: 539–549 Roizman B (1996) The function of herpes simplex virus genes: a primer for genetic engineering of novel vectors. Proc Natl Acad Sci U S A, 93: 11307–11312 Schmid DS, Rouse BT (1992) The role of T cell immunity in control of herpes simplex virus. Curr Top Microbiol Immunol, 179: 57–74 Senzer N, Kaufman H, Amatruda T, Nemunaitis M, Daniels G, Glaspy J, Goldsweig H, Coffin RS, Nemunaitis J (2009) Phase II clinical trial with a second generation, GM-CSF encoding, oncolytic herpesvirus in unresectable metastatic melanoma. Paper presented at the American Society of Clinical Oncology. Shimoyama S, Goshima F, Teshigahara O et al (2007) Enhanced efficacy of herpes simplex virus mutant HF10 combined with paclitaxel in peritoneal cancer dissemination models. Hepatogastroenterology, 54: 1038–1042 Simpson GR, Han Z, Liu B et al (2006) Combination of a fusogenic glycoprotein, prodrug activation, and oncolytic herpes simplex virus for enhanced local tumor control. Cancer Res, 66: 4835–4842 Song TJ, Eisenberg DP, Adusumilli PS et al (2006) Oncolytic herpes viral therapy is effective in the treatment of hepatocellular carcinoma cell lines. J Gastrointest Surg, 10: 532–542 Sundaresan P, Hunter WD, Martuza RL et  al (2000) Attenuated, replication-competent herpes simplex virus type 1 mutant G207: safety evaluation in mice. J Virol, 74: 3832–3841 Takakuwa H, Goshima F, Nozawa N et al (2003) Oncolytic viral therapy using a spontaneously generated herpes simplex virus type 1 variant for disseminated peritoneal tumor in immunocompetent mice. Arch Virol, 148: 813–825 Terada K, Wakimoto H, Tyminski E et  al (2006) Development of a rapid method to generate multiple oncolytic HSV vectors and their in  vivo evaluation using syngeneic mouse tumor models. Gene Ther, 13: 705–714 Teshigahara O, Goshima F, Takao K et al (2004) Oncolytic viral therapy for breast cancer with herpes simplex virus type 1 mutant HF 10. J Surg Oncol, 85: 42–47 Thelander L, Reichard P (1979) Reduction of ribonucleotides. Annu Rev Biochem, 48: 133–158 Thompson RL, Wagner EK, Stevens JG (1983) Physical location of a herpes simplex virus type-1 gene function(s) specifically associated with a 10 million-fold increase in HSV neurovirulence. Virology, 131: 180–192 Toda M, Rabkin SD, Martuza RL (1998) Treatment of human breast cancer in a brain metastatic model by G207, a replication-competent multimutated herpes simplex virus 1. Hum Gene Ther, 9: 2177–2185 Todo T, Feigenbaum F, Rabkin SD et  al (2000) Viral shedding and biodistribution of G207, a multimutated, conditionally replicating herpes simplex virus type 1, after intracerebral inoculation in aotus. Mol Ther, 2: 588–595 Todo T, Martuza RL, Rabkin SD et al (2001) Oncolytic herpes simplex virus vector with enhanced MHC class I presentation and tumor cell killing. Proc Natl Acad Sci U S A, 98: 6396–6401 Toyoizumi T, Mick R, Abbas AE et al (1999) Combined therapy with chemotherapeutic agents and herpes simplex virus type 1 ICP34.5 mutant (HSV-1716) in human non-small cell lung cancer. Hum Gene Ther, 10: 3013–3029 Tyminski E, Leroy S, Terada K et al (2005) Brain tumor oncolysis with replication-conditional herpes simplex virus type 1 expressing the prodrug-activating genes, CYP2B1 and secreted human intestinal carboxylesterase, in combination with cyclophosphamide and irinotecan. Cancer Res, 65: 6850–6857

230

R.L. Price et al.

Umene K (1986) Conversion of a fraction of the unique sequence to part of the inverted repeats in the S component of the herpes simplex virus type 1 genome. J Gen Virol, 67 (Pt 6): 1035–1048 Umene K, Eto T, Mori R et al (1984) Herpes simplex virus type 1 restriction fragment polymorphism determined using southern hybridization. Arch Virol, 80: 275–290 Ushijima Y, Luo C, Goshima F et al (2007) Determination and analysis of the DNA sequence of highly attenuated herpes simplex virus type 1 mutant HF10, a potential oncolytic virus. Microbes Infect, 9: 142–149 Varghese S, Newsome JT, Rabkin SD et  al (2001) Preclinical safety evaluation of G207, a replication-competent herpes simplex virus type 1, inoculated intraprostatically in mice and nonhuman primates. Hum Gene Ther, 12: 999–1010 Watanabe D, Goshima F, Mori I et al (2008) Oncolytic virotherapy for malignant melanoma with herpes simplex virus type 1 mutant HF10. J Dermatol Sci, 50: 185–196 Yamada Y, Kimura H, Morishima T et al (1991) The pathogenicity of ribonucleotide reductasenull mutants of herpes simplex virus type 1 in mice. J Infect Dis, 164: 1091–1097 Yamamura H, Hashio M, Noguchi M et al (2001) Identification of the transcriptional regulatory sequences of human calponin promoter and their use in targeting a conditionally replicating herpes vector to malignant human soft tissue and bone tumors. Cancer Res, 61: 3969–3977 Yazaki T, Manz HJ, Rabkin SD et al (1995) Treatment of human malignant meningiomas by G207, a replication-competent multimutated herpes simplex virus 1. Cancer Res, 55: 4752–4756 York IA, Roop C, Andrews DW et  al (1994) A cytosolic herpes simplex virus protein inhibits antigen presentation to CD8+ T lymphocytes. Cell, 77: 525–535

Chapter 13

Telomerase as a Target for Cancer Therapeutics Jerry W. Shay

Abstract  Telomerase, a ribonucleoprotein enzyme complex that includes a cellular reverse transcriptase component that adds DNA to the ends of chromosomes, is reactivated or upregulated in the vast majority of human advanced malignancies but is not expressed in most normal human tissues. Due to the selective expression in cancer cells but not most normal cells, many scientists have proposed that telomerase is a universal therapeutic target for human cancer. Most human tumors not only express telomerase but also have very short telomeres, whereas telomerase activity is absent, or is present at lower levels in normal tissues, which also generally have longer telomeres compared to tumor cell telomeres. The inhibition of telomerase, at least initially, should produce minimal toxicities to tissues and the mode of action of telomerase inhibitors predicts minimal side effects on normal stem cells that can express telomerase but have longer telomeres. Thus, telomerasebased therapies should be well tolerated and perhaps with minimal side effect on telomerase competent stem cells. This chapter will summarize the role of telomeres and telomerase in cancer and review the current status of ongoing telomerase clinical trials including gene therapy approaches. Keywords  Telomeres • Senescence • Immunotherapy • GRN163L • GV1001 • GRNVAC1 • Adenoviral suicide gene therapy • Oncolytic virus

1 Telomeres Human telomeres are repetitive, noncoding DNA [TTAGGG] structures at the ends of chromosomes that are bound by a series of single- and double-stranded DNAbinding proteins termed the shelterin complex (de Lange 2006). Telomeres shorten J.W. Shay (*) Department of Cell Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 7530-9039, USA e-mail: [email protected] J.A. Roth (ed.), Gene-Based Therapies for Cancer, Current Cancer Research, DOI 10.1007/978-1-4419-6102-0_13, © Springer Science+Business Media, LLC 2010

231

232

J.W. Shay

with each cell division in all cells including cancer cells, due to many factors including incomplete DNA lagging strand synthesis, end processing events and oxidative damage (de Lange 2006; von Zglinicki 2002). The shortening of telomeres would be predicted to occur in rapidly proliferating cells of the skin, gastrointestinal system, and blood (Shay and Wright 2006), but these tissues have mechanisms to transiently upregulate telomerase to slow down but not completely halt the rate of telomere loss (e.g., regulated telomerase activity). In contrast, the human embryonic stem cells and cancer cells have mechanisms to fully replace the progressively lost telomere repeats each cell cycle and thus maintain a steady state telomere length. At birth, human telomeres are approximately 15–20  kb in length (Wu et  al. 2003; Slagboom et  al. 1994) and shorten gradually throughout life in dividing cells, demonstrating that telomere length is a biological (as opposed to a calendar) replication-dependent timing mechanism. Thus, telomere length measurements in human cells provide a reasonable estimate of the approximate number of divisions that may be remaining. Once a critical shortened telomere length is attained, cell senescence is activated due to the uncapping of the telomere and recognition by the DNA damage response pathways (Shay and Roninson 2004; Campisi 2005; Campisi and d’Adda di Fagagna 2007). When a sufficient number of cells undergo senescence in a tissue or organ, a decline or loss of function of that tissue occurs that may contribute in substantial ways to what we recognize as aging. Cells that undergo senescence can do so by a variety of other mechanisms that may or may not be related to telomere shortening. For example, in addition to short/ dysfunctional telomeres due to repetitive cell divisions (replicative senescence), changes due to chromatin instability, DNA damage, and other stress signals may result in a more immediate cessation of cell growth (premature senescence, oncogene-induced senescence etc.) that is similar but not identical to replicative senescence (Shay and Roninson 2004; Campisi 2005; Campisi and d’Adda di Fagagna 2007). In addition, there is evidence supporting an inherited component to telomere length (Wu et al. 2003; Slagboom et al. 1994) and many correlative studies have demonstrated a connection between telomere length and aging. The progressive loss of telomeres throughout life has been measured in different human tissues and diseases (Brouilette et  al. 2003; von Zglinicki et  al. 2000; Benetos et al. 2001, 2004; Okuda et al. 2000; Samani et al. 2001; Panossian et al. 2003; Meeker et al. 2004; Rudolph et al. 2001; O’Sullivan et al. 2002; Wiemann et al. 2002; Farazi et al. 2003; Cawthon et al. 2003; Aviv 2004, 2006; Andrew et al. 2006; Valdes et al. 2005; Gardner et al. 2005; Nawrot et al. 2004; Martin-Ruiz et  al. 2005, 2006; Bischoff et  al. 2006; Jeanclos et  al. 2000; Unryn et  al. 2005; Epel et al. 2004; Honig et al. 2006; Rando 2006; Walne et al. 2005; Demissie et al. 2006; Fitzpatrick et  al. 2007). For example, shortened telomeres from cells in affected tissues have been reported in patients with Barrett’s esophagus, liver cirrhosis, myeloproliferative disorders, and ulcerative colitis (Rudolph et  al. 2001; O’Sullivan et al. 2002; Wiemann et al. 2002; Farazi et al. 2003). Other precancerous lesions such as prostatic and cervical intraepithelial neoplasias and ductal carcinoma in situ for breast cancer have also been shown to have critically shortened

13  Telomerase as a Target for Cancer Therapeutics

233

telomeres in situ (Meeker et al. 2004). One important question that remains difficult to answer is whether these greatly shortened telomeres in preneoplastic lesions promote the development of advanced disease or if the shortened telomeres and subsequent senescence act as a potent anticancer protection mechanism. One might predict that without additional cellular alterations, the DNA damage signals from telomere shortening (telomere uncapping) would be a very potent tumor suppressor/cell cycle checkpoint pathway, since the critically shortened telomeres could not easily be repaired in the absence of telomerase. Thus, replicative senescence and the many other induced forms of premature or oncogene-induced senescence stop cells from proliferating, thereby inhibiting progression to cancer. However, in the presence of alterations in important cell cycle checkpoint pathways, for example, p53 and the p16/pRB pathways, cells can continue to divide with critically shortened telomeres while still expressing activated DNA damage pathways (Wright et al. 1989; Shay and Wright 2005a, b). This bypass of normal senescence (M1) results in extended cellular lifespan and a progressive shortening of telomere lengths with each division until so many telomeres are so short that end–end chromosome fusions occur, producing chromosome breakage-fusion-bridge cycles (M2 or crisis), leading to chromosomal alterations, a hallmark of cancer. At M2 crisis, cells are initially balanced between altered cell cycle checkpoints encouraging cells to grow and terminally shortened telomeres preventing cell division. In this situation, the cells go into crisis (a balance between cell growth and cell death) and only a rare human cell that bypasses this stage can continue to divide. This very rare human cell with bypassed M1 escapes M2 only in 10−6 to 10−7 cells. Cells that escape crisis generally display telomere stability (usually at very short lengths) and the reactivation of telomerase (Wright et al. 1989; Shay and Wright 2005a, b).

2 Telomerase Telomerase adds telomeric (TTAGGG) repeats onto the chromosome ends compensating for the continued erosion of telomeres that occurs in its absence (de Lange et al. 1990; Blackburn 1992; Greider and Blackburn 1985; Cristofari and Lingner 2006). While the regulation of telomerase is still poorly understood, the human telomerase holoenzyme contains two essential components (Nakamura et al. 1997; Feng et al. 1995), a telomerase reverse transcriptase catalytic subunit (hTERT) (Nakamura et  al. 1997) and a functional (template) telomerase RNA (hTERC) (Feng et al. 1995). There are other telomerase-associated proteins (such as NOP10, NHP2, GAR1 and Dyskerin) that associate with the functional RNA component of telomerase some of which have been implicated in human diseases associated with telomere dysfunction (Mason et al. 2005; Yamaguchi et al. 2005; Mitchell et  al. 1999; Shay and Wright 1999, 2004; Vulliamy et  al. 2001; Heiss et al. 1998; Fogarty et al. 2003). In addition, p23/HSP90 associates with the catalytic hTERT component most likely as a molecular chaperone important in protein folding (Holt et al. 1999).

234

J.W. Shay

Telomerase is expressed in human embryonic and fetal cells and in adult male germline cells, but is undetectable in most normal somatic cells except at lower levels or transiently in proliferative cells of renewal tissues (Wright et al. 1996). For example, in resting lymphocytes, telomerase is not detected but it is in activated lymphocytes. Thus, telomerase activity in normal stem cells is low or repressed and in transit amplifying cells is active and then silenced in terminally differentiated cells. This mechanism of transiently upregulating telomerase likely slows down but does not prevent progressive telomere shortening over long periods of time. In normal somatic cells, even those with detectable telomerase activity, progressive telomere shortening occurs with each cell division, eventually leading to greatly shortened telomeres (Harley et al. 1990; Hastie et al. 1990; Lindsey et al. 1991). The introduction of the catalytic subunit of telomerase (hTERT) into telomerase silent cells is often sufficient to produce telomerase activity, elongate or maintain telomeres, and to result in the bypass of both M1/senescence and M2/crisis (Wright et  al. 1989; Shay and Wright 2005a; Bodnar et  al. 1998; Vaziri and Benchimol 1998). This demonstrates that the other components of the telomerase holoenzyme are present in normal telomerase silent cells and only the expression of the hTERT protein is absent in most normal cells. Thus, telomeres appear to count the number of times a cell has divided, and telomeres determine when cellular senescence (M1) and crisis (M2) occurs (Wright et al. 1989; Shay and Wright 2005a). Several human diseases of telomere dysfunction have recently been discovered (Mason et al. 2005; Yamaguchi et al. 2005; Mitchell et al. 1999; Shay and Wright 1999; 2004; Vulliamy et al. 2001; Heiss et al. 1998; Fogarty et al. 2003; Garcia et al. 2007; Tsakiri et al. 2007; Armanios et al. 2007), and individuals born with reduced levels of telomerase have significantly shortened telomeres often leading to disease progression earlier in life compared to normal individuals. These diseases often lead to telomere dysfunction in highly proliferative cells such as the hematopoietic cells of the bone marrow, resulting in diseases such as aplastic anemia and, in some instances, increased risk for the development of cancer (Mason et  al. 2005; Yamaguchi et  al. 2005; Mitchell et al. 1999; Shay and Wright 1999, 2004; Vulliamy et al. 2001; Heiss et al. 1998; Fogarty et  al. 2003). Thus, a more detailed knowledge of telomerase and telomere function may provide insights and perhaps therapeutic interventions into human diseases of telomere dysfunction.

3 Telomerase and Early Cancer Detection The potential of telomerase as a sensitive biomarker for screening, early cancer detection, prognosis, and in monitoring as an indication of residual disease have been well documented (Shay and Bacchetti 1997; Jakupciak et al. 2004; Shay et al. 1997; Herbert et al. 2003; Harley 2008; Shay 1998). The detection of telomerase activity has been evaluated using commercially available research assays on fresh or fresh frozen tumor biopsies, bodily fluids, and secretions. Almost all studies have shown that reactivation or upregulation of telomerase activity, its template

13  Telomerase as a Target for Cancer Therapeutics

235

RNA (hTERC) and catalytic protein component (hTERT) are associated with a higher percent of all cancer types investigated than most other cancer molecular diagnostic assays. The detection of lesions prior to the onset of tissue invasion would be one important goal of telomerase screening. The development of clinical telomerase diagnostic assays for monitoring patients that are approved by regulatory agencies such as the Food and Drug Administration are still undergoing additional validation and standardization studies (Jakupciak et  al. 2004; Shay et  al. 1997; Herbert et al. 2003; Harley 2008; Shay 1998).

4 Antitelomerase Cancer Therapy Since telomerase activity is detected in almost all advanced tumors (Table 1), the use of telomerase inhibitors may provide an effective and novel molecularly targeted approach for cancer therapy. Normal somatic cells that lack telomerase expression should be largely unaffected by antitelomerase therapy. Normal stem cells that when dividing express telomerase would also not be initially affected since most have significantly longer telomeres compared to cancer cell telomeres. Thus, it is believed there may be a therapeutic window of time where telomerase expressing tumor cells may be induced to undergo apoptosis before normal telomerase expressing cells are adversely affected. Antitelomerase therapies are likely to be most effective when used after tumor reduction surgery and in combination with other standard therapies such as chemotherapy and radiation therapy. In addition, the combination of novel targeted therapeutics, such as angiogenic inhibitors together with telomerase inhibitors, are especially attractive and are currently being tested in at least one ongoing clinical trial (see next section). In this instance, the angiogenic inhibitors would keep the tumor size small but dividing, while telomerase inhibitors would gradually lead to telomere shortening with each cell division resulting in the induction of apoptosis and potentially durable responses. Telomerase inhibitors may also be effective in reducing the risk of relapse by targeting the small numbers of telomerase-positive cancer cells in adjacent tissues not removed during tumor resection (Harley 2008; Shay 1998, 2003; White et al. 2004; Shay and Wright 2002, 2005c, 2007; Gellert et al. 2005; Shay and Keith 2008; Keith et al. 2007). While telomerase is downregulated in non dividing cells, even if there is a subset of more quiescent cancer cells, eventually they also would have to proliferate and thus in the presence of a telomerase inhibitor even these cells should eventually display progressive telomere shortening. There have been several recent reviews on this area of research (Harley 2008; Shay and Keith 2008; Keith et al. 2007; Shay and Wright 2007). Numerous telomerase inhibitor strategies have been developed that are in preclinical or various stages of clinical trials (Harley 2008; Shay 1998, 2003; White et al. 2004; Shay and Wright 2002, 2005c, 2007; Gellert et al. 2005; Shay and Keith 2008; Keith et al. 2007). Active clinical trials involving telomeres and telomerase strategies will be reviewed in the remainder of this chapter.

236

J.W. Shay Table 1  Telomerase activity in samples obtained from malignant tissues No. samples positive/ Telomerase Type of malignancy no. tested positive (%) Acute myeloid leukemia 47/64 73 Basal cell carcinoma 73/77 95 Bladder carcinoma 172/185 92 Breast carcinoma (ductal and lobular) 300/339 88 Cervical carcinoma 16/16 100 Chronic myeloid leukemia   Chronic 30/42 71   Blast 21/21 100 Colorectal carcinoma 123/138 89 Gastric carcinoma 72/85 85 Head and neck squamous cell carcinoma 112/120 86 Hepatocellular carcinoma 149/173 86 Lymphoma   Low grade 12/14 86   High grade 16/16 100 Melanoma 6/7 85 Neuroblastoma 99/105 94 Nonsmall cell lung carcinoma 98/125 78 Ovarian carcinoma 21/23 91 Pancreatic carcinoma 41/43 95 Prostate carcinoma 52/58 90 Renal carcinoma 95/115 83 Retinoblastoma 17/34 50 Small cell lung carcinoma 15/15 100 Squamous cell carcinoma 15/18 83 Data summarized from Shay and Bacchetti (1997)

5 Telomerase Immunotherapy Perhaps one of the most rapidly advancing areas of telomerase therapy involves immunotherapy or vaccines targeting telomerase (Vonderheide et  al. 2001a, b, 2004; Vonderheide 2002, 2007; Brunsvig et al. 2006; Su et al. 2005; Carpenter and Vonderheide 2006; Domchek et al. 2007; Danet-Desnoyers et al. 2005; Minev et al. 2000; Nair et  al. 2000; Nava-Parafa and Emems 2007; Mavroudis et  al. 2006; Bolonaki et al. 2007; Bernhardt et al. 2006). Telomerase is processed and presented in the context of HLA molecules at the cell surface and since telomerase is activated in the vast majority of cancers, it is an attractive candidate as a universal tumor antigen. In many types of human cancers, it has been determined that human hTERT-specific epitopes (amino acids 611–626, EARPALLTSRLRFIPK, and 540–548, ILAKFLHWL of the hTERT protein) are expressed on cancer cells but not on normal cells, such as activated lymphocytes or hematopoietic stem cells. The high-binding telomerase peptide which has received most attention is the HLA-A2

13  Telomerase as a Target for Cancer Therapeutics

237

restricted I540 epitope (ILAKFLHWL). Investigators have generated cytotoxic T lymphocytes (CTLs) specific for this peptide and then demonstrated that the CTLs lyse peptide-pulsed cells (Vonderheide et  al. 2001a; Minev et  al. 2000). Clinical trials involving patients with lung, prostate, breast, and pancreatic cancers have been conducted with excellent and encouraging early results (Brunsvig et al. 2006; Su et  al. 2005; Domchek et  al. 2007; Nava-Parafa and Emems 2007; Mavroudis et al. 2006; Bolonaki et al. 2007; Bernhardt et al. 2006). So far, patients have not had treatment-related serious adverse effects (such as autoimmune disease or bone marrow toxicity). A modification of the I540 high affinity peptide was made to reduce potential autoimmunity to self antigens. This involved making a low affinity epitope that allows escape from T-cell tolerance mechanisms. The affinity of such low affinity epitopes can be increased by deleting tyrosine at position 1 and replacing it with an arginine. One such peptide in clinical development is 572Y (Vx-001). Patients with advanced nonsmall cell lung cancer received sequential Y572 peptide vaccinations (Mavroudis et al. 2006; Bolonaki et al. 2007). None of the 22 patients demonstrated native TERT 572 specific IFN-gamma producing cells prevaccination, but these cells were present in 7/8 patients following the sixth vaccination and 9/10 patients had HLA A0201/TERT 572Y pentamer positive cells. Early immune responders had significantly longer time to progression and overall survival compared to immune nonresponders. In November 2007, Vaxon Biotech received European orphan drug designation for Vx-001 for the treatment of hTERT positive nonsmall cell lung cancer in HLA-A2 + patients and the company is planning further phase II development for both nonsmall cell lung cancer and for hepatocellular cancer. GV1001 is a telomerase-specific promiscuous Class II peptide vaccine that is currently in an advanced stage of clinical development. GV1001 is a 16 mer peptide vaccine representing amino acids 611–626 of hTERT (H-Glu-Ala-Arg-Pro-AlaLeu-Leu-Thr-Ser-Arg-Leu-Arg-Phe-Ile-Pro-Lys-OH). The peptide contains binding motifs that allow promiscuous binding to a broad array of Class II molecules. Clinical trials with GV1001 in two Phase II studies (Brunsvig et al. 2006; Bernhardt et al. 2006) have examined the immunogenicity and clinical activity of the vaccine (originally developed by Pharmexa, Denmark and now KAEL Co. Ltd. Korea, a company specializing in peptide vaccines). On the basis of these initial experiments, there is evidence that vaccination with the GV1001 peptide can stimulate T-helper cell reactivity to the GV1001 peptide presented in association with multiple Class II alleles. In addition, cells can recognize naturally processed hTERT protein as well as the naturally occurring CTL precursors against telomeraseexpressing malignant cells. In these early studies, evidence that an immune response could translate into a clinical benefit for patients vaccinated with GV1001 was obtained. Patients receiving an intermediate dose of GV1001 in the pancreatic cancer study had significantly improved overall survival compared with the lowdose group (8.6 vs. 4  months). The median survival for immune responders was 7.2 months compared with 2.9 months for the nonresponders. The drug has now achieved orphan drug status for the treatment of pancreatic cancer both in Europe and in the United States.

238

J.W. Shay

A Phase III clinical trial is now in progress for advanced pancreatic cancer (NCRI-sponsored UK Telovac trial, using GV1001, Gary Middleton, PI). GV1001 is supplied as a GMP-produced freeze dried peptide. GV1001 is reconstituted in normal saline and is given as an intradermal injection in combination with GM-CSF (75 mcgs per administration given 10–15  min before vaccination). The vaccine is administered in the right para-umbilical area and the vaccination schedule consists of three injections in week 1, weekly injections given in weeks 2, 3, 4, 6, and 10 and then booster injections given every 4 weeks (Gary Middleton, personnel communications). The TeloVac study is a 1:1:1 randomization to either Gemcitabine/Capecitabine chemotherapy (the control arm) or two experimental arms of either sequential or concomitant chemoimmunotherapy. The sequential arm consists of 8 weeks of Gemcitabine/Capecitabine chemotherapy followed by treatment with GV1001. If patients have either subjective or objective progression on the vaccine, chemotherapy is reinitiated provided the patient has not progressed on the first 8 weeks of chemotherapy. In the concurrent arm, Gemcitabine/Capecitabine and GV1001 are given together. The primary endpoint of the study is overall survival and the trial plans to recruit 1,110 patients. The first patient entered in March 2007 and the trial is recruiting well (Gary Middleton, personnel communications). A decision to include GV1001 into the treatment of pancreatic cancer patients in addition to chemotherapy is based on well-accepted evidence that chemotherapy provides improvements in both the quality of life and in overall survival. Indeed, a delay in the use of standard chemotherapy in a disease with rapid progression would be a suboptimal, perhaps unethical strategy. The current Phase III study should add to an emerging paradigm in cancer immunology that chemotherapy significantly augments immune responses. In addition to the Telovac Phase III clinical trial, GV1001 is also being tested in a phase II study called Heptovax in Germany, France, and Spain to evaluate the safety and immunogenicity of the vaccine in locally advanced or metastatic hepatocellular carcinoma patients. In the Hepatovax trial, all patients will be treated with a single initial dose of Cyclophosphamide to impact on T regulatory cells. A second phase II study, CT98, being conducted in Norway, is evaluating the safety and immunogenicity of the vaccine in patients with locally advanced NSCLC who have completed chemoradiation. The final approach to telomerase vaccination involves the use of purified DNAor RNA-transfected dendritic cells (immune cells that detect antigens and activate other immune cells). This has an advantage over vaccination with Class I peptides, since this approach encodes multiple epitopes for many HLA alleles and eliminates the need for HLA testing prior to vaccination. The approach taken by Geron Corporation (now in collaboration with Merck Corporation) uses a lysosomal targeting signal to channel antigen processing into the Class II pathway (Su et al. 2005). In an initial proof of principal study, 20 patients with metastatic prostate cancer were randomized to receive either hTERT or lysosome-associated membrane protein-1 (LAMP-1) mRNA transfected autologous mature DCs intradermally (Su et al. 2005). The development of hTERT-specific T cell responses was associated with the transient reduction in the number of PSA-expressing circulating tumor cells, an effect which disappeared as the immune response subsided. The LAMP hTERT vaccine is being clinically developed by Geron as GRNVAC1, which has entered phase II testing in AML patients in complete remission following

13  Telomerase as a Target for Cancer Therapeutics

239

chemotherapy. A second generation cancer immunotherapy (GRNVAC2) has recently been announced (www.Geron.com). Finally Geron and collaborators have reported the production of dendritic cells derived from human embryonic stem cells. These cells are scalable and exhibit the normal functions of naturally occurring human dendritic cells found in the bloodstream. These findings support the use of human embryonic stem cell-derived dendritic cells in therapeutic vaccine applications for cancer and other diseases. Substituting standardized dendritic cells for current approaches instead of using small numbers of dendritic cells obtained from individual patients may result in more cost-effective and reliable approaches to cancer immunotherapy (www.Geron.com). However, as with all trials using cells derived from human embryonic cells, safety considerations are a top priority. Finally, Merck recently initiated a Phase I clinical trial of V934/V935, a nondendritic cell-based cancer vaccine candidate targeting telomerase. The trial will assess the safety, tolerability and immunogenicity of the vaccine candidate in patients with solid tumors, including nonsmall cell lung cancer and prostate carcinoma. In summary, there are several ongoing randomized cancer vaccination trials associated with telomerase (Table  2). The development of this approach for a telomerase-based universal cancer vaccine is encouraging and may be even more effective when used to treat patients with less advanced disease.

6 GRN163L (Imetelstat), Oligonucleotide Enzyme Inhibitor Another major approach to inhibiting telomerase is an hTERC competitive telomerase (oligonucleotide-based) cancer therapy. This therapy is based on the 11-base template region of telomerase RNA (hTR, hTERC) that is a target for direct enzymatic inhibition of telomerase activity (Dikmen et  al. 2005, 2008; Pongracz et  al. 2003; Gryaznov et  al. 2007; Jackson et  al. 2007; Gellert et  al. 2006; Herbert et  al. 2005; Hochreiter et  al. 2006; Gomez-Millan et  al. 2007; Djojosubroto et  al. 2005; Ozawa et  al. 2004; Wang et  al. 2004; Akiyama et  al. 2003). Regardless of the conformation of the telomerase holoenzyme, the template region of hTERC (the site that encodes the sequence to be synthesized on the telomere) must be exposed for the enzyme to act and thus must be accessible to the targeted oligonucleotides. One such compound that has been developed is called GRN163L or Imetelstat (Geron Corp., Menlo Park, CA). It is in early stage clinical trials as a single agent in patients with chronic lymphocytic leukemia and solid tumors (Dikmen et  al. 2005, 2008; Pongracz et  al. 2003; Gryaznov et  al. 2007; Jackson et  al. 2007; Gellert et  al. 2006; Herbert et  al. 2005; Hochreiter et  al. 2006; Gomez-Millan et  al. 2007; Djojosubroto et  al. 2005; Ozawa et  al. 2004; Wang et al. 2004; Akiyama et al. 2003). GRN163L is also being tested in combination with standard doublet chemotherapy (Paclitaxel® and Carboplatin®) for advanced non small cell lung cancer, in combination with Paclitaxel® and Bevacizumab (Avastin®) for advanced breast cancer, and in combination with Bortezomib (Velcade®) for relapsed or refractory multiple myeloma (Table  3). The sequence of the GRN163L oligonucleotide is 5¢-Palm-TAGGGTTAGACAA-3¢,

Direct killing of telomerase positive cells

hTERT oncolytic virus (OBP-301)

Oncolys BioPharma, Tokyo, Japan (www.oncolys.com) John Numunaitis (www.marycrowleymedicalresearch.com)

Geron Corporation (www.geron.com)

Who is conducting clinical trials on the target Robert Vonderheide, U. Penn Medical (www.med.upenn.edu) Geron Corporation GRNVAC 1 and 2 (www.geron.com) Gary Middleton GV1001 (www.royalsurrey.nhs.uk)

TVAX tumor vaccine, hTERT human telomerase reverse transcriptase, hTR/hTERC human telomerase RNA

Telomerase inhibitor Telomere shortening

hTR/hTERC oligonucleotide (GRN163L)

Table 2  Telomerase targets and related therapies in clinical trials Expected outcome of intervention Telomerase target approach at target hTERT immunotherapy vaccine Tumor reduction Prevention of relapse (TVAX)

240 J.W. Shay

13  Telomerase as a Target for Cancer Therapeutics

241

Table 3  GRN163L ongoing or completed clinical trials •   Phase I solid tumor trial (safety, tolerability, MTD) – 2 U.S. sites open, GRN163L single agent •   Phase I/II CLL – 5 U.S. sites open GRN163L single agent •   Phase I/II Multiple Myeloma – 4 U.S. sites open – GRN163L + Bortezomib (Velcade®) with and without dexamethasone – Refractory or relapsed •   Phase I/II Breast Cancer – I U.S. site open – GRN163L + Paclitaxel and Bevacizumab (Avastin®) – Local recurrent or metastatic breast cancer •   Phase I/II NSCLC Lung Cancer – 4 U.S. sites open – GRN163L + Paclitaxel and Carboplatin Stage IIIb with pleural effusion, Stage IV, or recurrent disease

that is complementary to a 13 nucleotide-long region partially overlapping and extending by four nucleotides beyond the 5¢-boundary of the template region of hTERC. This lipidated 13-mer thio-phosphoramidate targets the hTERC component of telomerase, preventing it from forming an active complex with hTERT. Since GRN163L contains a lipid palmitate moiety, it can easily enter cells without uptake carriers. The thio-phosphoramidate backbone of GRN163L confers greatly enhanced stability and high affinity binding to telomerase while the lipid modification on GRN163L significantly improves its biodistribution. Most, but not necessarily all, telomerase inhibitors (see next section) may require a period of time to drive already short telomeres into a state of crisis and apoptotic cell death. During this treatment period, telomeres would gradually shorten but the tumor mass would also continue to increase. Thus, a telomerase inhibitor such as GRN163L may not be sufficiently effective by itself in patients with large tumor burdens, since cell death would only occur after many divisions had taken place (depending on the length of the shortest telomeres in the tumor tissue). Current chemotherapy approaches, when effective, result in a short-term reduction in tumor burden, but even with continued treatments tumor relapse and therapy resistance often occur and telomere length is not affected. However, using telomerase inhibitors such as GRN163L in combination with conventional or even other targeted therapeutic approaches that produce a setting of minimal residual disease should lead to progressive telomere shortening and perhaps durable responses leading to an overall improvement in survival. In summary, GRN163L is one of the first generation of oligonucleotide small molecule telomerase inhibitors for the treatment of cancer. Ongoing clinical trial results should provide valuable information on maximum tolerable doses and toxicity, hopefully leading to more advanced clinical trials to determine if there are decreases in time to progression or improvements in overall survival. An important challenge for basic and preclinical work in the future will be to develop the best strategies to design more effective clinical trials. The goal is to develop combination therapies to best enhance the shortening of telomeres to cause a more rapid decrease in cancer cell proliferation without affecting normal cell telomeres. Knowledge of

242

J.W. Shay

the factors influencing the recruitment to or action of telomerase on telomeres may enhance the efficacy of telomerase inhibitors (Shay and Wright 2005c; Seimiya et al. 2005; Seimiya 2006).

7 Telomerase-Associated Gene Therapy While the oligonucleotide approach with GRN163L is likely to require a substantial amount of treatment time to obtain durable responses, an alternate approach to reduce the lag time is to induce apoptotic pathways via gene therapy that can be coupled to telomerase activity. For example, one could utilize a central control mechanism that is responsible for regulating the expression of telomerase in the cell, and use it to drive a suicide gene or replication-competent adenoviral vector that will rapidly kill the telomerase-expressing cancer cells (Shay and Keith 2008; Keith et al. 2007). There are two general approaches to telomerase gene therapy; suicide gene therapy and oncolytic viral therapy.

8 Ad-hTR-NTR: A Telomerase Targeted Adenoviral Suicide Gene Therapy Vector The Ad-hTR-NTR adenoviral delivery of the suicide gene therapy construct consists of the activation of the nitroreductase enzyme (NTR) by the hTERC (telomerase functional RNA) promoter and the addition of a prodrug. Telomerase regulation of a suicide gene therapy approach is the novel component of this therapeutic plan. A phase I study of Ad-hTR-NTR is due to start in the near future. This will be a single dose, open label, Phase I study of intraperitoneal Ad-hTR-NTR, in patients with advanced inoperable intraabdominal cancer (Nicol Keith, personnel communications). This will include a number of different primary tumor types including ovarian, colon, pancreatic and gastric as well as patients with ascites (an accumulation of fluid in the abdominal cavity). These tumor types are known to express significant levels of hTERC; therefore, the expression of the suicide gene from the hTERC promoter should be strong. Because of the location and ascites, there will be opportunity for retrieving samples from patients to monitor the progress of the therapy and to collect valuable information that will contribute significantly to the further development of the therapeutic regimen that would not be easily available in other cancer models.

9 Telomerase Specific Oncolytic Virus This approach utilizes adenoviruses that have been manipulated or engineered to have oncolytic, or cancer-killing, properties, enabling them to selectively target and destroy cancer cells that express telomerase. The promoter region of the telomerase

13  Telomerase as a Target for Cancer Therapeutics

243

Fig. 1  Oncolytic viruses to target telomerase. Oncolytic viruses can be engineering to only replicate in telomerase expressing cells. Using the hTERT promoter driving critical adenoviral genes could result in highly selective replication in tumor cells while sparing telomerase-negative normal cells. To avoid affecting stem-like cells that also express telomerase, an oncolytic virus that incorporates more than one cancer specific pathway (e.g., alterations in RB/E2F or p53 in combination with the TERT promoter) could provide added safety against affecting normal telomeraseexpressing proliferative stem cells

(hTERT) gene regulating the replication of adenovirus permits selective viral propagation within cancer cells but not normal cells that do not express telomerase. Thus, telomerase-expressing tumor cells containing the telomerase-specific virus eventually rupture and die theoretically spreading the virus to adjacent cells. When these same engineered viruses infect normal somatic cells, there is no replication or killing effects. This approach is known as a Tumor-specific Replication competent ADdenoviral (hTERTp-TRAD) gene therapy approach and is discussed in Chap. 6 (Fig. 1).

10 Concluding Remarks The maintenance of telomeres by the cellular ribonucleoprotein enzyme telomerase is of well-documented importance for cancer (Granger et  al. 2002; Aisner et  al. 2000). While normal human tissues shorten telomeres throughout life, most human cancers possess short telomeres and maintain telomere length by expressing telomerase. Mice deficient in telomerase and humans with reduced telomerase activity develop progressive pathologies primarily by affecting the proliferative potential of tissues with high turnover. Telomerase is an attractive target for cancer diagnosis and therapy since it is expressed in ~90% of primary tumors cells while it is not expressed in most normal tissues (Yashima et  al. 1997; Ohyashiki et  al. 1997; Hiyama et al. 2001). Telomere dysfunction therefore represents a potent tumor suppressor

244

J.W. Shay

mechanism that limits the proliferative capacity of premalignant cells, thus at least initially preventing the unlimited growth that is a hallmark of cancer cells. Normal or premalignant cells that escape these proliferative limitations by activating or upregulating telomerase (or a rarer mechanism involving DNA recombination) can both continue to grow and also acquire additional mutations permitting more advanced disease progression such as distal metastasis. Targeting telomerase as a therapeutic approach for cancer would be expected to lead to telomere shortening and if maintained at high enough concentrations for sufficient time, could induce cancer cell death (as has been observed in some animal xenograft models). Several clinical trials are in progress using novel approaches to targeting telomerase and information on the utility of these approaches should be well established in a few years. While it is still too early to know the best methods to use telomerase inhibitors for treating patients with advanced disease, the ongoing trials will address questions of whether telomerase is an effective target in combination with the standard of care therapy. These may also indicate if there will be an additive or synergistic effect when a telomerase inhibitor is combined with chemotherapy. While it remains challenging to treat patients with advanced and metastatic cancer, the greatest utility may lie in using telomerase inhibitors as adjuvant therapy in earlier stage disease or even as a primary cancer preventive approach in high-risk patient populations, or in cancer patients with high risk for recurrence of disease. There are still many unanswered questions with respect to key safety concerns, such as the effect of telomerase inhibitors on normal stem cells that express some telomerase. While there is mounting evidence that cancer cells expressing stem cell markers also express telomerase activity, it is not known if they will be resistant to telomerase inhibition therapy. One issue that is currently being investigated is if cancer stem cells are more quiescent than the bulk of the more differentiated tumor cells. If this is correct, then telomerase inhibitors may be less effective. Clearly, there remains much to be discovered in this rapidly evolving and important area of cancer research. Acknowledgements  Supported in part by NCI SPORE CA70907 and CA127297.

References Aisner, DL, Wright, WE, Shay, JW. Telomerase regulation: not just flipping the switch. Curr Opin Genet Dev, 2000;12:80–5. Akiyama, M, Hideshima, T, Shammas, MA, et  al. Effects of oligonucleotide N3¢-->P5¢ thiophosphoramidate (GRN163) targeting telomerase RNA in human multiple myeloma cells. Cancer Res, 2003;63(19):6187–94. Andrew, T, Aviv, A, Falchi, M, et  al. Mapping genetic loci that determine leukocyte telomere length in a large sample of unselected, female sibling pairs. Am J Hum Genet, 2006;78:480–6. Armanios, MY, Chen, JJ, Cogan, JD, et al. Telomerase mutations in families with idiopathic pulmonary fibrosis. N Engl J Med, 2007;356:1370–2. Aviv, A. Telomeres and human aging: facts and fibs. Sci Aging Knowledge Environ, 2004;51:43–5.

13  Telomerase as a Target for Cancer Therapeutics

245

Aviv, A, Valdes, A, Gardner, JP, Swaminathan, R, Kimura, M, Spector, TD. Menopause modifies the association of leukocyte telomere length with insulin resistance and inflammation. J Clin Endocrinol Metab, 2006;91:635–40. Benetos, A, Okuda, K, Lajemi, M, et al. Telomere length as an indicator of biological aging: the gender effect and relation with pulse pressure and pulse wave velocity. Hypertension, 2001;37:381–5. Benetos, A, Gardner, JP, Zureik, M, et al. Short telomeres are associated with increased carotid atherosclerosis in hypertensive subjects. Hypertension, 2004;43:182–5. Bernhardt, SL, Gjertsen, MK, Trachsel, S, et al. Telomerase peptide vaccination of patients with non-resectable pancreatic cancer: a dose escalating phase I/II study. Br J Cancer, 2006 Dec 4;95(11):1474–82. Bischoff, C, Petersen, HC, Graakjaer, J, et al. No association between telomere length and survival among the elderly and oldest old. Epidemiology, 2006;17:190–4. Blackburn EH. Telomerases. Annu Rev Biochem, 1992;61:113–29. Bodnar, AG, Ouellete, M, Frolkis, M, et al. Extension of lifespan by introduction of telomerase in normal human cells. Science, 1998;279:349–52. Bolonaki, I, Kotsakis, A, Papadimitraki, E, Vaccination of patients with advanced non-small-cell lung cancer with an optimized cryptic human telomerase reverse transcriptase peptide. J Clin Oncol, 2007 Jul 1;25(19):2727–34. Brouilette, S, Singh, RK, Thompson, JR, Goodall, AH, Samani, NJ. White cell telomere length and risk of premature myocardial infarction. Arterioscler Throm Vasc Biol, 2003;23:842–6. Brunsvig, PF, Aamdal, S, Gjertsen, MK, et al. Telomerase peptide vaccination: a phase I/II study in patients with non-small cell lung cancer. Cancer Immunol Immunother, 2006;55(12): 1553–64. Campisi, J. Senescent cells, tumor suppression, and organismal aging: good citizen, bad neighbors. Cell, 2005;120:513–22. Campisi, J, d’Adda di Fagagna, F. Cellular senescence: when bad things happen to good cells. Nat Rev Mol Cell Biol, 2007;8:729–40. Carpenter, EL, Vonderheide, RH. Telomerase-based immunotherapy of cancer. Expert Opin Biol Ther, 2006;10:1031–9. Cawthon RM, Smith KR, O’Brien E, Sivatchenko A, Kerber RA. Association between telomere length in blood and mortality in people aged 60 years or older. Lancet, 2003;361:393–5. Cristofari, G, Lingner, J. The telomerase ribonucleoprotein particle. In: de Lange, T, Lundblad, V, and Blackburn, E, (Eds), Telomeres. Second Edition, Cold Spring Harbor Laboratory Press, NY, 2006; pp. 21–48. Danet-Desnoyers, GH, Luongo, JL, Bonnet, DA, Domchek, SM, Vonderheide, RH. Telomerase vaccination has no detectable effect on SCID-repopulating and colony-forming activities in the bone marrow of cancer patients. Exp Hematol, 2005;33:1275–80. de Lange, T. Mammalian Telomeres. In: de Lange, T, Lundblad, V, and Blackburn, E, (Eds), Telomeres. Second edition, Cold Spring Harbor Laboratory Press, NY, 2006; pp. 387–431. de Lange, T, Shiue, L, Meyers, RM, et al. Structure and variability of human chromosome ends. Mol Cell Biol, 1990:10:518–27. Demissie, S, Levy, D, Benjamin, EJ, et al. Insulin resistance, oxidative stress, hypertension, and leukocyte telomere length in men from the Framingham Heart Study. Aging Cell, 2006;5:325–30. Dikmen, ZG, Gellert, GC, Jackson, S, et al. In vivo inhibition of lung cancer by GRN163L – a novel human telomerase inhibitor. Cancer Res, 2005;65:7866–73. Dikmen, ZG, Wright, WE, Shay, JW, Gryaznov, SM. Telomerase targeted oligonucleotide thiophosphoramidates in T24-luc bladder cancer cells. J Cell Biochem, 2008;104:444–52. Djojosubroto, MW, Chin, AC, Go, N, et  al. Telomerase antagonists GRN163 and GRN163L inhibit tumor growth and increase chemosensitivity of human hepatoma. Hepatology, 2005;42(5):1127–36. Domchek, SM, Recio, A, Mick, R, et al. Telomerase-specific T-cell immunity in breast cancer: impact of vaccination on immunosurveillance. Cancer Res, 2007;67:10546–55.

246

J.W. Shay

Epel, ES, Blackburn, EH, Lin, J, et al. Accelerated telomere shortening in response to life stress. Proc Natl Acad Sci U S A, 2004;101:17312–5. Farazi, PA, Glickman, J, Jiang, S, Yu, A, Rudolph, KL, DePinho, RA. Differential impact of telomere dysfunction on initiation and progression of hepatocellular carcinoma. Cancer Res, 2003;63:5021–7. Feng, J, Funk, WD, Wang, S-S, et  al. The RNA component of human telomerase. Science, 1995;269:1236–41. Fitzpatrick, AL, Kronmal, RA, Gardner, JP, et al. Leukocyte telomere length and cardiovascular disease in the cardiovascular health study. Am J Epidemiol, 2007;165:14–21. Fogarty, PF, Yamaguchi, H, Wiestner, A, et  al. Late presentation of dyskeratosis congenital as apparently acquired aplastic anemia due to mutations in telomerase RNA. Lancet, 2003;362:628–30. Garcia, CK, Wright, WE, Shay, JW. Human diseases of telomerase dysfunction: insights into tissue aging. Nucleic Acids Res, 2007;35:7406–16. Gardner, JP, Li, S, Srinivasan, SR, Chen, W, et  al. Rise in insulin resistance is associated with escalated telomere attrition. Circulation, 2005;111:2171–7. Gellert, GC, Jackson, SR, Dikmen, G, Wright, WE, Shay, JW. Telomerase as a therapeutic target in cancer. Drug Discov Today, 2005;2:159–64. Gellert, GC, Dikmen, ZG, Wright, WE, Gryaznov, S, Shay, JW. Effects of a novel telomerase inhibitor, GRN163L, in human breast cancer. Breast Cancer Res Treat 2006;96:73–81. Gomez-Millan, J, Goldblatt, EM, Gryaznov, SM, Mendonca, MS, Herbert, BS. Specific telomere dysfunction induced by GRN163L increases radiation sensitivity in breast cancer cells. Int J Radiat Oncol Biol Phys, 2007 Mar 1;67(3):897–905. Granger, MP, Wright, WE, Shay, JW. Telomerase in cancer and aging. Critical Rev Oncol Hematol, 2002;41:29–40. Greider, CW, Blackburn, EH. Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell, 1985;43:405–13. Gryaznov, SM, Jackson, S, Dikmen, G, et  al. Oligonucleotide conjugate GRN163L targeting human telomerase as potential anticancer and antimetastatic agent. Nucleosides Nucleotides Nucleic Acids, 2007;26:1–3. Harley, CB. Telomerase and cancer therapeutics. Nat Rev Cancer, 2008;8:167–79. Harley, CB, Futcher, BA, Greider, CW. Telomeres shorten during aging of human fibroblasts. Nature, 1990;345:458–60. Hastie, ND, Dempster, M, Dunlop, MG, Thompson, AM, Green, DK, Allshire, RC. Telomere reduction in human colorectal carcinoma and with ageing. Nature, 1990;346:866–8. Heiss, NS, Knight, SW, Vulliamy, TJ, et al. X-linked dyskeratosis congenita is caused by mutations in a highly conserved gene with putative nucleolar functions. Nat Genet, 1998;19:32–8. Herbert, B-S, Shay, JW, Wright, WE. Analysis of telomeres and telomerase. In: Current protocols in cell biology. Wiley, Inc., New York, NY, 2003; pp. 18.6.6. Herbert, B-S, Gellert, GC, Hochreiter, A, et al. Lipid modification of oligonucleotide N3¢→P5¢ – thio-phosphoramidates enhances the potency of telomerase inhibition. Oncogene, 2005;24:5262–8. Hiyama, E, Hiyama, K, Yokoyama, T, et  al. Immunohistochemical detection of telomerase (hTERT) protein in human cancer tissues and a susbset of cells in normal tissues. Neoplasia, 2001;3:17–26. Hochreiter, AE, Xiao, H, Goldblatt, EM. Telomerase template antagonist GRN163L disrupts telomere maintenance, tumor growth, and metastasis of breast cancer. Clin Cancer Res, 2006 May 15;12(10):3184–92. Holt, SE, Aisner, DL, Baur, J, et al. Functional requirement of p23 and hsp90 in telomerase complexes. Genes Dev, 1999;13:817–26. Honig, LS, Schupf, N, Lee, JH, Tang, MX, Mayeux, R. Shorter telomeres are associated with mortality in those with APOE E4 and dementia. Ann Neurol, 2006;60:181–7. Jackson, SR, Zhu, C-H, Paulson, V, et al. Anti-adhesive effects of GRN163L – an oligonucleotide, N3¢-P5¢ thio-phosphoramidate targeting telomerase. Cancer Res, 2007;67:1121–9.

13  Telomerase as a Target for Cancer Therapeutics

247

Jakupciak, JP, Wang, W, Barker, PE, Srivastava, S, Atha, DH. Analytical validation of telomerase activity for cancer early detection – TRAP/PCR-CE and hTERT mRNA quantification assay for high-throughput screening of tumor cells. J Mol Diagn, 2004;6(3):157–65. Jeanclos, E, Schork, NJ, Kyvik, KO, Kimura, M, Skurnick, JH, Aviv, A. Telomere length inversely correlates with pulse pressure and is highly familial. Hypertension, 2000;36:195–200. Keith, WN, Thomson, CM, Howcroft, J, Maitland, NJ, Shay, JW. Seeding drug discovery: integrating telomerase cancer biology and cellular senescence to uncover new therapeutic opportunities in targeting cancer stem cells. Drug Discov Today, 2007;12:611–21. Lindsey, J, McGill, NI, Lindsey, LA, Green, DK, Cooke, HJ. In vivo loss of telomeric repeats with age in humans. Mutat Res, 1991;256:45–8. Martin-Ruis, C, Dickinson, HO, Key, B, Rowan, E, Kenny, RA, von Zgliniki, T. Telomere length predicts poststroke mortality, dementia, and cognitive decline. Ann Neurol, 2006;60:174–80. Martin-Ruiz, CM, Gussekloo, J, van Heemst, D, von Zglinicki, T, Westendrop, RG. Telomere length in white blood cells is not associated with morbidity or mortality in the oldest old: a population-based study. Aging Cell, 2005;4:287–90. Mason, PJ, Wilson, DB, Bessler, M. Dyskeratosis congenita – a disease of dysfunctional telomere maintenance. Curr Mol Med, 2005;5(2):159–70. Mavroudis, D, Bolonakis, I, Cornet, S. A phase I study of the optimized cryptic peptide TERT(572y) in patients with advanced malignancies. Oncology, 2006;70(4):306–14. Meeker, AK, Hicks, JL, Platz, EA, et al. Telomere length abnormalities occur early in the initiation of epithelial carcinogenesis. Clin Can Res, 2004;10:317–26. Minev, B, Hipp, J, Firat, H, et  al. Cytotoxic T cell immunity against telomerase reverse transcriptase in humans. Proc Natl Acad Sci U S A, 2000;97:4796–801. Mitchell, JR, Wood, E, Collins, KA. A telomerase component is defective in the human disease dyskeratosis congenita. Nature, 1999;402:551–5. Nair, SK, Heiser, A, Boczkowski, D, et al. Induction of cytotoxic T lymphocyte responses and tumor immunity against unrelated tumors using telomerase reverse transcriptase RNA transfected dendritic cells. Nat Med, 2000;6:1011–7. Nakamura, TM, Morin, GB, Chapman, KB, et  al. Telomerase catalytic subunit homologs from fission yeast and humans. Science, 1997;277:955–9. Nava-Parafa, P, Emems, LA. GV10001, an injectable telomerase peptide vaccine for the treatment of solid cancers. Curr Opin Mol Ther, 2007;9:490–7. Nawrot, TS, Staessen, JA, Gardner, JP, Aviv, A. Telomere length and possible link to X chromosome. Lancet, 2004;363:507–10. O’Sullivan, JN, Bronner, MP, Brentnall, TA, et al. Chromosomal instability in ulcerative colitis is related to telomere shortening. Nat Genet, 2002;32:280–4. Ohyashiki, K, Ohyashiki, JH, Nishimaki, J. et  al. Cytological detection of telomerase activity using an in situ telomeric repeat amplification protocol assay. Cancer Res, 1997;57:2100–3. Okuda, K, Khan, MY, Skurnick, J, Kimura, M, Aviv, H, Aviv, A. Telomere attrition of the human abdominal aorta: relationships with age and atherosclerosis. Atherosclerosis, 2000;152:391–8. Ozawa, T, Gryaznov, SM, Hu, LJ, et  al. Antitumor effects of specific telomerase inhibitor GRN163 in human glioblastoma xenografts. Neuro Oncol, 2004;6(3):218–26. Panossian, LA, Porter, VR, Valenzuela, HF, et al. Telomere shortening in T cells correlate with Alzheimer’s disease status. Neurobiol Aging, 2003;24:77–84. Pongracz, K, Li, S, Herbert, B-S, Pruzan, R, et  al. Novel short oligonucleotide conjugates as inhibitors of human telomerase. Nucleosides Nucleotides Nucleic Acids, 2003;22:1627–9. Rando, T. Prognostic value of telomere length: the long and short of it. Ann Neurol, 2006;60: 155–7. Rudolph, KL, Millard, M, Bosenberg, MW, DePinho, RA. Telomere dysfunction and evolution of intestinal carcinoma in mice and humans. Nat Genet, 2001;28:155–9. Samani, NJ, Boultby, R, Butler, R, Thompson, JR, Goodall, AH. Telomere shortening in atherosclerosis. Lancet, 2001;358:472–3.

248

J.W. Shay

Seimiya, H. The telomeric PARP, tankyrases, as targets for cancer therapy. Br J Cancer, 2006 Feb 13;94(3):341–5. Seimiya, H, Muramatsu, Y, Ohishi, T, Tsuruo, T, Tankyrase 1 as a target for telomere-directed molecular cancer therapeutics. Cancer Cell, 2005;7(1):25–37. Shay, JW. Telomerase in cancer: diagnostic, prognostic and therapeutic implications. Cancer J Scientific Am, 1998;4:26–34. Shay, JW. Telomerase therapeutics: telomeres recognized as a DNA damage signal. Clin Cancer Res, 2003;9:3521–5. Shay, JW, Bacchetti, S. A survey of telomerase in human cancer. Eur J Cancer, 1997;33:787–91. Shay, JW, Keith, WN. Targeting telomerase for cancer therapeutics. Br J Cancer, 2008;98:677–83. Shay, JW, Roninson, IB. Hallmarks of senescence in carcinogenesis and cancer therapy. Oncogene, 2004;23:2919–33. Shay, JW, Wright, WE. Telomeres in dyskeratosis congenita. Nat Genet, 1999;36:437–8. Shay, JW, Wright, WE. Telomerase: a target for cancer therapy. Cancer Cell, 2002;2:257–65. Shay, JW, Wright, WE. Mutant dyskerin ends relationship with telomerase. Science, 2004;286:2284–5. Shay, JW, Wright, WE. Senescence and immortalization: role of telomeres and telomerase. Carcinogenesis, 2005;26:867–74. Shay, JW, Wright, WE. Telomerase and human cancer. In: de Lange, T, Lundblad, V, and Blackburn, E, (Eds), Telomeres. Second Edition, Cold Spring Harbor Laboratory Press, NY, 2005, pp. 81–108. Shay, JW, Wright, WE. Mechanism-based combination telomerase inhibition therapy. Cancer Cell, 2005;7:1–2. Shay JW, Wright, WE. Telomerase therapeutics for cancer: challenges and new directions. Nat Rev Drug Discov, 2006;5:577–84. Shay, JW, Wright, WE. Hallmarks of telomeres in ageing research, J Pathol, 2007;211:114–23. Shay, JW, Werbin, H, Wright, WE. Telomerase assays in the diagnosis and prognosis of cancer. In: Telomeres and Telomerase (Ciba Foundation Symposium 211) Wiley, Chichester, 1997;211:148–60. Slagboom, PE, Droog, S, Boomsma, DI. Genetic determination of telomere size in humans: a twin study of three age groups. Am J Hum Genet, 1994;55:876–82. Su, Z, Dannull, J, Yang, BK, et  al. Telomerase mRNA-transfected dendritic cells stimulate antigen-specific CD8+ and CD4+ cell responses in patients with metastatic prostate cancer. J Immunol, 2005;174:3798–807. Tsakiri, KD, Cronkite, JT, Kuan, PJ, et al. Adult-onset pulmonary fibrosis caused by mutations in telomerase. Proc Nat Acad Sci U S A, 2007;104:7552–7. Unryn, BM, Cook, LS, Riabowol, KT. Paternal age is positively linked to telomere length of children. Aging Cell, 2005;4:97–101. Valdes, AM, Andrew, T, Gardner, JP, et  al. Obesity, cigarette smoking, and telomere length in women. Lancet, 2005;366:662–4. Vaziri, H, Benchimol, S. Reconstitution of telomerase activity in normal human cells leads to elongation of telomeres and extended replicative life span. Curr Biol, 1998;8:279–82. von Zglinicki, T. Oxidative stress shortens telomeres. Trends Biochem Sci, 2002;27:339–44. von Zglinicki, T, Serra, V, Lorenz, M. et al. Short telomeres in patients with vascular dementia: an indicator of low antioxidative capacity and a possible risk factors? Lab Invest, 2000;80:1739–47. Vonderheide, RH. Telomerase as a universal tumor-associated antigen for cancer immunotherapy. Oncogene, 2002;21:674–9. Vonderheide, RH. Universal tumor antigens for cancer vaccination: targeting telomerase for immunoprevention. Discov Med, 2007;7:103–8. Vonderheide, RH, Schultze, JL, Anderson, KS, et al. Equivalent induction of telomerase-specific cytotoxic T lymphocytes from tumor-bearing patients and healthy individuals. Cancer Res, 2001;61:8366–70.

13  Telomerase as a Target for Cancer Therapeutics

249

Vonderheide, RH, Anderson, KS, Hahn, WC, Butler, MO, Schultze, JL, Nadler, LM. Characterization of HLA-A3-restricted cytotoxic T lymphocytes reactive against the widely expressed tumor antigen telomerase. Clin Cancer Res, 2001;7:343–8. Vonderheide, RH, et al. Vaccination of cancer patients against telomerase induces functional antitumor CD8+ T lymphocytes. Clin Cancer Res, 2004;10:828–39. Vulliamy, T, Marrone, A, Goldman, F, et  al. The RNA component of telomerase is mutated in autosomal dominant dyskeratosis congenita. Nature, 2001;413:432–5. Walne, A, Marrone, A, Dokal, I. Dyskeratosis congenita: a disorder of defective telomere maintenance? Int J Hematol, 2005;82:184–9. Wang, ES, Wu, K, Chin, AC, et  al. Telomerase inhibition with an oligonucleotide telomerase template antagonist: in vitro and in vivo studies in multiple myeloma and lymphoma. Blood, 2004;103(1):258–66. White, L, Wright, WE, Shay, JW. Telomerase inhibitors. Trends Biotechnol, 2004;6(3):157–65. Wiemann, SU, Satyanarayana, A, Tsahuridu, M, Tillmann, HL, Zender, L, Klempnauer, J, et al. Hepatocyte telomere shortening and senescence are general markers of human liver cirrhosis. FASEB J, 2002;16:935–42. Wright, WE, Pereira-Smith, OM, Shay, JW. Reversible cellular senescence: a two-stage model for the immortalization of normal human diploid fibroblasts. Mol Cell Biol, 1989;9:3088–92. Wright, WE, Piatyszek, MA, Rainey, WE, Byrd, W, Shay, JW. Telomerase activity in human germline and embryonic tissues and cells. Dev Gen, 1996;18:173–9. Wu, X, Amos, CI, Zhu, Y, Zhao, H, Grossman, BH, Shay, JW, Swan, GE, Benowitz, NL, Luo, S, Spitz, MR. Telomere dysfunction: a potential cancer predisposition factor. J Natl Cancer Inst, 2003;95:1211–8. Yamaguchi, H, Calado, RT, Ly, H, et al. Mutations in TERT, the gene for telomerase reverse transcriptase, in aplastic anemia. N Engl J Med, 2005;352:1413–24. Yashima, K, Piatyszek, MA, Saboorian, HM, et  al. Telomerase activity and in situ telomerase RNA expression in malignant and non-malignant lymph node. J Clin Pathol, 1997;50:110–7.

Chapter 14

Gene Therapy for Sarcoma Keila E. Torres and Raphael E. Pollock

Abstract  Soft tissue sarcomas are a group of potentially devastating malignancies. Despite multidisciplinary treatment combining surgery, radiation therapy, and chemotherapy, the overall prognosis for sarcoma patients remains poor. Very few chemotherapeutic agents with meaningful efficacy: toxicity ratios are available, and the development of novel therapeutics will be critical if this currently unacceptable prognosis is to be improved. Much exciting progress in genetic profiling of sarcomas and molecular identification of constituent oncogenes and protein products has recently occurred, which will ultimately enable the development of sarcoma targeted therapies. In this chapter, we discuss the major advances in cytogenetics and the importance of genetic and molecular signatures in sarcoma diagnosis and treatment. In an attempt to create better treatments for cancers several strategies have evolved to administer therapeutic genes that inactivate oncogenes, restore tumor-suppressor genes, and enhance the native immune response. We explore some promising initial data regarding novel gene delivery systems, such as isolated limb perfusion which may be a useful promising technique in delivering growth-suppressing constructs into soft tissue sarcomas. Keywords  Soft tissue sarcoma • Cytogenetics • Expression signatures • Gene therapy

1 Introduction Soft tissue sarcoma comprises a heterogeneous group of rare malignancies consisting of more than 50 distinct histological subtypes that putatively share a common mesenchymal origin and account for approximately 1% of all adult malignancies. R.E. Pollock (*) Department of Surgical Oncology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Unit 444, Houston, TX, 77030, USA e-mail: [email protected] J.A. Roth (ed.), Gene-Based Therapies for Cancer, Current Cancer Research, DOI 10.1007/978-1-4419-6102-0_14, © Springer Science+Business Media, LLC 2010

251

252

K.E. Torres and R.E. Pollock

The American Cancer Society estimates that approximately 10,660 new cases of soft tissue sarcoma will be diagnosed in 2009 and that 3,820 patients will die from the disease (Jemal et  al. 2009). Effective treatment of sarcoma usually requires surgical excision with the addition of radiotherapy and/or chemotherapy. Although conventional therapeutic treatments can effectively reduce the overall tumor mass, they often fail to achieve a curative outcome. Very few chemotherapeutic agents with meaningful efficacy: toxicity ratios are available, and the development of novel therapeutics will be critical if this currently unacceptable prognosis is to be improved. However, progress in this regard has been impeded by the relative rarity of the disease, rendering it a challenge to assemble the requisite investigative resources to support dedicated sarcoma research teams. This problem is compounded by the remarkable histological and cytogenetic heterogeneity underlying the variable clinical behaviors of this tumor cluster. In turn, these issues negatively impact on the ability to generate the precise diagnoses needed for subtype-specific tumor staging, treatment planning, clinical trials accrual, and prognostic inferences needed in contemporary oncology practice. Against this backdrop, over the past several decades significant progress has been achieved due to the development of improved pathological diagnostic criteria, including the advent of molecular diagnostics, enhanced imaging techniques, including metabolic-based dynamic examinations, new surgical applications such as microvascular tissue transfer underlying advances in limb preservation, and the development of a somewhat broader chemotherapy armamentarium. These enhancements have been integrated into multidisciplinary treatment and research programs at the leading sarcoma centers world-wide, thereby providing a critical scaffold upon which alternative therapeutic initiatives such as sarcoma immunotherapy and gene therapy strategies can be actively investigated (Meyers et al. 2008; Mori et al. 2006; Witlox et al. 2007).

2 Cytogenetics of Soft Tissue Sarcoma One of the most significant advances in sarcoma diagnosis has been the development and introduction of molecular probes to detect consistent cytogenetic and molecular abnormalities (Table 1). The identification of numerous simple as well as complex karyotypic abnormalities in patients bearing different sarcoma subtypes suggests that these processes may be important in sarcomagenesis while also presenting prognostic insight (Ludwig 2008). From the molecular point of view, sarcomas can be divided into two major groups based on their genetic alterations. The former group consists of sarcomas showing specific, recurrent genetic alterations, and relatively simple karyotypes, such as Ewing sarcoma. The second group represents sarcomas with variable gene alterations and very complex karyotypes such as leiomyosarcomas (Yang et  al. 2009). Until recent years, gastrointestinal stromal tumors (GISTs) were not clearly delineated from leiomyosarcoma. Leiomyosarcoma only rarely expresses c-kit

14  Gene Therapy for Sarcoma Table 1  Cytogenetic and molecular abnormalities in sarcomas Tumor type Cytogenetic Bone sarcomas Ewing’s sarcoma/PNET t(11;22)(q24;q12) t(21;22)(q12;q22) t(7;22)(p22;q12) Soft tissue sarcomas Malignant fibrous histiocytoma 1q11, 3p12, 11p11, and 19p13 Myxoid/round cell t(12;16)(q13;p11) Liposarcoma t(12;22)(q13;q11±12) Synovial sarcoma t(x,18)(p11;q11) Rhabdomyosarcoma t(2;13)(q35±37;q14) Alveolar t(1;13)(p36;q14) Embryonal trisomy 2q Neuroblastoma del (1p) Desmoplastic small round cell t(11;22)(p13;q12) Myxoid chondrosarcoma t(9;22)(q31;q12) Clear cell sarcoma t(12;22)(q13±14;q12±13) EWS-ATF1 Dermatofibrosarcoma t(17,22)(q22;q13)

253

Molecular EWS-FLI1 EWS-ERG EWS-ETV1

CHOP-TLS CHOP-EWS SYT-SSX PAX3-FKHR PAX7-FKHR

EWS-WT1 EWS-TEC PDGFB-COL1A1

when compared to GISTs, in which the majority of cases are positive for c-kit protein as measured by immunohistochemical approaches (Meza-Zepeda et al. 2006; Trent et al. 2007). Soft tissue leiomyosarcomas show multiple gene alterations in tandem with these very complex karyotypes; these include numerous gains and losses of genetic material and/or function. Analysis of about 100 leiomyosarcomas revealed that most of the karyotypes were complex, and there were no consistent recurrent aberrations found at the chromosomal level (Wang et al. 2001). The most frequent losses were detected in 10q and 13q, regions where the tumor suppressor genes PTEN and Rb reside. Interestingly, high level amplifications in 17p were often found in small tumors. These amplifications were not common in the very large tumors, suggesting that the small and large tumors could represent distinct types of leiomyosarcoma. Furthermore, this finding may imply that large leiomyosarcomas do not necessarily progress from the small tumor type or at least do not derive from the same clones. Gains in 1q, 5p, 6q, and 8q were detected in large tumors (El-Rifai et  al. 1998). The oncogenes MYC (located on 8q24) and MYB (located on 6q22) are the most likely involved candidate oncogenes. Analysis of the relative chromosome copy number in 14 cases of leiomyosarcoma revealed frequent gains in 5p15, 8q24, 15q25-26, 17p, and Xp (Otano-Joos et al. 2000). Gain of 17p11-12 was also found in high-grade osteosarcoma as well as in gliomas (van Dartel et al. 2003). The putative oncogenes on 17p are not well characterized. One of the genes located in 17p region is COPS3 (17p11-12). COPS3 has been shown to target p53 protein for proteosome-mediated degradation in osteosarcoma (Henriksen et al. 2003). Interestingly, murine double minute protein 2 (MDM2) amplification or p53 mutation was not found in osteosarcomas that contained COPS3 amplification. Amplification of the COPS3 gene was proposed to target p53 protein for proteosome-mediated degradation in osteosarcoma in a

254

K.E. Torres and R.E. Pollock

manner similar to MDM2 (Henriksen et al. 2003). These findings suggest the possibility that osteosarcomas and leiomyosarcomas may share a common mechanism for inactivation of the p53 pathway. Mutations in the p53 tumor-suppressor gene have been identified as the most common genetic alterations in soft tissue sarcoma (Das et al. 2007; Latres et al. 1994). These gene mutations are more often observed in metastases than in primary tumors and in high-grade versus low-grade sarcomas. Patients with p53 mutations have a markedly decreased overall survival relative to those containing wildtype p53 genes. Therefore, these mutations are thought to have a considerable negative impact on both overall as well as sarcoma-specific survival (Pollock et al. 1998; Schneider-Stock et  al. 1999; Taubert et  al. 1996). Our investigations of autologous human primary and metastatic sarcoma have demonstrated that clonal expansion of p53-mutated cells in soft tissue sarcoma confers distinct metastatic advantages (Pollock et al. 1996). Furthermore, we have also demonstrated that p53 alterations in soft tissue sarcoma contribute to metastasis-promoting behaviors, including the loss of cell cycle control (Pollock et al. 1998), enhanced angiogenesis (Zhang et al. 2000), invasiveness (Liu et al. 2006), and chemoresistance (Zhan et al. 2001, 2005). Many sarcoma subtypes harbor characteristic chromosomal translocations or nonrandom mutations that can aid in their identification. The majority of the genes located at the chromosomal breakpoints that determine specific sarcoma-associated translocations result in fusion genes encoding aberrant transcription factors and/or transcriptional regulators that alter gene expression. For instance, the fusion of the collagen type I alpha 1 (COL1A1) gene with the platelet-derived growth factor beta-chain (PDGFB) gene (COL1-PDGFB) has been shown to result in overexpression of the growth factor PDGFB (Simon et al. 1997). This growth factor activates platelet derived growth factor receptor beta and platelet-derived growth factor receptor alpha, ultimately leading to unregulated cell growth. A novel treatment is to inactivate the growth factor or receptor at the cellular level. This possibility was confirmed by the remarkable clinical response of patients with dermatofibrosarcoma protuberans who were treated with imatinib, a PDGFR inhibitor (Maki et al. 2002; Rubin et al. 2002). It has been found that mutations or deletions in genes encoding tyrosine kinases can result in constitutive activation leading to unregulated cell growth. For example, more than 90% of gastrointestinal tumors (GISTs) are associated with mutations of the KIT and/or PDGFRA gene (Yang et  al. 2008). Mutations of these genes are believed to be critical in GIST tumorigenesis (Gunawan et  al. 2002; Lasota and Miettinen 2008; Wozniak et  al. 2007). Specific mutations of KIT and PDGFRA gene have been found to correlate with the specific cell morphology, histologic phenotype, metastasis, and prognosis (Lasota and Miettinen 2006; Penzel et  al. 2005; Singer et al. 2002). Furthermore, specific mutations in KIT and PDGFR have been found to lead to differential drug sensitivity (Chen et al. 2004a). Cytogenetic aberrations associated with these tumors thus far described include the loss of 1p, 13q, 14q, or 15q, the loss of heterozygosity of 22q, numeric chromosomal imbalances, and nuclear/mitochondrial microsatellite instability (Chen et al. 2004b; Kose

14  Gene Therapy for Sarcoma

255

et al. 2006; Yamashita et al. 2006). Molecular genetic aberrations include the loss of heterozygosity of p16 (INK4A) and p14 (ARF), methylation of p15(INK4B), homozygous loss of the Hox11L1 gene, and amplification of C-MYC, MDM2, EGFR1, and CCND1 (Gunawan et  al. 2002; Lasota and Miettinen 2006, 2008; Wozniak et al. 2007) . Mutation of the KIT gene results in constitutive activation of this tyrosine kinase (Rubin et  al. 2001) which can result in ligand-independent tyrosine kinase activity, receptor autophosphorylation, and induction of other downstream kinases (e.g., phosphatitidylinositol 3-kinase and mitogen-activated kinases) (Hirota et al. 1998; Lux et al. 2000; Sarlomo-Rikala et al. 1998), ultimately eventuating in unregulated GIST proliferation. Characteristic chromosomal translocations involving the EWSR1 gene have been identified in clear cell sarcomas (Antonescu et al. 2006; Coindre et al. 2006; Hisaoka et  al. 2008; Panagopoulos et  al. 2002). The most common translocation partner is the activating transcription factor-1 (ATF1) gene on chromosome 12q13, of which four chimeric types have been described (Coindre et  al. 2006; Hisaoka et  al. 2008; Panagopoulos et  al. 2002). A less common translocation partner is cyclic AMP responsive-binding protein (CREB1) located on chromosome 2q34 (Hisaoka et al. 2008). ATF1 and CREB1 (cyclic AMP responsive-binding protein) both encode basic leucine zipper transcription factors that are involved in cAMP and Cap2-induced transcriptional activation (Persengiev and Green 2003). Both EWSR1-ATF1 and EWSR1-CREB1 chimeric proteins are believed to play a critical role in clear cell sarcoma oncogenesis (Antonescu et  al. 2006; Panagopoulos et al. 2002). Analysis of clear cell sarcoma tumors from patients indicate that the EWSR1-ATF1 chimeric transcript type 1 is the most common chimeric transcript found, with many cases carrying multiple chimeric transcripts (Wang et al. 2009b). Furthermore, our group has found that EWSR1-CREB1 is not exclusive to gastrointestinal tract tumors, indicating the need of incorporating this variant in routine molecular testing of soft tissue clear cell sarcoma specimens if RT–PCR for EWSR1-ATF1 is negative (Wang et  al. 2009b). The potential prognostic significance of the different chimeric types remains to be defined in future studies. Chromosomal translocations involving EWSR1 gene have also been found in extraskeletal myxoid chondrosarcomas. The majority of extraskeletal myxoid chondrosarcomas harbor a balanced translocation t(9;22)(q22;q12) that fuses EWSR1 with NR4A3. We were able to identify the rearrangement of the EWSR1 locus in 14 (93%) of 16 cases of extraskeletal myxoid chondrosarcomas using fluorescence in situ hybridization (FISH; Wang et  al. 2008). In this study, the vast majority of extraskeletal myxoid chondrosarcomas were associated with a rearrangement at the EWSR1 locus (22q12). Most sporadic desmoids have been associated with activating mutations in exon 3 of the gene that encodes the cell adhesion cofactor and nuclear signaling factor, b-catenin (CTNNB; Miyoshi et al. 1998; Tejpar et al. 1999). Analysis of a large single cohort of sporadic desmoids demonstrated mutations in CTNNB1 in 85% of cases (Lazar et  al. 2008). A threefold increased risk of recurrence in desmoids was strongly associated with a specific CTNNB1 mutation in codon 45 (45F). This study suggests that genotyping of CTNNB1 exon 3 could be

256

K.E. Torres and R.E. Pollock

useful as a diagnostic test in equivocal situations such as in differentiating postsurgical scar from desmoid recurrence. In addition, such genotyping can also provide important prognostic insight regarding the risk of recurrence, and may therefore have bearing on selection of adjuvant therapeutic approaches. Conversely, desmoids arising in the setting of familial adenomatous polyposis (FAP) display germline inactivating mutations in adenosis polyposis coli (APC) tumor suppressor gene. Similar mutations in APC gene have been identified in a small subset of sporadic desmoid tumors (Alman et  al. 1997; Giarola et  al. 1998). Both APC and beta-catenin proteins are part of the Wnt signaling pathway. Activation of the Wnt/beta-catenin pathway is a hallmark of these tumors, and inhibition of this pathway may have clinical utility, as this appears to be the critical molecular event underlying desmoid tumor biology (Kotiligam et al. 2008). These chromosomal aberrations may occur alone or in combination with other genetic events and are believed to underlie malignant transformation. If a chromosomal alteration such as a nonrandom mutation can be connected to the development of a specific, aberrantly expressed protein, a potentially targetable molecular axis may be so identified. There is a growing awareness that more sensitive prognostication and therapeutic decision-making algorithms will need to incorporate relevant cytogenetic and molecular determinants. Furthermore, a new paradigm of classification, integrating the standard clinical and pathological criteria with molecular aberrations, may permit personalized prognosis and treatment.

3 Expression Signatures Much exciting progress in genetic profiling of sarcomas and molecular identification of constituent oncogenes and protein products has recently occurred, which will ultimately enable the development of sarcoma targeted therapies. Sarcoma expression profiles can be divided into two groups. One group consists of those that have consistent collections of upregulated and downregulated genes, thereby allowing specimen clustering such as with GIST (Nielsen et  al. 2002), synovial sarcoma (Nagayama et  al. 2002), and extraskeletal myxoid chondroma (Subramanian et al. 2005). The second group includes those that do not have genes that up- or downregulated, and therefore cannot be consistently grouped together by gene expression profiling such as pleomorphic sarcomas (Nielsen et al. 2002; Segal et al. 2003). Comparison of genes expressed in tumors and their precursor lesions can reveal important information about tumor progression. Malignant peripheral nerve sheath tumors, when compared with Schwann cells, exhibit a significant loss of expression of many genes related to Schwannian differentiation (SOX10, PMP22, NGFR, S100B) and concurrent upregulation of a smaller set of genes, many of which are markers of neural crest and mesenchymal stem cells (SOX9, TWIST1) (Miller et al. 2006). Using a large tissue microarray, our laboratory has shown that Ki67, vascular endothelial growth factor, p53, and pMEK are all overexpressed in malignant

14  Gene Therapy for Sarcoma

257

peripheral nerve sheath tumors (MPNST) compared to benign neurofibromas (Zou et al. 2009). In a multivariable analysis incorporating both molecular factors and traditional staging criteria, only tumor size and nuclear p53 expression were found to be independent prognosticators of MPNST outcome (Zou et  al. 2009). It has been found that neurofibromatosis-associated and sporadic cases of malignant peripheral nerve sheath tumor are remarkably similar on a molecular level (Holtkamp et al. 2004; Watson et al. 2004). Similar studies examining at malignant progression of chondrosarcoma have shown that this process correlates with an increased expression of genes that facilitate anaerobic metabolism, as well as a decreased production of matrix (Rozeman et al. 2005). This type of study can be used as a diagnostic tool by incorporating highly expressed genes for a particular tumor type, seeking to identify biomarkers that can be tested by immunohistochemistry. Microarray technology cannot only assist in the diagnosis of patients with sarcoma, but can also facilitate prognostication of patient outcomes. For example, analysis of gene expression profiles of Ewing sarcoma patients has led to the identification of a select set of genes that correlate with tumor resistance to chemotherapy in these individuals (Scotlandi et al. 2009). Molecular signatures allow the classification of sarcoma patients into high- and low-risk groups based on their clinical outcome. This classification has a practical value at diagnosis for selecting patients with sarcoma who are unresponsive to current treatments. Approaches such as these may enable definition of sarcoma molecular targets suitable for novel personalized therapy while also helping refine sarcoma staging algorithms. Microarrays have been a critical tool to identify new and relevant soft tissue sarcoma targetable loci. Nielsen et al. 2002 reported expression profiles for GIST involving Kit as a discriminator gene. Microarray approaches have been used to reveal involvement of the retinoic acid pathway in monophasic synovial sarcomas (Nielsen et al. 2003). It has been demonstrated that epidermal growth factor receptor (EGFR) is expressed in monophasic synovial sarcoma while erb-B2 is expressed in epithelial components of the biphasic synovial sarcoma variant (Allander et  al. 2002; Nielsen et al. 2002). The finding was further elaborated in in vitro studies demonstrating that EGFR blockade increased apoptosis, a p53-independent G(0)-G(1) cell cycle arrest, and decreased cyclin D1 expression (Ren et al. 2008). Additionally, in vivo studies using an EGFR tyrosine kinase inhibitor, Iressa plus doxorubicin markedly decreased the cell growth of HT1080 human fibrosarcoma cells in nude mice (Ren et al. 2008). These studies demonstrated that EGFR blockade combined with conventional chemotherapy results in antitumor activity in vitro and in vivo, suggesting the possibility that combining these synergistic treatments will improve sarcoma treatment. It has been demonstrated that ras protein has an active role in tumorigenesis. The ras protein is overexpressed in neurofibromatosis syndrome 1 (NF1), which confers a predisposition to the development of MPNST, a problem which affects as many as 10% of NF1 patients (Woods et al. 2002). It has been shown that inhibition of ras protein trafficking to the plasma cell membrane can be achieved using farnesylation inhibitors (Scappaticci and Marina 2001). Treatment with farnesylation

258

K.E. Torres and R.E. Pollock

inhibitors induces accumulation of cytoplasmic nonfarnesylated H-ras that is able to bind Raf and form inactive cytoplasmic ras/Raf complexes. The use of such targeted inhibitors may be therapeutically relevant in sarcomas overexpressing ras protein. Gene expression in leiomyosarcoma has been evaluated using microarray analysis arrays containing approximately 12,000 known genes and 48,000 expressed sequence tags (Skubitz and Skubitz 2003). Cyclin-dependent kinase (CDK) inhibitor 2A, diaphanous 3, doublecortin, calpain 6, interleukin-17B, and proteolipid 1 were overexpressed in uterine leiomyosarcoma compared with normal myometrium. In contrast several genes were found to be underexpressed in uterine leiomyosarcoma, including alcohol dehydrogenase 1A-polypeptide, alcohol dehydrogenase 1B polypeptide, insulin-like growth factor 1, c-jun, c-fos, and TU3A. Ragazzini et  al. (2004) analyzed whether the genetic amplification of CDK4, MDM2, GLI, and SAS genes of the 12q13-15 region in a group of soft tissue sarcomas correlated with overexpression of the protein products. This group observed that CDK4, MDM2, GLI and SAS were frequently altered and/or highly expressed in leiomyosarcomas and rhabdomyosarcomas, indicating that genes located at 12q13-15 may be important for tumorigenesis of these neoplasms. It was also found that most cases with CDK4, MDM2, or GLI gene alteration also demonstrated simultaneous high level of protein product expression. Conversely, another group found no MDM2 and CDK4 amplification in an additional leiomyosarcoma tissue collection so analyzed (Shimada et al. 2006). Therefore the role of MDM2 and CDK4 in leiomyosarcoma remains controversial and a subject for future elucidation. Another example of how overexpression of a specific protein can modulate tumor to chemoresistance is observed in cells overexpressing the Rad51 protein. Rad51 is believed to have a central role in homologous recombination. Excessive or uncontrolled homologous recombination poses a threat to genome integrity by inducing chromosome fusion, aberrant karyotype formation, and augmenting resistance to DNA damage-induced apoptosis (Raderschall et al. 2002a). Rad51 overexpression is sufficient to promote DNA strand exchange. High levels of Rad51 are associated with elevated rates of DNA recombination as well as enhanced resistance to DNA-damaging chemotherapies and/or ionizing radiation in several experimental tumor systems (Raderschall et  al. 2002b; Vispe et  al. 1998; Xu et al. 2005). Antisense strategies have been successfully used to attenuate Rad51mediated radioresistance in in  vitro and in  vivo (Ohnishi et  al. 1998; Taki et  al. 1996). We have shown that Rad51 protein is overexpressed in primary, recurrent, and metastatic human soft tissue sarcoma specimens of various histologic subtypes (Hannay et al. 2007b). In addition, inhibiting Rad51 expression using anti-Rad51 siRNA markedly increases chemosensitivity to low-dose doxorubicin. Interestingly, reintroduction of wtp53 into a human mutated p53 STS cell line (SKLMS-1 leiomyosarcoma) resulted in decreased Rad51 mRNA and protein expression. Examination of the Rad51 gene promoter suggested a putative activator protein 2 (AP2) binding site. AP2 is known to directly interact with p53. To confirm that AP2 binding to the Rad51 promoter leads to transcriptional repression, we transiently

14  Gene Therapy for Sarcoma

259

transfected SKLMS1 cells with a specific AP2 siRNA, leading to decreased AP2 expression in these cells and simultaneously increased Rad51 expression. The increase in Rad51 after AP2 inhibition in SKLMS1 cells harboring mutated p53 suggests that AP2 can also repress the Rad51 promoter in SKLMS1 cells in a wild type (wt) p53-independent manner. Taken together, these data suggest that p53induced Rad51 transcriptional repression is mediated by the binding of AP2 to the Rad51 promoter. Integration of gene expression profiles and cytogenetic profiles will provide additional information regarding the molecular basis of sarcoma development. While selective inhibition of signaling pathways holds great promise as a new strategy applicable to cancer therapy, it is important to bear in mind that there may be redundancy in signaling pathways or function such that abrogation of one might be obviated by the tumor shifting to an alternative mechanism, leading to therapeutic resistance. Consequently, it is more likely that the combinations of signal transduction inhibitors will lead to maximal and perhaps even synergistic constraints on tumor growth.

4 Gene Therapy Gene therapy offers an attractive approach to control specific genes and short nucleic acid sequences. The strategy is to transfer genetic material or nucleic acid constructs, such as plasmid DNA, viral RNA and DNA vectors, ribozymes, antisense molecules, decoy oligodeoxynucleotides, small interfering RNA (siRNA), and deoxyribozymes (DNAzymes) into cells in an attempt to achieve a therapeutic effect. The transfer of these gene therapy constructs can be achieved via several approaches, including viral-mediated vectors and nonviral-mediated vehicles using liposome delivery or DNA protein complexes. There have been some promising initial experiences utilizing these tools in the treatment of sarcoma.

5 Nonviral Vectors In 1996, Maelandsmo et al. (1996) constructed hammerhead ribozymes to target the calcium protein placental homolog (Cap1) gene, which has a putative role in enhancing the development of human cancer metastasis. This group demonstrated marked reduction of Cap1 transcript levels in vitro utilizing human osteosarcoma cell lines. While they were not able to demonstrate any in  vivo antiproliferation effects, they did observe a decrease in the metastatic burden in xenograft recipients treated with ribozyme targeted to Cap1. Some studies have explored interfering RNA (siRNA) for the treatment of osteosarcoma. For instance, expression of specific Ape1 siRNA was shown to inhibit tumor growth rate by as much as 62% in human osteosarcoma xenografts when

260

K.E. Torres and R.E. Pollock

combined with endostatin, an antiangiogenic agent (Wang et al. 2007). Ape1 was thought to enhance tumor sensitivity to antiangiogenic therapy in this preclinical experimental context. Additionally, in  vitro studies have demonstrated that the treatment with antivascular endothelial growth factor (anti-VEGF) siRNA reduces microvascular growth resulting in apoptosis of osteosarcoma tumors (Mei et al. 2008). The use of siRNA has the theoretical advantage of being resistant to nuclease degradation, thereby potentially allowing slightly longer therapeutic effects (Li et  al. 2006). Arrayed against this possible positive treatment consideration is the reality that naked RNA is usually unable to penetrate cellular lipid membranes and reach the cytoplasm of target cells. Moreover, the short half-life of siRNA and its rapid rate of excretion rate may contribute to the lack of durable effects if used as single application treatment (Dave and Pomerantz 2003). Wang et  al. (2009a) addressed this issue utilizing chitosan nanoparticle-mediated delivery of a short hairpin RNA (shRNA) expressing a vector to inhibit TGFB1 expression in the RD human rhabdomyosarcoma cell line. Knockdown of TGFB1 by shRNA resulted in a decrease in RD cell growth in vitro and a decrease of tumorigenicity in nude mice (Wang et al. 2009a). These results support chitosan nanoparticle-mediated delivery as a potentially valuable gene therapy vehicle. DNAzymes are highly sequence-specific RNases-independent nucleotides which bind and cleave RNA (Breaker and Joyce 1994; Papachristou et al. 2003), and are also a potential tool for cancer therapy. Immunohistochemical studies have indicated increased levels of activated c-Jun and c-Fos in osteosarcoma (Papachristou et al. 2003). Dass et al. (2008a) demonstrated the use of chitosan nanoparticles as delivery system for DNAzyme designed against the c-Jun transcript, resulting in knockdown of c-Jun, thereby inhibiting the osteosarcoma cell growth. The same group was also able to demonstrate that c-Jun knockdown sensitizes osteosarcoma cells to doxorubicin (Dass et al. 2008b).

6 Viral Vectors There have been significant improvements in gene therapy technology involving the development of third-generation lentiviral vectors, adenoviral vectors, and encapsulated methods of delivering naked DNA (Dubensky et al. 2000; Vigna and Naldini 2000). In particular, adenoviral-mediated gene therapy using the tumor suppressor gene p53 has demonstrated promise in preclinical sarcoma models (Milas et al. 2000). Over the years, several strategies targeted in cancer have evolved to administer therapeutic genes that inactivate oncogenes, restore tumor-suppressor genes and enhance the native immune response. Numerous investigations have focused on p53 gene therapy, aiming to restore the important tumor-suppressor functions of this molecule. Thirty to fifty percent of adult soft-tissue sarcomas contain genetic mutations in the p53 gene (Das et  al. 2007; Latres et  al. 1994; Schneider-Stock et  al. 1999). In vitro studies using SKLMS-1 cells, a human-derived leiomyosarcoma cell

14  Gene Therapy for Sarcoma

261

line with a codon 245 p53 point mutation, indicated that stable transfectants expressing wild-type (wt) p53 exhibit reduced tumor growth and clonogenicity, and decreased tumorigenicity in severe combined immunodeficient (SCID) mice (Milas et  al. 2000; Pollock et al. 1998). These results encouraged the examination of p53 gene restoration in sarcomas by treating mice bearing tumors induced by subcutaneous infection of the SKLMS-1 cells with an adenoviral-mediated wt p53 gene delivery system. We were able to demonstrate that wt p53 sensitizes soft tissue sarcoma cells to doxorubicin by downregulating multidrug resistance-1 protein expression (Zhan et  al. 2001). The use of this strategy in conjunction with systemic chemotherapy could conceivably result in synergistic promotion of apoptosis in sarcoma. To test the above in vitro and in vivo findings, our group has developed an isolated limb perfusion (ILP) model in the sarcoma-bearing nude rat (Hannay et al. 2007a). This regional approach allows for the delivery of high gradients of biochemotherapy directly to the tumor in a concentrated intensity that is not feasible using standard systemic drug delivery systems. The feasibility of regional ILP delivery of potentially therapeutic adenovirus-based gene therapy against human soft tissue sarcoma was tested in a soft tissue sarcoma-xenograft nude rat hind limb model, modeling a traditional chemotherapy technique in widespread practice. An incompetent adenovirus bearing FLAG-tagged wild-type p53 and a fiber-modified, replication selective oncolytic adenovirus was administered into human leiomyosarcoma xenografts by ILP, utilizing escalating doses of the constructs. After 72 h of treatment, expression of FLAGp53 was confirmed by reverse transcriptionpolymerase chain reaction. Diffuse upregulation of p21CIP1/WAF1 was observed in ILP-treated tumors. These experiments demonstrated that the reintroduction of functional wild-type p53 into soft tissue sarcoma cells harboring a p53 mutation is possible when delivered via ILP. ILP delivery of therapeutic viruses theoretically has a number of inherent advantages over systemic or intratumoral delivery; these include minimization of systemic exposure to the therapeutic agent so delivered, attainment of higher regional concentrations of the therapeutic agent, and treatment of in-transit disease. This novel gene delivery system may be useful in delivering growth-suppressing constructs to cells bearing p53 mutations, and suggest the possibility of combining p53 gene therapy with current multimodality treatment options to improve sarcoma patient outcomes. Cell cycle regulatory proteins are an additional potential novel target for gene therapy. The CDK inhibitor p27 has been shown to be downregulated in myxoid and round-cell liposarcomas (Oliveira et al. 2000). This low expression of p27 correlated with decreased metastasis-free and overall survival. Theoretically, if the expression of this factor could be increased, an aggressive sarcoma phenotype could potentially be altered. Rexin-G, a target gene therapy vector bearing a cytocidal dominant negative cyclin G1 construct, is currently being tested simultaneously in three phase I/II clinical trials for chemotherapy-resistant metastatic sarcoma, pancreatic cancer, and breast cancer (Chawla et al. 2009). It is also being tested in one phase II study for chemotherapy resistant metastatic osteosarcoma (Chawla et al. 2009), preliminary studies suggesting that Rexin-G is well tolerated and may have activity in controlling tumor growth.

262

K.E. Torres and R.E. Pollock

Transcription factors of the E2F family are essential for cell cycle transition from the G1 to the S phase. While all the E2F transcription factors can induce proliferation and differentiation, only E2F-1 is known to effectively induce apoptosis in a broad variety of cancer cells (Phillips and Vousden 2001). Vorburger et  al. (2005) investigated the efficacy of E2F-1 gene therapy in leiomyosarcoma in vitro and in vivo. A replication-deficient adenovirus carrying the E2F-1 gene (Ad5E2F) was used to induce E2F-1 overexpression in the p53-mutated leiomyosarcoma cell line SKLMS-1. E2F-1 overexpression led to cell apoptosis following infection with Ad5E2F. In vivo experiments revealed that the treatment of SKLMS-1 tumorbearing BALB/c mice with intratumoral injections of Ad5E2F viral particles resulted in significant inhibition of tumor growth compared with control animals; complete disappearance of all tumors was observed in two of seven Ad5E2F-treated mice. Furthermore, these investigations demonstrate that adenovirus-mediated overexpression of E2F-1 resulted in upregulation of the protein kinase PKR, a kinase known to regulate several transcription factors involved in cell growth, differentiation, proliferation, and induction of apoptosis. Several challenges must be confronted when using viral vectors for gene transfer. Viruses are not endogenous to the human body; their introduction can induce the immune system to recognize viral capsid proteins, resulting in adverse immunological side effects (Hartman et al. 2008). Additionally, viral genetic material will not always incorporate in the host genome as desired resulting in oncogenesis, as has been previously observed in several well-known patient episodes (Hacein-BeyAbina et al. 2003; Kaiser 2007; Tack et al. 2006). Despite these adverse events, viral vectors remain attractive due to their high transfection rate and rapid transcription of material that has been inserted into the viral genome. However, there are additional concerns that will need to be addressed prior to the clinical utilization of viral vectors in sarcoma treatment. These include viral proinflammatory effects, technical difficulties inherent in attempting to incorporate foreign DNA into viral genomes, sporadic wild-type mutations of viral constructs, and the potential for oncogenesis.

7 Summary The use of gene therapy for the treatment of sarcomas will be greatly accelerated by an enhanced understanding of the biology of these tumors. Insight into the expression of the antiapoptotic factors and cell cycle regulatory proteins will enable the design of gene therapy strategies to replace or antagonize these factors in sarcoma cells. Selective delivery of genes to sarcoma cells with long-term gene expression remains a great challenge and a frontier for future scientific research. It is also necessary to define and verify more appropriate clinical parameter to assess efficacy in sarcoma treatment. Tumor shrinkage in the form of partial or complete response is the standard measure used to define chemoefficacy. However, since gene therapies may convert cancer into a chronic process without total eradication

14  Gene Therapy for Sarcoma

263

of disease, tumor stabilization, and freedom from progression maybe more meaningful parameters of clinical impact. Discovery of gene expression and cytogenetic profiles may provide additional information regarding the molecular basis of sarcomagenesis while presenting even more specific targets for therapy. More sensitive prognostication and therapeutic decision-making algorithms will require the incorporation of such relevant cytogenetic and molecular determinants. New paradigms of classification, integrating standard clinical and pathological criteria with molecular aberrations, will also facilitate personalized prognosis and even treatment. Moreover, the utilization of novel gene delivery systems such as ILP may be useful in delivering growth-suppressing constructs into soft tissue sarcomas. Hopefully, the use of these novel approaches coupled with current multimodality treatment options may improve the therapeutic options for sarcoma patients, thereby enhancing their overall prognostic outlook.

References Allander, S.V., Illei, P.B., Chen, Y., Antonescu, C.R., Bittner, M., Ladanyi, M., and Meltzer, P.S. (2002). Expression profiling of synovial sarcoma by cDNA microarrays: association of ERBB2, IGFBP2, and ELF3 with epithelial differentiation. Am J Pathol 161, 1587–1595. Alman, B.A., Li, C., Pajerski, M.E., Diaz-Cano, S., and Wolfe, H.J. (1997). Increased beta-catenin protein and somatic APC mutations in sporadic aggressive fibromatoses (desmoid tumors). Am J Pathol 151, 329–334. Antonescu, C.R., Nafa, K., Segal, N.H., Dal Cin, P., and Ladanyi, M. (2006). EWS-CREB1: a recurrent variant fusion in clear cell sarcoma--association with gastrointestinal location and absence of melanocytic differentiation. Clin Cancer Res 12, 5356–5362. Breaker, R.R., and Joyce, G.F. (1994). A DNA enzyme that cleaves RNA. Chem Biol 1, 223–229. Chawla, S.P., Chua, V.S., Fernandez, L., Quon, D., Saralou, A., Blackwelder, W.C., Hall, F.L., and Gordon, E.M. (2009). Phase I/II and phase II studies of targeted gene delivery in vivo: intravenous Rexin-G for chemotherapy-resistant sarcoma and osteosarcoma. Mol Ther 17, 1651–1657. Chen, L.L., Trent, J.C., Wu, E.F., Fuller, G.N., Ramdas, L., Zhang, W., Raymond, A.K., Prieto, V.G., Oyedeji, C.O., Hunt, K.K., et al. (2004a). A missense mutation in KIT kinase domain 1 correlates with imatinib resistance in gastrointestinal stromal tumors. Cancer Res 64, 5913–5919. Chen, Y., Tzeng, C.C., Liou, C.P., Chang, M.Y., Li, C.F., and Lin, C.N. (2004b). Biological significance of chromosomal imbalance aberrations in gastrointestinal stromal tumors. J Biomed Sci 11, 65–71. Coindre, J.M., Hostein, I., Terrier, P., Bouvier-Labit, C., Collin, F., Michels, J.J., Trassard, M., Marques, B., Ranchere, D., and Guillou, L. (2006). Diagnosis of clear cell sarcoma by realtime reverse transcriptase-polymerase chain reaction analysis of paraffin embedded tissues: clinicopathologic and molecular analysis of 44 patients from the French sarcoma group. Cancer 107, 1055–1064. Das, P., Kotilingam, D., Korchin, B., Liu, J., Yu, D., Lazar, A.J., Pollock, R.E., and Lev, D. (2007). High prevalence of p53 exon 4 mutations in soft tissue sarcoma. Cancer 109, 2323–2333. Dass, C.R., Khachigian, L.M., and Choong, P.F. (2008a). c-Jun Is critical for the progression of osteosarcoma: proof in an orthotopic spontaneously metastasizing model. Mol Cancer Res 6, 1289–1292.

264

K.E. Torres and R.E. Pollock

Dass, C.R., Khachigian, L.M., and Choong, P.F. (2008b). c-Jun knockdown sensitizes osteosarcoma to doxorubicin. Mol Cancer Ther 7, 1909–1912. Dave, R.S., and Pomerantz, R.J. (2003). RNA interference: on the road to an alternate therapeutic strategy! Rev Med Virol 13, 373–385. Dubensky, T.W., Jr., Liu, M.A., and Ulmer, J.B. (2000). Delivery systems for gene-based vaccines. Mol Med 6, 723–732. El-Rifai, W., Sarlomo-Rikala, M., Knuutila, S., and Miettinen, M. (1998). DNA copy number changes in development and progression in leiomyosarcomas of soft tissues. Am J Pathol 153, 985–990. Giarola, M., Wells, D., Mondini, P., Pilotti, S., Sala, P., Azzarelli, A., Bertario, L., Pierotti, M.A., Delhanty, J.D., and Radice, P. (1998). Mutations of adenomatous polyposis coli (APC) gene are uncommon in sporadic desmoid tumours. Br J Cancer 78, 582–587. Gunawan, B., Bergmann, F., Hoer, J., Langer, C., Schumpelick, V., Becker, H., and Fuzesi, L. (2002). Biological and clinical significance of cytogenetic abnormalities in low-risk and highrisk gastrointestinal stromal tumors. Hum Pathol 33, 316–321. Hacein-Bey-Abina, S., Von Kalle, C., Schmidt, M., McCormack, M.P., Wulffraat, N., Leboulch, P., Lim, A., Osborne, C.S., Pawliuk, R., Morillon, E., et al. (2003). LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 302, 415–419. Hannay, J., Davis, J.J., Yu, D., Liu, J., Fang, B., Pollock, R.E., and Lev, D. (2007a). Isolated limb perfusion: a novel delivery system for wild-type p53 and fiber-modified oncolytic adenoviruses to extremity sarcoma. Gene Ther 14, 671–681. Hannay, J.A., Liu, J., Zhu, Q.S., Bolshakov, S.V., Li, L., Pisters, P.W., Lazar, A.J., Yu, D., Pollock, R.E., and Lev, D. (2007b). Rad51 overexpression contributes to chemoresistance in human soft tissue sarcoma cells: a role for p53/activator protein 2 transcriptional regulation. Mol Cancer Ther 6, 1650–1660. Hartman, Z.C., Appledorn, D.M., and Amalfitano, A. (2008). Adenovirus vector induced innate immune responses: impact upon efficacy and toxicity in gene therapy and vaccine applications. Virus Res 132, 1–14. Henriksen, J., Aagesen, T.H., Maelandsmo, G.M., Lothe, R.A., Myklebost, O., and Forus, A. (2003). Amplification and overexpression of COPS3 in osteosarcomas potentially target TP53 for proteasome-mediated degradation. Oncogene 22, 5358–5361. Hirota, S., Isozaki, K., Moriyama, Y., Hashimoto, K., Nishida, T., Ishiguro, S., Kawano, K., Hanada, M., Kurata, A., Takeda, M., et  al. (1998). Gain-of-function mutations of c-kit in human gastrointestinal stromal tumors. Science 279, 577–580. Hisaoka, M., Ishida, T., Kuo, T.T., Matsuyama, A., Imamura, T., Nishida, K., Kuroda, H., Inayama, Y., Oshiro, H., Kobayashi, H., et al. (2008). Clear cell sarcoma of soft tissue: a clinicopathologic, immunohistochemical, and molecular analysis of 33 cases. Am J Surg Pathol 32, 452–460. Holtkamp, N., Reuss, D.E., Atallah, I., Kuban, R.J., Hartmann, C., Mautner, V.F., Frahm, S., Friedrich, R.E., Algermissen, B., Pham, V.A., et al. (2004). Subclassification of nerve sheath tumors by gene expression profiling. Brain Pathol 14, 258–264. Jemal, A., Siegel, R., Ward, E., Hao, Y., Xu, J., and Thun, M.J. (2009). Cancer statistics, 2009. CA Cancer J Clin 59, 225–249. Kaiser, J. (2007). Clinical research. Death prompts a review of gene therapy vector. Science 317, 580. Kose, K., Hiyama, T., Tanaka, S., Yoshihara, M., Yasui, W., and Chayama, K. (2006). Nuclear and mitochondrial DNA microsatellite instability in gastrointestinal stromal tumors. Pathobiology 73, 93–97. Kotiligam, D., Lazar, A.J., Pollock, R.E., and Lev, D. (2008). Desmoid tumor: a disease opportune for molecular insights. Histol Histopathol 23, 117–126. Lasota, J., and Miettinen, M. (2006). KIT and PDGFRA mutations in gastrointestinal stromal tumors (GISTs). Semin Diagn Pathol 23, 91–102. Lasota, J., and Miettinen, M. (2008). Clinical significance of oncogenic KIT and PDGFRA mutations in gastrointestinal stromal tumours. Histopathology 53, 245–266.

14  Gene Therapy for Sarcoma

265

Latres, E., Drobnjak, M., Pollack, D., Oliva, M.R., Ramos, M., Karpeh, M., Woodruff, J.M., and Cordon-Cardo, C. (1994). Chromosome 17 abnormalities and TP53 mutations in adult soft tissue sarcomas. Am J Pathol 145, 345–355. Lazar, A.J., Tuvin, D., Hajibashi, S., Habeeb, S., Bolshakov, S., Mayordomo-Aranda, E., Warneke, C.L., Lopez-Terrada, D., Pollock, R.E., and Lev, D. (2008). Specific mutations in the betacatenin gene (CTNNB1) correlate with local recurrence in sporadic desmoid tumors. Am J Pathol 173, 1518–1527. Li, C.X., Parker, A., Menocal, E., Xiang, S., Borodyansky, L., and Fruehauf, J.H. (2006). Delivery of RNA interference. Cell Cycle 5, 2103–2109. Liu, J., Zhan, M., Hannay, J.A., Das, P., Bolshakov, S.V., Kotilingam, D., Yu, D., Lazar, A.F., Pollock, R.E., and Lev, D. (2006). Wild-type p53 inhibits nuclear factor-kappaB-induced matrix metalloproteinase-9 promoter activation: implications for soft tissue sarcoma growth and metastasis. Mol Cancer Res 4, 803–810. Ludwig, J.A. (2008). Personalized therapy of sarcomas: integration of biomarkers for improved diagnosis, prognosis, and therapy selection. Curr Oncol Rep 10, 329–337. Lux, M.L., Rubin, B.P., Biase, T.L., Chen, C.J., Maclure, T., Demetri, G., Xiao, S., Singer, S., Fletcher, C.D., and Fletcher, J.A. (2000). KIT extracellular and kinase domain mutations in gastrointestinal stromal tumors. Am J Pathol 156, 791–795. Maelandsmo, G.M., Hovig, E., Skrede, M., Engebraaten, O., Florenes, V.A., Myklebost, O., Grigorian, M., Lukanidin, E., Scanlon, K.J., and Fodstad, O. (1996). Reversal of the in vivo metastatic phenotype of human tumor cells by an anti-CAPL (mts1) ribozyme. Cancer Res 56, 5490–5498. Maki, R.G., Awan, R.A., Dixon, R.H., Jhanwar, S., and Antonescu, C.R. (2002). Differential sensitivity to imatinib of 2 patients with metastatic sarcoma arising from dermatofibrosarcoma protuberans. Int J Cancer 100, 623–626. Mei, J., Gao, Y., Zhang, L., Cai, X., Qian, Z., Huang, H., and Huang, W. (2008). VEGF-siRNA silencing induces apoptosis, inhibits proliferation and suppresses vasculogenic mimicry in osteosarcoma in vivo. Exp Oncol 30, 29–34. Meyers, P.A., Schwartz, C.L., Krailo, M.D., Healey, J.H., Bernstein, M.L., Betcher, D., Ferguson, W.S., Gebhardt, M.C., Goorin, A.M., Harris, M., et al. (2008). Osteosarcoma: the addition of muramyl tripeptide to chemotherapy improves overall survival--a report from the Children’s Oncology Group. J Clin Oncol 26, 633–638. Meza-Zepeda, L.A., Kresse, S.H., Barragan-Polania, A.H., Bjerkehagen, B., Ohnstad, H.O., Namlos, H.M., Wang, J., Kristiansen, B.E., and Myklebost, O. (2006). Array comparative genomic hybridization reveals distinct DNA copy number differences between gastrointestinal stromal tumors and leiomyosarcomas. Cancer Res 66, 8984–8993. Milas, M., Yu, D., Lang, A., Ge, T., Feig, B., El-Naggar, A.K., and Pollock, R.E. (2000). Adenovirus-mediated p53 gene therapy inhibits human sarcoma tumorigenicity. Cancer Gene Ther 7, 422–429. Miller, S.J., Rangwala, F., Williams, J., Ackerman, P., Kong, S., Jegga, A.G., Kaiser, S., Aronow, B.J., and Frahm, S. (2006). Large-scale molecular comparison of human schwann cells to malignant peripheral nerve sheath tumor cell lines and tissues. Cancer Res 66, 2584–2591. Miyoshi, Y., Iwao, K., Nawa, G., Yoshikawa, H., Ochi, T., and Nakamura, Y. (1998). Frequent mutations in the beta-catenin gene in desmoid tumors from patients without familial adenomatous polyposis. Oncol Res 10, 591–594. Mori, K., Redini, F., Gouin, F., Cherrier, B., and Heymann, D. (2006). Osteosarcoma: current status of immunotherapy and future trends (Review). Oncol Rep 15, 693–700. Nagayama, S., Katagiri, T., Tsunoda, T., Hosaka, T., Nakashima, Y., Araki, N., Kusuzaki, K., Nakayama, T., Tsuboyama, T., Nakamura, T., et  al. (2002). Genome-wide analysis of gene expression in synovial sarcomas using a cDNA microarray. Cancer Res 62, 5859–5866. Nielsen, T.O., Hsu, F.D., O’Connell, J.X., Gilks, C.B., Sorensen, P.H., Linn, S., West, R.B., Liu, C.L., Botstein, D., Brown, P.O., et  al. (2003). Tissue microarray validation of epidermal growth factor receptor and SALL2 in synovial sarcoma with comparison to tumors of similar histology. Am J Pathol 163, 1449–1456.

266

K.E. Torres and R.E. Pollock

Nielsen, T.O., West, R.B., Linn, S.C., Alter, O., Knowling, M.A., O’Connell, J.X., Zhu, S., Fero, M., Sherlock, G., Pollack, J.R., et al. (2002). Molecular characterisation of soft tissue tumours: a gene expression study. Lancet 359, 1301–1307. Ohnishi, T., Taki, T., Hiraga, S., Arita, N., and Morita, T. (1998). In vivo and in vivo potentiation of radiosensitivity of malignant gliomas by antisense inhibition of the RAD51 gene. Biochem Biophys Res Commun 245, 319–324. Oliveira, A.M., Nascimento, A.G., Okuno, S.H., and Lloyd, R.V. (2000). p27(kip1) protein expression correlates with survival in myxoid and round-cell liposarcoma. J Clin Oncol 18, 2888–2893. Otano-Joos, M., Mechtersheimer, G., Ohl, S., Wilgenbus, K.K., Scheurlen, W., Lehnert, T., Willeke, F., Otto, H.F., Lichter, P., and Joos, S. (2000). Detection of chromosomal imbalances in leiomyosarcoma by comparative genomic hybridization and interphase cytogenetics. Cytogenet Cell Genet 90, 86–92. Panagopoulos, I., Mertens, F., Debiec-Rychter, M., Isaksson, M., Limon, J., Kardas, I., Domanski, H.A., Sciot, R., Perek, D., Crnalic, S., et al. (2002). Molecular genetic characterization of the EWS/ATF1 fusion gene in clear cell sarcoma of tendons and aponeuroses. Int J Cancer 99, 560–567. Papachristou, D.J., Batistatou, A., Sykiotis, G.P., Varakis, I., and Papavassiliou, A.G. (2003). Activation of the JNK-AP-1 signal transduction pathway is associated with pathogenesis and progression of human osteosarcomas. Bone 32, 364–371. Penzel, R., Aulmann, S., Moock, M., Schwarzbach, M., Rieker, R.J., and Mechtersheimer, G. (2005). The location of KIT and PDGFRA gene mutations in gastrointestinal stromal tumours is site and phenotype associated. J Clin Pathol 58, 634–639. Persengiev, S.P., and Green, M.R. (2003). The role of ATF/CREB family members in cell growth, survival and apoptosis. Apoptosis 8, 225–228. Phillips, A.C., and Vousden, K.H. (2001). E2F-1 induced apoptosis. Apoptosis 6, 173–182. Pollock, R., Lang, A., Ge, T., Sun, D., Tan, M., and Yu, D. (1998). Wild-type p53 and a p53 temperature-sensitive mutant suppress human soft tissue sarcoma by enhancing cell cycle control. Clin Cancer Res 4, 1985–1994. Pollock, R.E., Lang, A., Luo, J., El-Naggar, A.K., and Yu, D. (1996). Soft tissue sarcoma metastasis from clonal expansion of p53 mutated tumor cells. Oncogene 12, 2035–2039. Raderschall, E., Bazarov, A., Cao, J., Lurz, R., Smith, A., Mann, W., Ropers, H.H., Sedivy, J.M., Golub, E.I., Fritz, E., et  al. (2002a). Formation of higher-order nuclear Rad51 structures is functionally linked to p21 expression and protection from DNA damage-induced apoptosis. J Cell Sci 115, 153–164. Raderschall, E., Stout, K., Freier, S., Suckow, V., Schweiger, S., and Haaf, T. (2002b). Elevated levels of Rad51 recombination protein in tumor cells. Cancer Res 62, 219–225. Ragazzini, P., Gamberi, G., Pazzaglia, L., Serra, M., Magagnoli, G., Ponticelli, F., Ferrari, C., Ghinelli, C., Alberghini, M., Bertoni, F., et al. (2004). Amplification of CDK4, MDM2, SAS and GLI genes in leiomyosarcoma, alveolar and embryonal rhabdomyosarcoma. Histol Histopathol 19, 401–411. Ren, W., Korchin, B., Zhu, Q.S., Wei, C., Dicker, A., Heymach, J., Lazar, A., Pollock, R.E., and Lev, D. (2008). Epidermal growth factor receptor blockade in combination with conventional chemotherapy inhibits soft tissue sarcoma cell growth in vivo and in vivo. Clin Cancer Res 14, 2785–2795. Rozeman, L.B., Hameetman, L., van Wezel, T., Taminiau, A.H., Cleton-Jansen, A.M., Hogendoorn, P.C., and Bovee, J.V. (2005). cDNA expression profiling of chondrosarcomas: Ollier disease resembles solitary tumours and alteration in genes coding for components of energy metabolism occurs with increasing grade. J Pathol 207, 61–71. Rubin, B.P., Schuetze, S.M., Eary, J.F., Norwood, T.H., Mirza, S., Conrad, E.U., and Bruckner, J.D. (2002). Molecular targeting of platelet-derived growth factor B by imatinib mesylate in a patient with metastatic dermatofibrosarcoma protuberans. J Clin Oncol 20, 3586–3591. Rubin, B.P., Singer, S., Tsao, C., Duensing, A., Lux, M.L., Ruiz, R., Hibbard, M.K., Chen, C.J., Xiao, S., Tuveson, D.A., et al. (2001). KIT activation is a ubiquitous feature of gastrointestinal stromal tumors. Cancer Res 61, 8118–8121.

14  Gene Therapy for Sarcoma

267

Sarlomo-Rikala, M., Kovatich, A.J., Barusevicius, A., and Miettinen, M. (1998). CD117: a sensitive marker for gastrointestinal stromal tumors that is more specific than CD34. Mod Pathol 11, 728–734. Scappaticci, F.A., and Marina, N. (2001). New molecular targets and biological therapies in sarcomas. Cancer Treat Rev 27, 317–326. Schneider-Stock, R., Onnasch, D., Haeckel, C., Mellin, W., Franke, D.S., and Roessner, A. (1999). Prognostic significance of p53 gene mutations and p53 protein expression in synovial sarcomas. Virchows Arch 435, 407–412. Scotlandi, K., Remondini, D., Castellani, G., Manara, M.C., Nardi, F., Cantiani, L., Francesconi, M., Mercuri, M., Caccuri, A.M., Serra, M., et al. (2009). Overcoming resistance to conventional drugs in Ewing sarcoma and identification of molecular predictors of outcome. J Clin Oncol 27, 2209–2216. Segal, N.H., Pavlidis, P., Antonescu, C.R., Maki, R.G., Noble, W.S., DeSantis, D., Woodruff, J.M., Lewis, J.J., Brennan, M.F., Houghton, A.N., et al. (2003). Classification and subtype prediction of adult soft tissue sarcoma by functional genomics. Am J Pathol 163, 691–700. Shimada, S., Ishizawa, T., Ishizawa, K., Matsumura, T., Hasegawa, T., and Hirose, T. (2006). The value of MDM2 and CDK4 amplification levels using real-time polymerase chain reaction for the differential diagnosis of liposarcomas and their histologic mimickers. Hum Pathol 37, 1123–1129. Simon, M.P., Pedeutour, F., Sirvent, N., Grosgeorge, J., Minoletti, F., Coindre, J.M., TerrierLacombe, M.J., Mandahl, N., Craver, R.D., Blin, N., et al. (1997). Deregulation of the plateletderived growth factor B-chain gene via fusion with collagen gene COL1A1 in dermatofibrosarcoma protuberans and giant-cell fibroblastoma. Nat Genet 15, 95–98. Singer, S., Rubin, B.P., Lux, M.L., Chen, C.J., Demetri, G.D., Fletcher, C.D., and Fletcher, J.A. (2002). Prognostic value of KIT mutation type, mitotic activity, and histologic subtype in gastrointestinal stromal tumors. J Clin Oncol 20, 3898–3905. Skubitz, K.M., and Skubitz, A.P. (2003). Differential gene expression in leiomyosarcoma. Cancer 98, 1029–1038. Subramanian, S., West, R.B., Marinelli, R.J., Nielsen, T.O., Rubin, B.P., Goldblum, J.R., Patel, R.M., Zhu, S., Montgomery, K., Ng, T.L., et  al. (2005). The gene expression profile of extraskeletal myxoid chondrosarcoma. J Pathol 206, 433–444. Tack, F., Bakker, A., Maes, S., Dekeyser, N., Bruining, M., Elissen-Roman, C., Janicot, M., Brewster, M., Janssen, H.M., De Waal, B.F., et  al. (2006). Modified poly(propylene imine) dendrimers as effective transfection agents for catalytic DNA enzymes (DNAzymes). J Drug Target 14, 69–86. Taki, T., Ohnishi, T., Yamamoto, A., Hiraga, S., Arita, N., Izumoto, S., Hayakawa, T., and Morita, T. (1996). Antisense inhibition of the RAD51 enhances radiosensitivity. Biochem Biophys Res Commun 223, 434–438. Taubert, H., Meye, A., and Wurl, P. (1996). Prognosis is correlated with p53 mutation type for soft tissue sarcoma patients. Cancer Res 56, 4134–4136. Tejpar, S., Nollet, F., Li, C., Wunder, J.S., Michils, G., dal Cin, P., Van Cutsem, E., Bapat, B., van Roy, F., Cassiman, J.J., et al. (1999). Predominance of beta-catenin mutations and betacatenin dysregulation in sporadic aggressive fibromatosis (desmoid tumor). Oncogene 18, 6615–6620. Trent, J.C., Lazar, A.J., and Zhang, W. (2007). Molecular approaches to resolve diagnostic dilemmas: the case of gastrointestinal stromal tumor and leiomyosarcoma. Future Oncol 3, 629–637. van Dartel, M., Leenstra, S., Troost, D., and Hulsebos, T.J. (2003). Infrequent but high-level amplification of 17p11.2 approximately p12 in human glioma. Cancer Genet Cytogenet 140, 162–166. Vigna, E., and Naldini, L. (2000). Lentiviral vectors: excellent tools for experimental gene transfer and promising candidates for gene therapy. J Gene Med 2, 308–316. Vispe, S., Cazaux, C., Lesca, C., and Defais, M. (1998). Overexpression of Rad51 protein stimulates homologous recombination and increases resistance of mammalian cells to ionizing radiation. Nucleic Acids Res 26, 2859–2864.

268

K.E. Torres and R.E. Pollock

Vorburger, S.A., Hetrakul, N., Xia, W., Wilson-Heiner, M., Mirza, N., Pollock, R.E., Feig, B., Swisher, S.G., and Hunt, K.K. (2005). Gene therapy with E2F-1 up-regulates the protein kinase PKR and inhibits growth of leiomyosarcoma in vivo. Mol Cancer Ther 4, 1710–1716. Wang, D., Zhong, Z.Y., Li, M.X., Xiang, D.B., and Li, Z.P. (2007). Vector-based Ape1 small interfering RNA enhances the sensitivity of human osteosarcoma cells to endostatin in vivo. Cancer Sci 98, 1993–2001. Wang, R., Lu, Y.J., Fisher, C., Bridge, J.A., and Shipley, J. (2001). Characterization of chromosome aberrations associated with soft-tissue leiomyosarcomas by twenty-four-color karyotyping and comparative genomic hybridization analysis. Genes Chromosomes Cancer 31, 54–64. Wang, S.L., Yao, H.H., Guo, L.L., Dong, L., Li, S.G., Gu, Y.P., and Qin, Z.H. (2009a). Selection of optimal sites for TGFB1 gene silencing by chitosan-TPP nanoparticle-mediated delivery of shRNA. Cancer Genet Cytogenet 190, 8–14. Wang, W.L., Mayordomo, E., Zhang, W., Hernandez, V.S., Tuvin, D., Garcia, L., Lev, D.C., Lazar, A.J., and Lopez-Terrada, D. (2009b). Detection and characterization of EWSR1/ATF1 and EWSR1/CREB1 chimeric transcripts in clear cell sarcoma (melanoma of soft parts). Mod Pathol 22, 1201–1209 Wang, W.L., Mayordomo, E., Czerniak, B.A., Abruzzo, L.V., Dal Cin, P., Araujo, D.M., Lev, D.C., Lopez-Terrada, D., and Lazar, A.J. (2008). Fluorescence in situ hybridization is a useful ancillary diagnostic tool for extraskeletal myxoid chondrosarcoma. Mod Pathol 21, 1303–1310. Watson, M.A., Perry, A., Tihan, T., Prayson, R.A., Guha, A., Bridge, J., Ferner, R., and Gutmann, D.H. (2004). Gene expression profiling reveals unique molecular subtypes of Neurofibromatosis Type I-associated and sporadic malignant peripheral nerve sheath tumors. Brain Pathol 14, 297–303. Witlox, M.A., Lamfers, M.L., Wuisman, P.I., Curiel, D.T., and Siegal, G.P. (2007). Evolving gene therapy approaches for osteosarcoma using viral vectors: review. Bone 40, 797–812. Woods, S.A., Marmor, E., Feldkamp, M., Lau, N., Apicelli, A.J., Boss, G., Gutmann, D.H., and Guha, A. (2002). Aberrant G protein signaling in nervous system tumors. J Neurosurg 97, 627–642. Wozniak, A., Sciot, R., Guillou, L., Pauwels, P., Wasag, B., Stul, M., Vermeesch, J.R., Vandenberghe, P., Limon, J., and Debiec-Rychter, M. (2007). Array CGH analysis in primary gastrointestinal stromal tumors: cytogenetic profile correlates with anatomic site and tumor aggressiveness, irrespective of mutational status. Genes Chromosomes Cancer 46, 261–276. Xu, Z.Y., Loignon, M., Han, F.Y., Panasci, L., and Aloyz, R. (2005). Xrcc3 induces cisplatin resistance by stimulation of Rad51-related recombinational repair, S-phase checkpoint activation, and reduced apoptosis. J Pharmacol Exp Ther 314, 495–505. Yamashita, K., Igarashi, H., Kitayama, Y., Ozawa, T., Kiyose, S., Konno, H., Kazui, T., Ishikawa, S., Aburatani, H., Tanioka, F., et al. (2006). Chromosomal numerical abnormality profiles of gastrointestinal stromal tumors. Jpn J Clin Oncol 36, 85–92. Yang, J., Du, X., Chen, K., Ylipaa, A., Lazar, A.J., Trent, J., Lev, D., Pollock, R., Hao, X., Hunt, K., et al. (2009). Genetic aberrations in soft tissue leiomyosarcoma. Cancer Lett 275, 1–8. Yang, J., Du, X., Lazar, A.J., Pollock, R., Hunt, K., Chen, K., Hao, X., Trent, J., and Zhang, W. (2008). Genetic aberrations of gastrointestinal stromal tumors. Cancer 113, 1532–1543. Zhan, M., Yu, D., Lang, A., Li, L., and Pollock, R.E. (2001). Wild type p53 sensitizes soft tissue sarcoma cells to doxorubicin by down-regulating multidrug resistance-1 expression. Cancer 92, 1556–1566. Zhan, M., Yu, D., Liu, J., Glazer, R.I., Hannay, J., and Pollock, R.E. (2005). Transcriptional repression of protein kinase Calpha via Sp1 by wild type p53 is involved in inhibition of multidrug resistance 1 P-glycoprotein phosphorylation. J Biol Chem 280, 4825–4833. Zhang, L., Yu, D., Hu, M., Xiong, S., Lang, A., Ellis, L.M., and Pollock, R.E. (2000). Wild-type p53 suppresses angiogenesis in human leiomyosarcoma and synovial sarcoma by transcriptional suppression of vascular endothelial growth factor expression. Cancer Res 60, 3655–3661. Zou, C., Smith, K.D., Liu, J., Lahat, G., Myers, S., Wang, W.L., Zhang, W., McCutcheon, I.E., Slopis, J.M., Lazar, A.J., et al. (2009). Clinical, pathological, and molecular variables predictive of malignant peripheral nerve sheath tumor outcome. Ann Surg 249, 1014–1022.

Index

A Adenoviruses (Ads) based gene therapy, 194, 261 Delta-24-RGD, 23–24 description, 21–22 E1A products, 22 E2F-1 overexpression, 262 oncolytic virus, 242–243 ONYX-015, 22–23 retargeting adapter-based, 145 Ad5 vectors, 144–145 cancer gene therapy, 143 delivery, adjunct technologies, 149 description, 141–142 gene expression, cellular control, 146–148 genetic modifications, 146 immunotherapy, 143 life cycle and genomic organization, 142–143 virotherapy, 144 Ad-mda-7/IL-24 metastatic melanoma dacarbazine (DTIC), 188 IL-2 based regimens, 188–189 phase II intratumoral injection clinical results, 191 cutaneous lesions, 190 peripheral immune system, 192–193 pro-apoptotic and anti-proliferative effects, 191–192 phase I/II trial gene transfer intralesional injection and multiple biopsies, 189 monotherapy protocol, 189–190 TUNEL assay, 190 Alternative lengthening of telomeres (ALT), 81

Androgen suppression therapy (AST), 34 Antiangiogenesis therapy, lentiviral vectors, 168–169 B Brain tumors ICP47, 209 malignant gliomas causes, 17–18 clinical trials testing, 18, 19 viral vectors, 18 oncolytic viruses adeno, 21–24 HSV-1, 24–25 measles, 26 NDV, 26–27 reovirus, 25–26 replication-deficient viral vectors Ad-p53, 18, 20 HSVtk/GCV gene therapy, 20–21 tk-deficient HSV1, 206 C Cancer gene therapy vectors Ad-based, 143 HSV1 vectors, 207 lentiviruses (See Lentiviruses) Carcinogenesis, 168 Cell-based vaccines allogenic tumor vaccine, 43 GVAX cells, 43–44 immune infiltrates, 43 prednisone, 44 Chemotherapeutic drugs dacarbazine, 188 immune activation, hTERT promoter-driven, 85

269

270 Chemotherapeutic drugs (cont.) vs. myeloprotection, lentiviral vector, 169 stable lentiviral transduction, HCT116 colon cancer cells, 168 SV40 Tag expression, 97 Conditionally replication adenoviral vectors (CRads) advantages, 127 human MSC effect, 127–128 Cytotoxic T lymphocytes (CTLs) generation, MM-bearing mice, 100 induction, IFN-g, 85 interleukins, 126 MHC I expression, 99 peptide-pulsed cells, 237 D DCs. See Dendritic cells Delayed type hypersensitivity (DTH), 43 Delta-24-RGD, 23–24 Dendritic cells (DCs) Ad5 vectors, 149 characteristics, 85 description, 12 human, 239 IL, 126 production, 239 skin-derived (sDC), 167 telomerase vaccination, 238 DOTAP: cholesterol (DOTAP:Chol.) FUS1-expressing plasmid vector, 73 mda7/IL-24, 194–196 systemic therapy, 197 DTH. See Delayed type hypersensitivity E Enzyme/prodrug gene therapy replication-competent advantages, 37 efficacy, 40 oncolytic, 44–45 phase 1 trials, 39 PSADT lengthening, 38 PSA responses, 37 salvage therapies, 38–39 replication-defective biopsy results, 35 HSV-1 TK, 34–35 nitroreductase, 36 osteocalcin promoter, 36 suicide gene therapy, 35

Index F Familial adenomatous polyposis (FAP), 256 FUS1 gene DOTAP:Chol. nanoparticles, 195, 196 inactivation, 72 nanoparticle, 73 3p21.3-deficient NSCLC cells, 72, 73 G Gancyclovir (GCV) cytotoxic gancyclovir triphosphate, 166 hrR3, 208 HSVtk’s effect, 99 HSV1 tumor cells, 205 production, 206 tk gene, 210 Gastrointestinal stromal tumors (GISTs) expression profiles, 256, 257 leiomyosarcoma, 252–253 GCV. See Gancyclovir Gene-directed enzyme prodrug therapy (GDEPT), 20 Gene silencing gene replacement and, 168 and RNAi, 52, 168 siRNA-mediated, 10–11 GISTs. See Gastrointestinal stromal tumors GV1001, telomerase immunotherapy Heptovax, 238 pancreatic cancer, 238 phase II studies, 237 H Herpes simplex virus-1 (HSV-1) clinical trials, 212–213 cyclophosphamide, 221–222 extracellular matrix, 222–223 G207 anti-tumor efficacy, rodents, 210 beta galactosidase, 209–210 humans, 210–211 genome, 204–205 HF10 description, 218–219 paclitaxel, 219 skin nodules, 219–220 HSV1716 adverse events, 217 759 base pair deletion, 211 electron microscopy and DNA fragmentation, 214 gliomas, 215, 216

Index head and neck squamous cell carcinoma, 215, 217 intracerebral inoculation, 215 ICP 47 host immunity, 208–209 US11 viral gene, 209 ICP 34.5/RL1 amino acids, 206 transcriptionally targeted viruses, 207 virus replication, 206–207 NV1020/RV7020 antitumor efficacy, 218 description, 217 oncolytic viruses, 24–25 OncoVex gm-csf doses, 220–221 head and neck, 221 Us11gene, 220 properties, 206 ribonucleotide reductase/ICP 6, 208 TK, 205–206 Herpes simplex virus (HSV) HSV-1, 24–25 HSVtk/GCV gene therapy, 20–21 HSV-1. See Herpes simplex virus-1 hTERT promoter-driven oncolytic adenovirus immune activation chemotherapeutic drugs, 85 DCs, 85 structure E1A/E4, 82 E1B gene, 82 telomelysin replication, 82 in vivo antitumor effects, 83–84 I IFN. See Interferons IFN-b. See Interferon-b IL. See Interleukins ILP. See Isolated limb perfusion Immunotherapy vectors, 143 Interferon-b (IFN-b) melanoma apoptosis, 188 MSC as cellular delivery system, 124 Interferons (IFN), 125–126 Interleukin-24 gene therapy, melanoma Ad mda-7/IL-24 metastatic melanoma, 188–189 phase II intratumoral injection, 190–193 phase I/II trial, advanced solid tumors, 189–190

271 cytokine properties low amino acid sequence homology, 184 skin biology, 185 expression, melanocytes and melanoma immunohistochemical analysis, 185 loss, 186 non-viral nanoparticle-based gene delivery systems clinical trials, 195–196 DOTAP, chol mda7/IL-24, 194–195 tumor suppressor properties Ad-mda/IL-24, intratumoral administration, 183 anti-angiogenic activity, 184 human cancer cells, 182 mda-7/IL-24 gene, 181–182 tumor suppressor, re-expression bystander antitumor effect, 186–187 co-administration, 188 induced cell death, 187 viral delivery phase II trial, 193 recombinant adeno-associated virus, 194 Interleukins (IL) IL-24 gene therapy (See Interleukin-24 gene therapy, melanoma) MSC, 126–127 Isolated limb perfusion (ILP) growth-suppressing constructs, soft tissue sarcomas, 263 regional delivery, 261 L Leiomyosarcoma c-kit expression, 252–253 E2F-1 overexpression, 262 gene expression, 258 human-derived, 260–261 Lentiviral immunotherapy host immune response, 166–167 TAA, 167 Lentiviral vectors, lentiviruses See Lentiviruses applications anti-angiogenesis therapy, 168–169 gene transfer efforts, 166 immunotherapy, 166–167 myelo-protection, chemotherapeutics, 169 replacement and silencing, gene, 168 suicide gene therapy, 166 gene transfer clinical trials, 169–170 HIV-1 derived

272 Lentiviral vectors, lentiviruses See Lentiviruses (cont.) description, 159 design and improvement, 160–163 non-HIV-1, 163–164 packaging system, 160 production, 164–165 pseudotyping, 164 Lentiviruses description, 157 genome and structure function, 158 proteins, 157 life cycle DNA flap, 159 gp120, 158–159 reverse transcriptase, 159 M Malignant mesothelioma (MM) description, 95 IFN-b cytokine effect, 101 Malignant pleural mesothelioma (MPM) anti-angiogenesis, 98 apoptosis induction CRI1 gene, 98 downstream inducers, 97 p16INK4A mutations, 97 p53 mutations, 96–97 REIC/Dickkopf-3 (Dkk-3), 97–98 cytokine gene therapy Ad.IFN-b dose escalation, 105 Ad.IFN vector, 105–106 intratumoral cytokine gene delivery, 104 vero cells, 105 gene therapy, target, 96 immuno-gene therapy anti-mesothelioma effects, 101 intraperitoneal b-galactosidase delivery, 100 protein gene-65 delivery, 100 MM, description, 95 suicide-gene therapy dose-related intratumoral HSVtk gene transfer, 103 E1/E4-deleted adenoviral vector, 104 GCV, 99 HSVtk DNA transfer, 99–100 immunogenic killing, 99 tumor-selective oncolytic viral vectors, replication, 101–102 Measles virus characteristics, 26 live-attenuated, 102

Index Mesenchymal stem/stromal cells (MSC) alternative mesenchymal tissues, 129–130 cell vehicles, cancer, 124–125 chemokines and growth factor antagonists, 128 CRads advantages, 127 human MSC effect, 127–128 fibroblasts and stromal precursors, 117–118 IFN, 125–126 IL, 126–127 migratory factors EGFR, 121–122 wound repair, 121 stem cells antitumor proteins, 123 IFNb, 124 lung tumor nodules, 123 NSC, 122 stroma cancer induced stroma, 116 description, 114 desmoplastic reaction, 116 precusor cells, 120 TAF and EMT, 116–117 target, BM-derived cells, 119–120 TFG-b signaling, 115 suicide genes, 128–129 TRAIL, 129 tropism, wounds and tumors, 118–119 tumor cell-centric, 113–114 MM. See Malignant mesothelioma MPM. See Malignant pleural mesothelioma MSC. See Mesenchymal stem/stromal cells N Newcastle disease virus (NDV), 26–27 O Off-target effects non-specific shRNA, 59 siRNA immunologic effect, 58–59 specific shRNA, 58 siRNA, 58 OncoVex gm-csf doses, 220–221 head and neck, 221 prodrug arming, 222 Us11gene, 220

Index P PDGFR. See Platelet-derived growth factor receptor Personalized cancer therapy delivery strategies, 59–60 RNAi shRNA, 55–57 siRNA, 53–55 signal transduction networks, 52–53 SiRNA vs. shRNA Dicer/Drosha expression, RNAi effector suitability, 57 off-target effects, 58–59 p53 gene adenoviral-mediated gene therapy, 260 cisplatin, 69 gene replacement adenovirus, 66, 67 biomarkers, 67 lung cancer, 65–66 neck cancer, 66 radiation therapy, 69–71 retrovirus vector, 66 therapeutic benefit, 65 mutations, 254 pathway regulation, 65 restoration, sarcomas, 261 PKR. See Protein kinase R Platelet-derived growth factor receptor (PDGFR) differential drug sensitivity, 254 RTKs, 24 Prostate cancer, gene therapy enzyme/prodrug replication-competent adenoviruses, 36–40 replication-defective adenoviruses, 34–36 replication-competent, oncolytic adenoviruses CG7870, 45 CV706, intraprostatic injection, 44–45 vaccine-based cell, 43–44 Poxvirus, 41–43 Protein kinase R (PKR) as anti-viral protective mechanism, 24 cellular, 207 lung cancer cells, 182 mediated translation shutoff, 207 R Radiation therapy, p53 gene replacement adenovirus p53 expressing vector, 70–71 Ad-p53 injection, 70, 71 biopsies, 70

273 NPC, 71 NSCLC, 72–73 Receptor tyrosine kinases (RTKs), 24 Reovirus, 25–26 Replication competent lentiviruses (RCL), 160 Retroviral vector-producing cells (RVPCs), 21 RNA interference (RNAi) biodistribution and target modulation, 10–11 clinical trials, 11–12 delivery strategies, clinical translation, 59–60 gene regulatory therapy description, 2 development, 3 siRNA, 2–3 genetic dysregulation, 2 off-target effects classification, 8–9 non-specific, 9–10 search engines, siRNA design and validation, 9 sequences, siRNA, 9 processing steps characterization, 3 Dicer and Drosha levels, 3, 4 shRNA (See Short hairpin RNA (shRNA)) siRNA chemical modifications, 5, 6 delivery systems, 5, 7 local delivery, 4 minicells, 8 naked, 5 RISC, 53–54 and shRNA, 8 vs. shRNA, 55–59 synthetic, 55 RNA-interfering silencing complex (RISC), 53–54 RTKs. See Receptor tyrosine kinases RVPCs. See Retroviral vector-producing cells S Sarcoma, gene therapy constructs, 259 expression signatures leiomyosarcoma, 258 malignant peripheral nerve sheath tumor, 256–257 microarray technology, 257 profile divisions, 256 Rad51 protein, 258–259 ras protein, 257–258 SKLMS1 cells, 259

274 Sarcoma, gene therapy (cont.) non-viral vectors Cap1 gene, 259 immunohistochemical studies, 260 siRNA use, 259–260 soft tissue, cytogenetics chromosomal aberrations, 256 EWSR1 and CREB1, 255 leiomyosarcoma, 252–253 molecular abnormalities, 253 molecular probes, 252 17p11-12 gain, 253–254 p53 mutations, 254 sporadic desmoids, 255–256 unregulated cell growth, 254–255 telomelysin, 83 viral vectors E2F family, 262 ILP model, 261 p53 gene, 260 Rexin-G, 261 SCLC. See Small-cell lung cancer Short hairpin RNA (shRNA) bi-functional cleavage-dependant and-independent expression, 56–57 expression unit design, 56 dicer-mediated endonucleolytic cleavage, 55 vs. SiRNA comparative efficacy, 57 Dicer/Drosha expression, 57 off-target effects, 58–59 synthesis, 55 Small-cell lung cancer (SCLC), 71–72 Small interfering RNA (siRNA) chemical modifications, 5, 6 chemosensitivity, 258 clinical trials, 11–12 delivery systems, 5, 7 local delivery, 4 minicells, 8 naked, 5 nuclease degradation, resistance, 260 off-target effects classification, 8–9 non-specific, 9–10 search engines, design and validation, 9 sequences, siRNA, 9 osteosarcoma, 259–260 RISC assembly, 53–54 cleavage independent, 54–55 component, 53

Index and shRNA, 8 vs. shRNA comparative efficacy, 57 Dicer/Drosha expression, 57 off-target effects, 58–59 Suicide-gene therapy adenoviral vector, 242 dose-related intratumoral HSVtk gene transfer, 103 E1/E4-deleted adenoviral vector, 104 GCV, 99 HSVtk DNA transfer, 99–100 immunogenic killing, 99 lentiviral vectors, 160 MSC, 128–129 syngeneic murine MM models, 102 T TAA. See Tumor-associated antigens TAF. See Tumor-associated fibroblasts Targeted oncolytic adenovirus Ad5CMV-p53, 80 cytotoxic efficacy, 80–81 description, 79–80 oncolytic vectors, 80 telomerase-specific cancer therapeutics, hTERT promoter-driven, 81–85 clinical application, 88 ex vivo imaging, GFP fluorescence, 86–87 hTERT promoter-driven GFPexpressing, 86 in vivo imaging, GFP fluorescence, 87 transcriptional cancer, telomerase activity ALT, 81 enzyme, 81 Telomerase activity, transcriptional cancer targeting, 81 Ad-hTR-NTR, adenoviral suicide gene therapy vector, 242 anti-telomerase cancer therapy activity, 236 normal somatic cells, 235 associated gene therapy, 242 clinical application, 88 components, human, 233 early cancer detection, 234–235 GFP fluorescence ex vivo imaging, human circulating tumor cells, 86–87 lymph node micrometastasis, in vivo imaging, 87

Index GRN163L, oligonucleotide enzyme inhibitor apoptotic cell death, 241 clinical trial, 241–242 sequence, 239, 241 template region, 239 hTERT promoter-driven GFP-expressing, 86 immune activation, 85 preclinical studies, 82–84 structure, 81–82 immunotherapy dendritic cells, 238–239 GV1001, 237–238 Heptovax, 238 HLA molecules, 236–237 low affinity epitopes, 237 nondendritic cell-based cancer vaccine, 239 specific oncolytic virus adenoviruses, 242 hTERTp-TRAD gene therapy, 243 telomeres (See Telomeres) Telomeres bypass, 233 human, 231–232 length, 232 senescence, 232, 233 shortened, 232–233 Thymidine kinase (TK) description, 205–206 HSVtk/GCV gene therapy, 20 tk-deficient virions, 206 TRAIL. See Tumor necrosis factor related apoptosis inducing ligand Transductional targeting adapter-based, 145 Ad genome, 147 cryptic transcription, 147 DNA regulatory sequences, 147 genetic modifications, 146

275 Translational targeting, 148 Tumor-associated antigens (TAA), 143, 167 Tumor-associated fibroblasts (TAF), 116 Tumor necrosis factor related apoptosis inducing ligand (TRAIL) antitumor effects, 122 MSC, 129 Tumor-specific replication competent Adenoviral (hTERTp-TRAD) gene therapy, 243 Tumor suppressor gene therapy gene replacement chemotherapy, 69 DNA damaging agents, 68–69 p53, radiation therapy, 69–71 lung cancers, 64 malignant growth, 63 metastases cationic immunoliposome system, 73–74 FUS1 gene, 72 nanoscale synthetic particles, 72 NSCLC, 72–73 p53 protein expression, 71 3p21.3 region genes, 73 SCLC, 71–72 p53 gene product, 64–65 gene replacement, 65–68 pathway regulation, 65 protein, 64 V Vaccine-based gene therapy, Poxvirus advantages, 41 recombinant, 42 vaccinations, 41–42 Vesicular stomatitis virus G glycoprotein (VSVG), 164