adenoviruses. basic biology to gene therapy

MEDICAL INTELLIGENCE UNIT 15 Prem Seth Adenoviruses: Basic Biology to Gene Therapy R.G LANDES C OM PA N Y MEDICAL INTELLIGENCE UNIT 15 Adenoviruses: Basic Biology to Gene Therapy Prem Seth, Ph.D Human Gene Therapy Research Institute Des Moines, Iowa, U.S.A and Medicine Branch National Cancer Institute National Institutes of Health Bethesda, Maryland, U.S.A R.G LANDES COMPANY AUSTIN, TEXAS U.S.A MEDICAL INTELLIGENCE UNIT Adenoviruses: Basic Biology to Gene Therapy R.G LANDES COMPANY Austin, Texas, U.S.A Copyright ©1999 R.G Landes Company All rights reserved No part of this book may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher Printed in the U.S.A Please address all inquiries to the Publishers: R.G Landes Company, 810 South Church Street, Georgetown, Texas, U.S.A 78626 Phone: 512/ 863 7762; FAX: 512/ 863 0081 ISBN: 1-57059-584-4 While the authors, editors and publisher believe that drug selection and dosage and the specifications and usage of equipment and devices, as set forth in this book, are in accord with current recommendations and practice at the time of publication, they make no warranty, expressed or implied, with respect to material described in this book In view of the ongoing research, equipment development, changes in governmental regulations and the rapid accumulation of information relating to the biomedical sciences, the reader is urged to carefully review and evaluate the information provided herein Library of Congress Cataloging-in-Publication Data Adenoviruses: basic biology to gene therapy / [edited by] Prem Seth p cm (Medical intelligence unit) Includes bibliographical references and index ISBN 1-57059-584-4(alk paper) Adenoviruses Genetic vectors Gene therapy I Seth, Prem, II Series [DNLM: Adenoviridae Gene Therapy methods Genetic Vectors QW 165.5.A3 A2323 1999] QR396.A343 1999 579.2'443 dc21 DNLM/DLC 99-32566 for Library of Congress CIP PUBLISHER’S NOTE Landes Bioscience produces books in six Intelligence Unit series: Medical, Molecular Biology, Neuroscience, Tissue Engineering, Biotechnology and Environmental The authors of our books are acknowledged leaders in their fields Topics are unique; almost without exception, no similar books exist on these topics Our goal is to publish books in important and rapidly changing areas of bioscience for sophisticated researchers and clinicians To achieve this goal, we have accelerated our publishing program to conform to the fast pace at which information grows in bioscience Most of our books are published within 90 to 120 days of receipt of the manuscript We would like to thank our readers for their continuing interest and welcome any comments or suggestions they may have for future books Michelle Wamsley Production Manager R.G Landes Company CONTENTS Section I: Discovery and Structure of Adenoviruses Discovery and Classification of Adenoviruses Harold S Ginsberg Discovery of Adenoviruses Classification 2 Adenovirus Capsid Proteins John J Rux and Roger M Burnett Virion Architecture Major Coat Proteins Minor Coat Proteins 14 Future Directions 15 Organization of the Adenoviral Genome 17 Jane Flint Organization of Coding Sequences 18 Other Important Features 26 Sequences That Fulfill Multiple Functions 27 Conclusion 27 Section II: Adenovirus Life Cycle Entry of Adenovirus into Cells 31 Prem Seth Binding of Adenovirus to the Cell Receptor, and its Entry into the Endosomes 31 Adenovirus-Mediated Lysis of Endosome Membrane: Role of Low pH and Penton Base 33 Vectorial Movement of the Adenovirus into the Nucleus 33 Conclusion 35 Early Gene Expression 39 Philip E Branton Adenovirus Genes and Products 39 Early Region 1A (E1A) 40 Regulation of Gene Expression by E1A Products 42 Early Region 1B (E1B) 46 Early Region (E2) 50 VA RNA and Regulation of Protein Synthesis 50 Early Region (E3) 50 gp19K 50 Early Region (E4) 51 Adenoviruses and Adenoviral Products as Therapeutic Agents 54 Conclusion 55 Adenovirus DNA Replication 59 Muralidhara Ramachandra and R Padmanabhan Viral Genome and the Origin of DNA Replication 59 E2 Region and Its Regulation 59 Viral Replication Proteins 60 Cellular Factors Required for Replication 62 Initiation and Elongation of DNA Replication 63 Conclusion 65 Adenovirus Late Gene Expression 69 Julie Boyer and Gary Ketner Structure of the Late RNAs 69 Transcriptional Activation 72 Non-MLTU Late Proteins 73 Regulation of Polyadenylation 73 Regulation of Splicing 74 Nuclear Organization 74 mRNA Export 75 Inhibition of Translation of Host mRNA 76 Conclusion 76 Role of Endoprotease in Adenovirus Infection 79 Joseph Weber Adenovirus Assembly 85 Susanne I Schmid and Patrick Hearing Assembly Intermediates 85 Incomplete Particles of Adenovirus 85 Polar Encapsidation of Adenovirus DNA 86 Cis-acting Sequences Involved in Packaging Specificity 86 Trans-acting Components May Be Involved in Packaging 88 Virus Release from Infected Cells 88 Section III: Adenoviral Vectors for Gene Therapy: Preclinical Research 10 Development of Adenoviral Vectors for Gene Therapy 91 Dai Katayose and Prem Seth Recombinant Adenoviral Vectors 91 Adenovirally-Mediated Enhancement of DNA Delivery and the Concepts of Molecular Conjugates 96 Conclusion 99 11 Adenoviral Vectors for Cancer Gene Therapy 103 Prem Seth, Yu Katayose, and Amol N.S Rakkar Direct: Toxic Transgene Products 104 Indirect: Immunomodulation Through Recombinant Adenoviral Vectors 111 Other Novel Strategies 113 Conclusion 115 12 Adenoviral Vectors for Cardiovascular Diseases 121 Noel M Caplice, Timothy O’Brien, and Robert D Simari Vector Requirements for Cardiovascular Disease 121 Comparisons with Other Vectors 122 Potential for Toxicity 122 Specific Enhancements of Adenoviral Vectors for Cardiovascular Targets 124 Preclinical and Clinical Studies of Cardiac Gene Transfer Using Adenoviral Vectors 124 Preclinical Studies of Vascular Gene Transfer Using Adenoviral Vectors 125 Conclusion 126 13 IAP-Based Gene Therapy for Neurodegenerative Disorders 129 Stephen J Crocker, Daigen Xu, Charlie S.Thompson, Peter Liston, and George S Robertson The IAP Gene Family 130 Function of IAP Proteins 130 IAP Gene Therapy for Stroke 131 IAP Gene Therapy for Optic Neurodegeneration 132 IAP Gene Therapy for Parkinson’s Disease 133 Prospects for IAP-based Gene Therapy 135 14 Adenovirus Vectors for Therapeutic Gene Transfer to Skeletal Muscles 139 Josephine Nalbantoglu, Basil J Petrof, Rénald Gilbert, and George Karpati 15 Adenovirus-Mediated Gene Transfer: Applications in Lipoprotein Research 147 Silvia Santamarina-Fojo and Marcelo J.A Amar Analysis of Gene Function in Lipoprotein Metabolism 147 Gene Replacement Therapy in Animal Models of Hyperlipidemia and Atherosclerosis 148 Expression of Genes that Modulate Lipid Metabolism by Enhancing Alternative Lipoprotein Pathways 149 Structure-Function Analysis of Proteins Modulating Lipoprotein Metabolism 150 16 Correction of Serum Protein Deficiencies with Recombinant Adenoviral Vectors 157 James N Higginbotham and Prem Seth α1-antitrypsin Deficiency 157 Factor VIII and Factor IX Deficiency 158 Erythropoietin Deficiency 160 Other Potential Uses of Adenovirally-Delivered Serum Protein 160 Conclusion 161 17 Adenoviral Vectors for Vaccines 163 Bernard Klonjkowski, Caroline Denesvre, and Marc Eloit Several Deletion Mutants with Different Properties Can be Used 163 Efficacy and Safety of Adenovirus-Vectored Vaccines 165 Comparison of Replicative and Nonreplicative Viruses 167 Mechanisms of Immune Response Induction by Recombinant Adenoviruses 167 Prospects for Use 169 Conclusion 171 Section IV: Targetable Adenoviral Vectors 18 Strategies to Adapt Adenoviral Vectors for Gene Therapy Applications 175 Joanne T Douglas, Meizhen Feng, and David T Curiel The Generation of Targeted Adenoviral Vectors by Immunological Modifications of the Fiber Protein 175 Achievement of Long-Term Heterologous Gene Expression via Adenoviral Vectors 177 19 Adenovirus-AAV Combination Strategies for Gene Therapy 183 Krishna J Fisher Adenovirus Vector Development 183 Adenovirus-AAV Blueprint 184 Conclusion 189 20 Transcriptional and Promoter-Driven Control of Adenovirus-Mediated Gene Expression 191 Yoko Yoshida and Hirofumi Hamada Transcriptional and Promoter-Driven Targeting of Adenoviral Vectors 191 Tetracycline-Inducible System for Adenoviral Vectors 192 VSVG-Pseudotyped Retroviral Packaging System Through Adenovirus-Mediated Inducible Gene Transduction 198 Future Applications 198 MEDICAL INTELLIGENCE UNIT 15 Adenoviruses: Basic Biology to Gene Therapy Prem Seth, Ph.D Human Gene Therapy Research Institute Des Moines, Iowa, U.S.A and Medicine Branch National Cancer Institute National Institutes of Health Bethesda, Maryland, U.S.A R.G LANDES COMPANY AUSTIN, TEXAS U.S.A MEDICAL INTELLIGENCE UNIT Adenoviruses: Basic Biology to Gene Therapy R.G LANDES COMPANY Austin, Texas, U.S.A Copyright ©1999 R.G Landes Company All rights reserved No part of this book may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher Printed in the U.S.A Please address all inquiries to the Publishers: R.G Landes Company, 810 South Church Street, Georgetown, Texas, U.S.A 78626 Phone: 512/ 863 7762; FAX: 512/ 863 0081 ISBN: 1-57059-584-4 While the authors, editors and publisher believe that drug selection and dosage and the specifications and usage of equipment and devices, as set forth in this book, are in accord with current recommendations and practice at the time of publication, they make no warranty, expressed or implied, with respect to material described in this book In view of the ongoing research, equipment development, changes in governmental regulations and the rapid accumulation of information relating to the biomedical sciences, the reader is urged to carefully review and evaluate the information provided herein Library of Congress Cataloging-in-Publication Data Adenoviruses: basic biology to gene therapy / [edited by] Prem Seth p cm (Medical intelligence unit) Includes bibliographical references and index ISBN 1-57059-584-4(alk paper) Adenoviruses Genetic vectors Gene therapy I Seth, Prem, II Series [DNLM: Adenoviridae Gene Therapy methods Genetic Vectors QW 165.5.A3 A2323 1999] QR396.A343 1999 579.2'443 dc21 DNLM/DLC 99-32566 for Library of Congress CIP Adenoviruses: Basic Biology to Gene Therapy 300 Table 32.1 Preliminary results from a Phase p53 gene therapy clinical trial for ovarian cancer; cont Patient No No Doses Dose (Viral) Particles) Chemotherapy Transgene Expression 216 2.5 x 1013 IV Carbo/Taxol + (C1) 20 2.5 x 1013 IV Cisplatin/ Taxol - (C1),+(C2) 21 2.5 x 1013 IV Carbo/ Taxol + (C1 and 2) 22 2.5 x 1013 IV Carbo/ Taxol +/- (C1)* 23 7.5 x 1013 IV Carbo/Taxol Pending NR= sample degraded, no results; BQL= below quantifiable levels; ND= not done; C=dosing cycle; Patient 20 was allergic to carboplatin *Ascites positive for transgene expression, tumor biopsy negative in ascites, one decreased CA-125, and one short lived CT-objective decrease in tumor mass Future development of this treatment modality remains promising Conclusion Seldom does such a new and exciting therapeutic category of drug make it into the clinic We are just starting to evaluate the extent to which p53 gene therapy can achieve clinically meaningful outcomes and add to our currently inadequate cancer treatments Key to this discussion is the definition of appropriate clinical endpoints for gene therapy trials Development in an unprecendented area results in reliance on endpoints used in the past to justify approval of more traditional forms of cancer therapy Historically, improvement in overall or disease-free survival has been the clinical “gold standard” Most practitioners would acknowledge that there are other meaningful endpoints which guide them in the care of their patients Endpoints such as improvement in quality of life and response rate may translate into an improvement in signs and symptoms of the disease Other surrogates, such as improvement in tumor markers like CA-125, which parallels tumor burden, are significant to the patient yet more difficult to prove to regulatory authorities In the gene therapy arena, other potentially meaningful endpoints include the ability to express the transgene, and downstream effects such as tumor cell apoptosis It must still be determined whether clinically meaningful results and regulatory requirements could include the combination of a surrogate marker, such as CA-125, and cellular apoptosis Survival studies are extremely long and significantly delay the introduction of new therapeutics to the patient population There is ample evidence that the use of replacement gene therapy, either alone, or in combination with chemotherapy, translates into anticancer effects The preliminary clinical results of transgene expression hold out hope that future applications in the clinic will result in improvement in the current response and survival rates for cancer patients p53 Tumor Suppressor Gene Therapy in Ovarian and Other Peritoneal Cancers 301 Acknowledgments The authors would like to acknowledge the contribution of the clincial investigators and their patients who have participated in this clincial program and the reported data, especially Dr Richard Buller Abbreviations p53null p53mut p53wt moi pn AUC no p53 protein expressed mutant p53 protein expressed wild type p53 expressed, but not necessarily functional; ciu, cellular infectious units multiplicity of infection = ciu/cell viral particles mg/ml/min = “area under the curve” of the carboplatin serum concentration versus time plot Because elimination o fcarboplatin is almost entirely dependent on renal glomerular filtration rate (=creating clearance rate in ml/min), carboplatin dosing is basedon the projected area under the curve of the pharmacokinetic plot for each individual patient based on direct measurement of their creatine clearance or an indirect estimate of renal function based on patient age, mass, and serum creatinine concentration References Wills KN, Maneval D., Menzel P et al Development and characterization of recombinant adenoviruses encoding human p53 for gene therapy of cancer Human Gene Ther 1994; 5:1079-1088 Nielsen LL, and Maneval DC p53 tumor suppressor gene therapy for cancer Cancer Gene Ther 1998; 5:52-63 Nielsen LL, Gurnani M, Syed J et al Recombinant E1-deleted adenovirus-mediated gene therapy for cancer: Efficacy studies with p53 tumor suppressor gene and liver histology in mouse tumor xenograft models Human Gene Ther 1998a; 9:681-694 Grace M, Nuovo G, Johnson RC et al Solid tumor penetration of SCH58500 (p53 adenovirus) after intraperitoneal dosing as assessed by immunohistochemistry, p53 RT-PCR in situ, and laser scanning cytometry Proc Amer Assoc Gene Ther 1998; 1:8a Lowe SW Cancer therapy and p53 Curr Opin Oncol 1995; 7:547-553 Donaldson KL, Goolsby GL, and Wahl AF Cytotoxicity of the anticancer agents cisplatin and taxol during cell proliferation and the cell cycle Int J Cancer 1994; 57:847-855 Wahl AF, Donaldson KL, Fairchild C et al Loss of normal p53 function confers sensitization to taxol by increasing G2/M arrest and apoptosis Nature Med 1996; 2:72-79 Nielsen LL, Lipari P, Dell J et al Adenovirus-mediated p53 gene therapy and paclitaxel have synergistic efficacy in models of human head and neck, ovarian, prostate, and breast cancer Clin Cancer Res 1998b; 4:835-846 Gurnani M, Dell J, Lipari P et al Adenovirus-mediated p53 gene therapy has greater efficacy when combined DNA-damaging agents against human head and neck, ovarian, breast, and prostate cancer Cancer Chemother Phar, 1999; (In press) 10 Berenbaum MC What is synergy? Pharmacol Rev 1989; 41:93-141 11 Batra RK, Olsen JC, Hoganson DK et al Retroviral gene transfer is inhibited by chondroitin sulfate proteoglycans/glycosaminoglycans in malignant pleural effusions Journal of Biological Chemistry 1997; 272:11736-11743 12 Bergelson JM, Cunningham JA, Droguett G et al Isolation of a common receptor for coxsackie B viruses and adenoviruses and Science 1997; 275:1320-1323 CHAPTER 33 Adenoviral Gene Therapy for Malignant Pleural Mesothelioma Daniel H Sterman, Larry R Kaiser, and Steven M Albelda M alignant mesothelioma is a primary neoplasm of the mesothelial lining of the pleural (80%) or peritoneal cavities (19.5%) It has been linked conclusively to prior exposure to asbestos and may also be associated with certain genetic predispositions and past viral exposures, including SV40 Although relatively rare, mesothelioma accounts for approximately 3000 deaths per year in the United States To date, standard treatment for mesothelioma (including surgical resection, radiation therapy, and chemotherapy) have not proven effective in significantly prolonging patient survival.1,2 A number of characteristics make this tumor an attractive target for gene therapy First is the absence of any currently effective therapy Second is its unique accessibility in the pleural space for vector delivery, biopsy, and subsequent analysis of treatment effects A surgical “debulking” procedure to remove gross disease, followed by gene therapy to remove residual disease, would thus be technically feasible Third, local extension of disease, rather than distant metastases, is responsible for the morbidity and mortality associated with this neoplasm Thus, unlike other more widespread neoplasms, small increments of improvement in local control could engender significant improvements in palliation or survival Based on this rationale, we have recently conducted a phase clinical trial using an E1/E3-deleted replication-incompetent adenovirus carrying the herpes simplex virus thymidine kinase (HSVtk) gene aimed at treating mesothelioma.3 This approach serves as a model for treatment of other localized malignancies such as ovarian, bladder, and brain carcinoma Gene Therapy Using the Herpes Simplex Thymidine Kinase Gene One prominent approach in current experimental cancer gene therapy is the introduction of toxic or “suicide” genes into tumor cells, facilitating their destruction (molecular chemotherapy) One such “suicide” gene approach involves the transduction of a neoplasm with a cDNA encoding for an enzyme, such as the HSVtk gene, that would render its cells sensitive to a “benign” drug, such as ganciclovir (GCV), by converting the “prodrug” to a toxic metabolite.4 GCV is an acyclic nucleoside that is poorly phosphorylated by mammalian cells and is thus normally relatively non-toxic After being converted to GCV-monophosphate by HSVtk , however, it is rapidly converted to ganciclovir triphosphate by mammalian kinases Ganciclovir triphosphate is a potent inhibitor of DNA polymerase and a toxic analog that competes with nucleosides for DNA replication.5 One important feature of the HSVtk/GCV system is that not every cell within a tumor needs to be transduced This so-called “bystander” effect was demonstrated in in vitro experiments and subsequently Adenoviruses: Basic Biology to Gene Therapy, edited by Prem Seth ©1999 R.G Landes Company 304 Adenoviruses: Basic Biology to Gene Therapy in experiments where complete tumor regression was noted in animals after GCV treatment when only 10-20% of the tumor cells contained the HSVtk gene.6 Early experiments with the HSVtk gene involved the use of retroviral vectors (i.e., ref 7); however, our group and others have produced replication-deficient, recombinant adenoviral vectors encoding the HSVtk gene and shown that this vector, in combination with GCV, could eradicate tumor cells in vitro and in in vivo models of localized tumors.8-12 Preclinical Data: Animal and Toxicity Studies Based on experiments showing that replication-deficient adenovirus efficiently transduced mesothelioma cells both in tissue culture and in animal models8,9 and that infection with an adenovirus containing the HSVtk gene driven by the Rous sarcoma virus promoter (Ad.RSVtk ) rendered human mesothelioma cells sensitive to doses of GCV that were 2-4 logs lower that the doses required to kill cells infected with control virus,8 the Ad.RSVtk vector has been used to successfully treat established human mesothelioma tumors and human lung cancers growing within the peritoneal cavities of SCID mide.9 Marked decreases in tumor size have also been seen in an intrapleural rat model of syngeneic mesothelioma; however, survival increases have been more modest in this system.10 These in vitro and in vivo experimental results have been confirmed by other independent investigators.12 Based on this efficacy data in animals, we conducted preclinical toxicity testing for submission to the DNA Recombinant Advisory Committee of the NIH and the Federal Drug Administration (FDA) Rats and baboons were given high doses of virus intrapleurally followed by intraperitoneal administration of GCV at the same proposed dose for initial use in the clinical trial Toxicity was limited to localized inflammation of the pleural and pericardial surfaces.13 Clinical Data: Results from Phase I Clinical Trial On the strength of these animal studies, a phase I clinical trial for patients with mesothelioma began in November, 1995 at the University of Pennsylvania Medical Center in conjunction with Penn’s Institute for Human Gene Therapy The results of this trial were recently reported.3 The purpose of the phase I trial was to determine the maximally tolerated dose of Ad.RSVtk virus instilled into the pleural space, to evaluate the biological effects of therapy, and to evaluate, in preliminary fashion, any response rate.14 Patients were eligible for this study if they had a histologically proven diagnosis of malignant pleural mesothelioma, were not candidates for resection, had an ECOG performance status of 0, 1, or 2, and had the presence of at least some residual pleural space The protocol was designed as a dose escalation study, starting with a vector dose of x109 plaque forming units (pfu) and increasing in half log intervals to the current dose level of x1012 pfu On day of the study, patients 1-15 underwent videothoracoscopy for tissue acquisition, confirmation of diagnosis and placement of a chest tube For patients 16-21, a chest tube was inserted at the bedside with no pretreatment biopsies taken On day 2, the Ad.RSVtk viral vector, diluted in 50-100 ml normal saline, was instilled via the thoracostomy tube Three days later (on study day 5), a repeat videothoracoscopy was performed for tumor specimen acquisition The following morning (day 6), intravenous GCV was initiated at mg per kg given over one hour twice daily for 14 days At the completion of the 14 day GCV course, patients were discharged for outpatient follow-up Throughout the study, the patients were carefully evaluated for evidence of toxicity, viral shedding, immune responses to the virus and radiographic evidence of tumor response Adenoviral Gene Therapy for Malignant Pleural Mesothelioma 305 As summarized in Table 33.1, between November 1995 and November 1997, 21 (16 male, female) patients were enrolled in the study; none left the study prior to completion The ages of the patients ranged from 37 to 74 years with a median age of 66 years All stages and histologic subtypes of mesothelioma were represented Clinical toxicities of the Ad.RSVtk /GCV gene therapy were minimal and a maximally-tolerated dose (MTD) was not achieved Four non-dose-limiting toxicities were commonly noted: fever, liver enzyme abnormalities, myelosuppression, and skin rash Temperature elevations to 100-102˚F within 6-12 hours of vector instillation were seen in 20 of 21 patients, but with spontaneous defervescence after 48-72 hours and without associated hemodynamic or respiratory compromise In two of the three patients treated at the highest dose level, we noted some transient hypotension occurring within 1-4 hours of receiving vector Thirteen of 21 patients demonstrated minor abnormalities of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma glutamyltransferase (GGT) No patients developed elevated bilirubin or prothrombin time, or clinical evidence of hepatotoxicity Minor hematological toxicity was common, with 17 of 21 patients demonstrating a decline in hemoglobin, but only two patients required blood transfusions There was only one episode of moderate neutropenia and one episode of lymphopenia Both resolved spontaneously and without infectious complications About half the patients developed a vesicular skin rash at the site of the chest tube site The etiology of these eruptions are not known for certain but are thought to be similar to contact dermatitis No shedding of recombinant adenovirus was detected via an antibody fluorescent unit (AFU) assay from sterile swabs of the nares, rectum and urethra In addition, we found no evidence of adenoviral or HSVtk DNA using PCR techniques in the tumor or in any of the distant organ samples obtained from seven patients at the time of autopsy Detectable gene transfer (Table 33.1) has been documented in 12 of 20 evaluable patients in a dose-dependent fashion by either DNA polymerase chain reaction (PCR) (Fig 33.1A), RT-PCR, in situ hybridization (Fig 33.1B,C), and immunohistochemistry (IHC) (Fig 33.1D, E), the latter method utilizing a murine monoclonal antibody directed against HSVtk Once the dose level of 3.2 x 1011 was achieved, all patients except one demonstrated evidence of tk protein on post-treatment biopsies via IHC, with positive staining of tumor cells as deep as 40-50 cell layers below the mesothelial surface (Fig 33.1D, E) Strong anti-adenoviral humoral and cellular immune responses have been noted These include acute neutrophil-predominant intratumoral inflammation in the post-treatment biopsy sections, generation of high titers of anti-adenoviral neutralizing antibodies in serum and pleural fluid, significant increases in inflammatory cytokine production (TNF-α, IL-6) in pleural fluid, generation of serum antibodies against adenoviral structural proteins, and increased lymphocyte proliferative responses to adenoviral proteins.15 As in most phase I trials, the actual clinical effects of Ad.RSVtk/GCV gene therapy upon the patients’ tumors has been difficult to gauge This is made more difficult because of the heterogeneity of our patient population in terms of age, stage, histology, and vector dose Chest radiography and CT scanning, although quite sensitive for detecting tumor progression, are poor in determining therapy-related response in mesothelioma Given these caveats, with a median follow-up of approximately 12 months, 12 of 21 patients have died, with no fatal complications attributable to the gene therapy protocol (Table 33.1) Although no definite tumor regressions were noted, three of the 21 patients remain clinically stable, with no evidence of tumor growth on serial chest radiographs and chest CT scans Of those three patients without evidence of progression, all presented with early-stage mesothelioma (Stage IA/IB) Adenoviruses: Basic Biology to Gene Therapy 306 Table 33.1 Results of PENN phase clinical trial using Ad.RSVtk Meso Patient Stage/Cell Type Vector Dose (pfu) Status Survival s/p Rx (months) Gene Transfer (PCR/IHC) 62/M IA/E• x 109 progressed 28 - 56/M III/E x 109 deceased - 69/M III/B x 109 deceased 20 + 66/M II/E 3.2 x 109 deceased 11 - 71/M IA/E 3.2 x 109 stable 24 + 71/M II/B x 1010 deceased + 70/M II/E x 1010 deceased - 60/M II/E x 1010 progressed 21 + 74/M II/B 3.2 x 1010 deceased * 10 60/M III/E 3.2 x 1010 deceased - 11 37/F IV/E x 1011 deceased 16 - 12 37/M III** x 1011 deceased - 13 65/F III/E x 1011 deceased 10 + 14 66/F IA/E 3.2 x 1011 progressed 18 + 15 60/M IV/B 3.2 x 1011 deceased + 16 69/M IB/E 3.2 x 1011 deceased + 17 70/F IB/E 3.2 x 1011 progressed 11 + 18 69/F IB/E 3.2 x 1011 progressed 11 + 19 72/M II/E x 1012 stable + 20 65/M II/E x 1012 progressed + 21 67/M IA/S x 1012 stable + *Patient 009 was unable to have the follow-up thorascopic biopsy; **Patient 012 had a pseudomesotheliomatoid adenocarcinoma; •E-Epithelioid; B-Biphasic; S-Sarcomatoid Adenoviral Gene Therapy for Malignant Pleural Mesothelioma 307 123 bp markers Meso 013 Pre Meso 013 Pre Meso 013 Post Meso 013 Post Meso 013 Post Meso 014 Pre Meso 014 Post (Apex) Meso 014 Post (Lateral) 10 Meso 014 Post (Diaphragm) 11 Meso 014 Post (Skin Bx) 12 H20 Negative Control 13 tk DNA Positive Control 14 123 bp markers Fig.33.1 Transgene detection in the Ad.tk gene therapy trial (A) PCR detection of HSVtk DNA from pre- and post-vector delivery pleural biopsies of Patients 13 and 14 A 536 bp fragment is detected in the positive control lane as well as the post-treatment specimens from patients 13 and 14, including three diverse intrapleural locations for patient 14 No HSVtk DNA was detectable by ethidium bromide gel or Southern blot from any of the pretreatment samples or a skin biopsy obtained from the chest tube site after virus instillation (B) Photomicrographs (x200) of in situ hydridization assay performed with antisense oligonucleotide probes on post-vector biopsy specimens from Patient 13 Arrowhead demonstrates positivity for HSVtk mRNA in tumor cells 10-20 cell layers from pleural edge (C) Similar section hybridized with HSVtk mRNA sense control probe (D,E) Immunohistochemical staining of tumor biopsy from Patient 16 with the anti-HSVtk monoclonal antibody mixture at x100 (D) and x200 (E) Black staining denotes transgene expression Note strong nuclear staining in some cells 308 Adenoviruses: Basic Biology to Gene Therapy Problems and Future Approaches The recently completed phase trial has clearly demonstrated that delivery of large doses of an adenoviral vector to the pleural space is well tolerated and resulted in significant gene transfer to the surface and upper layers of tumor nodules Our major challenge for future will be to opimize gene delivery We plan to approach this problem in a number of ways Since gene transfer appears to be dose related, one strategy will be to deliver higher doses of vector To accomplish this, we plan to continue dose escalation; however, we are switching to an E1/E4-deleted “third generation” HSVtk -expressing vector The main advantages of this vector will be lower production costs (due to lower levels of replication-competent adenovirus) and potentially lower hepatoxicity16 and less immunogenicity Preclinical in vitro, in vivo, and toxicity studies indicate that this new vector performs almost identically to the “first generation” Ad.RSVtk A second approach to optimize efficacy will involve tumor debulking and lavage of pleural space prior to gene therapy treatment to maximize intrathoracic vector to tumor cell ratios A phase trial to study the toxicity of delivering virus using this approach is planned for the near future Future investigation might also focus on more efficient HSVtk mutant enzymes or drugs (such as retinoids) that might enhance bystander effects.17,18 One future strategy that might be particularly efficient in increasing gene delivery to mesothelioma cells could be the use of replicating viral vectors which have the capability of killing tumors by primary viral lysis and/or via delivery of therapeutic genes to cancer cells.19 Promising viruses in this regard are tumor-selective replication-competent adenoviruses (see ref 20 and chapter 21) Conclusion Cancer gene therapy is still in its infancy Even though clinical trials of gene therapy for mesothelioma have begun, it is important to realize the preliminary nature of these studies Although it is very unlikely that any of these early trials will result in practical therapies for advanced tumors, well-designed trials that are aimed at testing specific hypotheses and generating useful information about issues such as toxicity, gene transfer, and immune responses will be important first steps that must be taken for the advancement of cancer gene therapy As more information is obtained about tumor immunology and biology, and as better vectors are developed, gene therapy will almost certainly play a key role in the treatment of mesothelioma in the next decade Acknowledgments The authors would like to acknowledge the many collaborators and colleagues whose work was described here This includes past and present members of the Thoracic Oncology Laboratory (Dr Kunjlata Amin, Dr Leslie Litzky, Dr Katherine Molnar-Kimber, Dr Roy Smythe, Dr Harry Hwang, Dr Claude El-Kouri, Dr Ashraf Elshami, Dr John Kucharczuk, Dr Nabil Rizk, Dr Michael Chang), members of Penn’s Institute for Human Gene Therapy (Dr Stephen Eck, Dr Joseph Hughes, Dr Nelson Wivel, and its Director, Dr James Wilson), and the Penn Cancer Center (Dr Joseph Treat, Adri Ricio, and Dr John Glick) This work was supported by Grant No P01 CA66726 from the National Cancer Institute and Grant MO1-RR00040 to the General Clinical Research Center of the University of Pennsylvania Medical Center from the National Institutes of Health Support was also obtained from the National Gene Vector Laboratories, the Nicolette Asbestos Trust, and the Samuel H Lunenfeld Charitable Foundation Institutional support was provided by the University of Pennsylvania Cancer Center Adenoviral Gene Therapy for Malignant Pleural Mesothelioma 309 References Antman KH Natural history and epidemiology of malignant mesothelioma Chest 1993; 103:373S Aisner J Current approach to malignant mesothelioma of the pleura Chest 1995; 107:332S Sterman DH, Treat J, Litzky LA et al Adenovirus-mediated herpes simplex virus thymidine kinase gene delivery in patients with localized malignancy: Results of a phase clinical trial in malignant mesothelioma Human Gene Ther 1998; 9:1083 Tiberghien P Use of suicide genes in gene therapy J Leuk Biol 1994; 56:203 Matthews T, Boehme R Antiviral activity and mechanism of action of ganciclovir Rev of Infect Dis 1988; 10:S490 Pope IM, Poston GJ, Kinsella AR The role of the bystander effect in suicide gene therapy Euro J Cancer 1997; 33(7)1005-1016 Culver KW, Ram Z, Wallbridge S et al In vivo gene transfer with retroviral vector-producer cells for treatment of experimental brain tumors Science 1992; 256:1550 Smythe WR, Hwang HC, Amin KM et al Use of recombinant adenovirus to transfer the herpes symplex virus thymidine kinase (HSVtk) gene to thoracic neoplasms: An effective in vitro drug sensitization system Cancer Res 1994;54:2055 Smythe WR, Hwang HC, Elshami AA et al Successful treatment of experimental human mesothelioma using adenovirus transfer of the herpes simplex-thymidine kinase gene Ann of Surg 1995; 222:78-86 10 Elshami A, Kucharczuk J, Zhang H et al Treatment of pleural mesothelioma in an immunocompetent rat model utilizing adenoviral transfer of the HSV-thymidine kinase gene Hum Gene Ther 1996; 7:141-148 11 Chen SH, Shine HD, Goodman JC et al Gene therapy for brain tumors: Regression of experimental gliomas by adenovirus-mediated gene transfer in vivo Proc Natl Acad Sci USA 1994; 91:3054 12 Esandi MC, van Someren GD, Vincent AJPE et al Gene therapy of experimental malignant mesothelioma using adenovirus vectors encoding the HSVtk gene Gene Therapy 1997; 4:280-298 13 Kucharczuk JC, Raper S, Elshami AA et al Safety of intrapleurally administered recombinant adenovirus carrying herpes simplex thymidine kinase cDNA followed by ganciclovir therapy in non-human primates Human Gene Therapy 1996; 7: 2225-2233 14 Treat J, Kaiser LR, Litzky L et al Treatment of advanced mesothelioma with the recombinant adenovirus H5.01RSVTK: A phase trial Human Gene Therapy 1996; 7:2047-2057 15 Molnar-Kimber KL, Sterman DH, Chang M et al Impact of preexisting and induced humoral and cellular immune responses in an adenoviral-based gene therapy phase clinical trail for localized malignancy Human Gene Ther 1998; 9:2121 16 Gao G-P, Yang Y, Wilson JM Biology of adenovirus vectors with E1 and E4 deletions for liver-directed gene therapy J Virol 1996; 70:8934-8943 17 Black ME, Newcomb TG, Wilson HMP et al Creation of drug-specific herpes simplex virus type thymidine kinase mutants for gene therapy Proc Natl Acad Sci USA 1996; 93:3525-3529 18 Park JY, Elshami AA, Amin K et al Retinoids augment the bystander effect in vitro and in vivo in herpes simplex virus thymidine kinase/ganciclovir-mediated gene therapy Gene Therapy 1997; 9:909-917 19 Russell SJ Replicating vectors for gene therapy of cancer: Risks, limitations and prospects Euro J of Cancer 1994; 30A:1165-1175 20 Rodriguez R, Schuur ER, Lim HY et al Prostate attenuated replication competent adenovirus (ARCA) CN706: A selective cytotoxic for prostate-specific antigen-positive prostate cancer cells Ca Res 1997; 57:2559-2563 Index Symbols C α-fetoprotein promoter/enhancer (AFP) 114, 191, 192 12S mRNA 40, 42 13S mRNA 47 CAAT box 73 CAAT transcription factor 59 Carbamyl phosphate synthetase, aspartate transcarbamylase and dihyroorotase (CAD) 61, 67 Cancer vaccine 111 Caspase recruitment domain (CARD) 130 Cardiac myocytes 121, 122, 124, 125 Caspase recruitment domain 130 Cytosine deaminase (CD) 108, 114, 285, 287 CD34+ stem cells 32 CD8+ T cell 166 Carcinoembryonic antigen (CEA) promoter 192 Cellular protein kinase R 76 Cystic Fibrosis transmembrane conductance regulator (CFTR) 265,273-277 Chemotherapy 114 Chicken embryo lethal orphan (CELO) virus 17 Chimeric vector 178, 180 Christmas disease (see also hemophilia A) 158 Chronic lymphocytic leukemia 208 Clinical trials 287, 293-295, 308 Chronic lymphocytic leukemia (CLL) 208 Coated pits 31-34 Colon-colorectal liver metastases (CLM) 285-278 Complement 253-255 Coxsackie and adenovirus receptor (CAR) 32 Cre-lox recombination 96 Cyclic AMP response element binding protein (CREB) 218 Cystic fibrosis 262, 265, 268, 273, 285 Cystic fibrosis transmembrane conductance regulator (CFTR) 265, 273-277 Cytokeratin K18 80 Cytosine deaminase (CD) 108, 114, 285, 287 Cytotoxic T lymphocytes (CTLs) 221, 243, 251 A AAV 178, 184, 185, 189 AAV provirus 184, 185 Acute Respiratory Disease 1, Ad epitopes 251 Ad-p53 288 Ad.AAV 184, 185 Ad2 hexon Adenoid cells 1, Adenoviral hnRNPs 76 Adenoviral receptors 31, 32 Adenoviral tropism 175 Adenovirus capsid 5, Airway epithelium 251, 252 Allograft rejection 125 Anaplastic large cell lymphoma 208 Anemia 157, 160 Angiogenesis 103-105, 113 Antisense vectors 109 Antitrypsin deficiency 157 Apolipoprotein 147 Apoptosis 39, 44-48, 51, 53-55, 125, 126, 129-133, 135, 169 ATF 42, 43, 52 Atherosclerosis 122, 126, 147-149, 151 ATM gene 231 Aviadenoviruses AVP 79, 82 B Bax 104 Becker dystrophin minigene 139 Bone marrow 207, 209-211, 214 Breast cancer 191, 209, 211-213 Burkitt’s lymphoma 208 Bystander effect 104, 105, 108 Adenoviruses: Basic Biology to Gene Therapy 312 D H Death domain (DD) 245, 248 Dendritic cells (DC) 112, 113, 209 Diabetes mellitus 157, 161 Dihydrofolate reductase 218 DNA binding protein (DBP) 73, 88, 95 DNA encapsidation 85, 86 DNA polymerase 218 DNA Recombinant Advisory Committee 304 Dystrophin gene transfer 140 Hemagglutination Hematopoietic stem cell (HSC) 207, 209-213 Hemophilia A (see also Christmas disease) 159, 160 Hemophilia B 160 Hepatocellular carcinoma (HCC) 285-288, 291 Herpes simplex virus thymidine kinase (HSVtk) 108 Herpes virus 231 Hexon (polypeptide II) High density lipoproteins (HDL) 147-150 Histone acetyltransferase 218, 246 Histone deacetylase 222 Hodgkin’s disease 208 Homologous recombination 92, 94, 95 Human AAT locus 267 Hyperlipidemia 147-149 E E1-/E4- adenoviral vectors 95 E1-deleted recombinant adenoviral vectors 92 E1A genes 18 E1a mRNA 21, 40 E1A protein 18, 40, 41-44, 53 E1A transcription unit 18, 26 E1b mRNA 24 E1B transcription unit 27 E1B-19K 48, 51, 53, 55 E1B-55K 47, 50, 52, 53, 55 E2 encoded proteins 95 E2 transcription unit 18, 21, 27 E2F 18, 43, 44, 53 E2F transcription family 233 E3 gp19k 243, 248 E4 proteins 267 E4 region 95 Early region 1A (E1A) 18, 19, 21, 24, 26, 35, 39, 40-48, 51-55 Early region 1B (E1B) 18, 19, 21, 22, 24, 27, 39, 43, 46-48, 50-55 Endopeptidase 79, 82 Endosomes 31, 33, 34, 35 Endothelial cells (EC) 121, 122, 168, 169 I Icosahedral capsid 18 IFN-α 253, 255 IFN-β 253, 255 IFN-γ 255, 257, 258 IGFBP3 104 IgG isotype 261 Immune response 122, 124, 126, 140, 141, 150, 163, 164, 166, 167, 169-171 Innate immunity 252, 253, 255, 258 Interferon (IFN) 253 Integrins 32 Inverted terminal repeat (ITR) 17, 18, 26 K K7 80 F Factor IX deficiency 160 Fas 104 Fas ligand 245 Fibroblast growth factor receptor (FGF-1) 104 G Gap junctions 108 Gene targeting 232, 233 Glial cell ine-derived neurotrophic factor (GDNF) 134 Glucuronidase 248 Granzymes 245 L L1-52K scaffolding protein 79 Leptin 161 Liposomes 97 Lipolysacharide (LPS) 252 Lipoprotein receptor-related protein (LRP) 147, 148 LXCXE motif 218 Lysosomotropic agents 33 Index 313 M R Major late transcription unit (MLTU)19, 21, 25, 27,69, 71-75 MAPK 257 Mastadenoviruses Mesothelioma 303-305, 308 Metalloproteinase enyzmes (MMPs) 113 Microtubules 33 MLP-driven transcription 72 MLTF/USF 72 mRNA splicing 2, 24, 40 Multiple myeloma 208 Muscular dystrophy 139 Myeloid cells 207, 208 RAD51 genes 231 Radiotherapy 114 Raf-1 257 ras gene 109 Rb tumor suppressor 28, 43 Receptor internalization and degradation (RID) 246-248 REC2 gene 231 Recombinant adenoviruses 91, 92, 94-96, 99, 104, 112 Recombinant adeno-associated virus (rAAV) vector 184, 185, 189, 190 Rep minigene 185 Replication competent adenoviruses (RCA) 94 Replication competent retroviruses (RCR) 180 Replication competent viruses 96 Retinoblastoma (Rb) gene 105 Retroviruses 97 Ribozymes 109, 110, 118 RIDα 246, 247 RIDβ 246, 247 RNA polymerase III 25, 27, 50 N Natural immunity 252, 253 Natural killer (NK) cells 241, 243, 248, 253, 258 Neuroblastoma 211 Neutralization assays 2, NFκB 222-224, 246 Nuclear factor I (NF-I) 59 Nuclear localization signals 35 Nuclear pores 35 Nude mice 265, 279, 280 O Orphan receptors 222 Ovarian cancer 293, 295 P p300/CBP family 44 p53 44-49, 53-55 Papain 82 Penton base 5, 6, 11, 12, 15, 32-34 Penton base (polypeptide III) Penton base (polypeptide III) Perforin 245, 253 Phospholipid transfer protein (PLTP)147 pIX 19, 27 Polyadenylation 69, 74, 93 Preterminal protein (pTP) 21, 50, 59, 79 Primary hematopoeitic cells 32 Promyelocytic leukemia gene (PML) 75, 77, 107, 118 Proselin 97 Protease 61, 79-82 Prostate-specific antigen (PSA) promoter 192 S Sarcolemma 139, 140, 142 Saturation binding kinetics 31 SCID mice 294, 304 Serine proteinase inhibitor 157 Serpin 157 Shuttle vector 92, 93, 96 SIV gag p55 antigen 166 Splicing 2, 22, 24, 40, 42, 47, 50, 52 Squamous cell carcinoma 203 Suicide gene 108, 113 Surfactant proteins A (SP-A) 255 Surfactant proteins D (SP-D) 255 314 Adenoviruses: Basic Biology to Gene Therapy T V TATA box-binding protein (TBP) 60 T-cell receptor 243 Terminal protein (TP) 17, 21, 26, 27, 35, 50 TFIID 42 TGF-α 255 TGF-β 255 Th2 cells 264 Thrombospondin 105 Thymidine kinase 285 Thyroid receptor 104 tk 285, 287, 305 TNF 112, 114, 239-241, 243, 245, 246, 247, 248, 253, 255, 257, 259 Transcription factors 59, 63, 72 Tripartite late leader 73 ts1 79, 81 Tumor necrosis factor (TNF) 112, 114, 239-241, 243, 245- 248, 253, 255, 257, 259 Tumor suppressor genes 96, 104, 108, 113 Type I DNA topoisomerase 59, 63 VA RNA genes 25, 27 Vascular endothelial cells 121 Vascular endothelial derived growth factor (VEGF) 103 Vascular smooth muscle cells 122, 124 Very low density lipoproteins (VLDL) 147-150 Viral assembly intermediate 85 Viral cysteine proteases 82 Viral ITRs 92 U USF 19 Utrophin 141-143 Y Yeast artificial chromosome (YAC) 92 ... Adenoviruses: Basic Biology to Gene Therapy, edited by Prem Seth ©1999 R.G Landes Company 2 Adenoviruses: Basic Biology to Gene Therapy not have to wait until then,” and he telephoned Dr Rowe and told... (Fig 2.2), but showed that Adenoviruses: Basic Biology to Gene Therapy, edited by Prem Seth ©1999 R.G Landes Company Adenoviruses: Basic Biology to Gene Therapy A B Fig 2.1 Adenovirus virion (A)... human subgroup C archetype Adenoviruses: Basic Biology to Gene Therapy, edited by Prem Seth ©1999 R.G Landes Company 18 Adenoviruses: Basic Biology to Gene Therapy Organization of Coding Sequences