Cancer Gene Therapy by Viral and Non-viral Vectors E-Book

• Herausgeber: John Wiley & Sons
• Kategorie: Fachliteratur
• Serie: Translational Oncology
• Sprache: Englisch

Seitenzahl: 465

Veröffentlichungsjahr: 2014

Beschreibung

Provides expert, state-of-the-art insight into the current progress of viral and non-viral gene therapy Translational medicine has opened the gateway to the era of personalized or precision medicine. No longer a one-size-fits-all approach, the treatment of cancer is now based on an understanding of underlying biologic mechanisms and is increasingly being tailored to the molecular specificity of a tumor. This book provides a comprehensive overview of the pertinent molecular discoveries in the cancer field and explains how these are being used for gene-based cancer therapies. Designed as a volume in the Translational Oncology book series, Cancer Gene Therapy by Viral and Non-viral Vectors deals with the practice of gene-therapy, with reference to vectors for gene expression and gene transfer, as well as viral therapy. It covers the history and current and future applications of gene transfer in cancer, and provides expert insight on the progress of viral and non-viral gene therapy with regard to delivery system, vector design, potential therapeutic genes, and principles and regulations for cancer gene therapy. Presented in three parts, Cancer Gene Therapy by Viral and Non-viral Vectors covers: Delivery Systems * Translational Cancer Research: Gene Therapy by Viral and Non-viral Vectors * Retroviruses for Cancer Therapy * DNA Plasmids for Non-viral Gene Therapy of Cancer * Cancer Therapy with RNAi delivered by Non-viral Membrane/Core Nanoparticles Targeted Expression * Cancer Gene Therapy by Tissue-specific and Cancer-targeting Promptors * MicroRNAs as Drugs and Drug Targets in Cancer Principles of Clinical Trials in Gene Therapy * Regulatory issues for Manufacturers of Viral Vectors and Vector-transduced Cells for Phase I/II Trials * US Regulations Governing Clinical Trials in Gene Therapy * Remaining Obstacles to the Success of Cancer Gene Therapy Focusing on speeding the process in clinical cancer care by bringing therapies as quickly as possible from bench to bedside, Cancer Gene Therapy by Viral and Non-viral Vectors is an absolutely vital book for physicians, clinicians, researchers, and students involved in this area of medicine.

Leseprobe

Contents

Cover

Translational Oncology

Title page

Copyright page

List of Contributors

Series Foreword

Preface

Reference

Part 1: Delivery Systems

Chapter 1: Translational Cancer Research

Adenovirus

Oncolytic Adenoviruses for Treatment of Cancer

Vaccinia Virus

Herpes Virus

Parvovirus

Final Remarks

References

Chapter 2: Retroviruses for Cancer Therapy

Introduction

Overview of Retroviral Vectors

Gammaretroviral and Alpharetroviral Vectors

Foamyviral (Spumaviral) Vectors

HIV-1-Derived Lentiviral Vectors

Evolution of HIV-1-Derived Lentiviral Vectors

Retroviral Vector-Mediated RNAi Therapy: From Basic Science to Clinical Trial

Concluding Remarks and Challenges

Acknowledgments

References

Chapter 3: DNA Plasmids for Non-viral Gene Therapy of Cancer

Delivery

Expression of Transgenes from DNA Plasmid

Safety

Therapeutic Applications

Summary

References

Chapter 4: Cancer Therapy with RNAi Delivered by Non-viral Membrane/Core Nanoparticles

Introduction

Barriers

Overcoming Delivery Barriers

Putting it All Together: Model Examples of Innovative and Successful NPs

Conclusions

Acknowledgments

References

Part 2: Targeted Expression

Chapter 5: Cancer Gene Therapy by Tissue-specific and Cancer-targeting Promoters

Introduction

Cancer-Specific Promoters

Cancer-Targeting Promoters

Engineered Systems that Enhance Promoter Activity

Conclusion

References

Chapter 6: MicroRNAs as Drugs and Drug Targets in Cancer

Introduction

MiRNA and its Functioning Mechanism

MiRNA and Cancers

MiRNA in Other Diseases

MiRNA Therapeutics

Challenges

Outlook

References

Part 3: Principles of Clinical Trials in Gene Therapy

Chapter 7: Regulatory Issues for Manufacturers of Viral Vectors and Vector-transduced Cells for Phase I/II Trials

Introduction

Development of Good Manufacturing Practices

cGMP Requirements

cGMP Compliance

Practical Compliance: One Center’s Experience

Conclusions

Acknowledgments

References

Chapter 8: US Regulations Governing Clinical Trials in Gene Therapy

Introduction

NIH OBA and RAC

IBC

IRB

FDA

Protocol

Consent Form

Multisite Protocols

Conclusion

References

Chapter 9: Remaining Obstacles to the Success of Cancer Gene Therapy

Introduction

Drug-Development Pathways

Broadening the Appeal of Complex Biologics

Clinical Trial Design and Execution

Conceptual Limitations

Conclusions

References

Index

End User License Agreement

List of Tables

Chapter 1

Table 1.1 The main characteristics of the viruses discussed in this chapter.

Chapter 3

Table 3.1 Ongoing clinical trials targeting B-cell malignancies infusing T cells (i) genetically modified ex vivo using the Sleeping Beauty (SB) system to express a CD19-specific chimeric antigen receptor and (ii) propagated on engineered artificial antigen-presenting cells.

Chapter 4

Table 4.1 Barriers that hinder delivery efficiency and necessitate strategies to overcome them.

Table 4.2 NP characteristics.

Chapter 6

Table 6.1 Overview of the major miRNAs involved in cancer.

Chapter 8

Table 8.1 Assessment of risk of delayed adverse events in a gene therapy study. Source: US Food and Drug Administration [26].

Table 8.2 Integration properties of current commonly used gene therapy vectors in clinical trials.Source: US Food and Drug Administration [26].

Chapter 9

Table 9.1 Therapeutic applications of cancer gene therapy.

Table 9.2 Advantages and disadvantages of cancer gene therapy vectors.

List of Illustrations

Chapter 1

Figure 1.1 Schematic diagram representing the different kinds of adenovirus-derived vectors used for gene therapy. (A) Wild-type adenovirus is able to replicate and kill all permissive cells. (B) The E1 gene is replaced by the expression cassette; this vector can infect all permissive cells but they cannot replicate unless E1 is not transcomplemented by the packaging cell line. (C) All viral genes are deleted except ITRs and the packaging signal. These vectors can infect all permissive cells but they cannot replicate. (D) Oncolytic adenoviruses. These viruses have been engineered to selectively replicate in and kill cancer cells.

Figure 1.2 Transcriptional targeting. Simplified schematic illustrating the strategies used to achieve transcriptional targeting of tumor cells. (A) For example, a viral genome is modified to not be able to counteract the defense mechanisms that a normal cell turns on following a viral infection. (B) Tumor cells are defective of such mechanisms hence the virus can have its normal life cycle. (C) Tumor-specific promoter can initiate virus DNA replication, starting its life cycle.

Figure 1.3 Transductional targeting. Schematic diagram representing the strategies used for transductional targeting. (A) Tumor expressing a specific receptor can be infected and killed by an adenovirus that infects through the same receptor, e.g. adenovirus serotype 5 and Coxackie adenovirus receptor-expressing tumor. (B) Some tumors do not express Coxackie adenovirus receptors hence they are not susceptible to infection by Ad5. (C) Viruses bearing the knob from a different serotype (chimera viruses) are used to overcome the lack of Coxackie adenovirus receptors.

Chapter 2

Figure 2.1 Schematic representations of retrovirus genomes and HIV-1 life cycle. (A) A simple retrovirus genomic RNA. (B) HIV-1 genomic RNA. In addition to three indispensable retroviral genes (gag, pol, and env), the HIV-1 genome also contains six accessory genes (tat, rev, vpr, vif, vpu, and nef). PBS, primer-binding site. (C) Life cycle of HIV-1. Early stage: the HIV-1 envelope protein recognizes specific receptors on the surface of target cells to mediate entry and release of HIV-1 virion into the cytoplasm. After entry, the viral RNA is reverse transcribed into cDNA, imported into the host cell nucleus, and integrated into the host genome to become a provirus. Late stage: using the host cell transcription and translation machinery, the provirus is transcribed and the transcripts are exported to cytoplasm where they are translated into viral packaging proteins or used to generate the viral genome of next-generation virus. These viral components are then assembled into mature virions that exit the host cell through budding, to become next generation viruses.

Figure 2.2 Scheme of lentiviral vectors derived from HIV-1. (A) The first-generation vector consists of three plasmid constructs. HIV-1 envelope glycoprotein is substituted by VSV-G. (B) On the basis of the first-generation vectors, the second-generation vectors exclude the four accessory genes (vpr, vif, vpu, and nef) of HIV-1. (C) The third-generation vector further deletes the HIV-1 accessory gene tat and is composed of four plasmid constructs. Rev is expressed by a separate plasmid construct and SIN LTRs are included in transgene plasmid construct.

Figure 2.3 Schematic representation of lentiviral vector production and transduction. Transgene plasmid construct and packaging plasmids are cotransfected into 293 T cells for viral production. Then viral particles are collected by ultracentrifugation, resuspended, and titrated to determine the needed amount for target cell infection.

Chapter 3

Figure 3.1 Model of electroporation leading to channel formation and DNA transfer. (A) DNA is present in the proximity or in direct contact with cell membranes prior to the application of an electric field. (B) Upon application of an electric current, the cells elongate along electric field lines. Consequently, the curvature radii at the apices of the aligned cells increase and the internal and external charges redistribute to form a transmembrane potential. Subsequent discharge of electric potential across the membrane leads to the formation of conical pores that permit ion flux. (C) Under the continuous influence of electric field currents, the pores coalesce to form larger inverted hydrophilic channels that facilitate the passage of nucleic acid. Intracellular transport of nucleic acid may be passive or may be facilitated by the application of electrophoretic currents.

Figure 3.2 Mechanism of hydrodynamic gene delivery. (A) Tight junctions between endothelial cells form the vascular wall surrounded by hepatocytes in undisrupted liver tissue. (B) Upon hydrodynamic injection of naked DNA solution, the rapid increase in vascular volume disrupts the tight endothelial cell junctions, placing the hepatocytes in direct contact with the circulation. Transient pores form in the cell membranes allowing naked DNA in circulation to enter the cytosol. Other models suggest entry of DNA into the cytosol via vesicles created by macropinocytosis.

Figure 3.3 Non-viral genomic integration systems for stable expression of therapeutic genes. These systems are based on the cotransfection of expression cassettes and transposase genes to facilitate genomic integration. These genes may be on separate vectors, as shown, or encoded on the same plasmid. In the Sleeping Beauty system, the expression cassette is flanked by inverted terminal repeat/direct repeat (IR/DR) sequences. Transposase binds to the IR/DR elements forming a synaptic complex that is transposed into genomic TA dinucleotide sites. The piggyBac expression cassette is flanked by inverted terminal repeat (ITR) elements that target TTAA sequences in the target cell genome. The phiC31 integrase system is based on recombination between two attachment (att) sites: attB on the vector and so-called pseudo-attP genomic sites that are similar to native bacterial attP sequences. The stably integrated expression cassette is flanked by the newly recombined att sites (attR and attL).

Chapter 4

Figure 4.1 Cytoplasmic double-stranded DNA binds to Dicer, which cleaves it into a short oligonucleotide. Upon binding with R2D2/TRBP and Ago2, one RNA strand is discarded and the other is used as the template strand that is complementary to the target mRNA. mRNA then binds to this guide strand and is cleaved and released, while the siRNA guide strand remains a part of the RNA-induced silencing complex (RISC) and is able to bind and cleave additional mRNA.

Figure 4.2 Depictions of mushroom, brush, and collapsed conformations. (A) When s > 2r, PEG molecules do not sterically hinder each other and are free to spread out close to the NP surface. (B) When s < 2r, the PEG molecules are closer together and their interaction causes them to extend out from the NP. (C) When the NP curvature is increased, the same PEG density on the NP surface provides less shielding because the distal ends of each PEG molecule are now farther from each other. (D) High-density PEG may collapse on the surface of NPs.

Figure 4.3 Mechanisms of endosomal escape. (A) Ion-pair formation. Cationic lipids in the NP form ion pairs with anionic lipids in the endosome, leading to destabilization and fusion of both membranes. (B) Electrostatic interaction. Highly charged cationic lipids (or other particles) in the NP disrupt the anionic endosome. (C) The proton sponge effect. A proton sponge molecule (e.g. PEI) acts as a buffer while proton pumps in the endosomal membrane actively pump in H+ and Cl−. Therefore, the pH in the endosome does not drop, and more H+ and Cl− are pumped in, followed by water moving with its osmotic gradient. The increased pressure in the endosome ruptures it and allows the release of siRNA. (D) Osmotic rupture. A solid precipitate (e.g. calcium phosphate, shown by CaP) dissolves in the acidic endosome, increasing the osmotic pressure in the endosome, and ushering in water moving with its osmotic gradient. The increased pressure in the endosome ruptures it and allows the release of siRNA.

Figure 4.4 Hypothetical diagram of LCP with a collapsed and entangled PEG layer. A hollow calcium phosphate core (middle) can encapsulate siRNA, DNA, drug, or peptide. An asymmetric lipid bilayer, where the lipids in the inner leaflet differ from those in the outer leaflet, surrounds the core and is functionalized with PEG (green) and targeting ligands (yellow). Ligands may also be entangled, allowing only a fraction to be available for receptor binding. Source: B. DiPrete, unpublished diagram.

Figure 4.5 Schematic diagram of an AuNP complex. (A) The fully functionalized NP in systemic circulation. (B) Degradation of ketal linkages causes individual PEG-functionalized AuNPs to release and dissipate. (C) Exposed cationic core facilitates enhanced endocytosis and proton sponge effect, and is further degraded in the endosome to release siRNA. Source: Adapted from Shim 2010 [55]. Copyright 2012 American Chemical Society.

Chapter 5

Figure 5.1 Schematic of transcriptional amplification modules. (A) The TSTA system comprises of a two-step transcription process. In the first step of the system, a synthetic transcription factor, GAL4–VP2 is expressed under a tissue-/cancer-specific promoter. This transcription activator then binds to five GAL4 DNA-binding sites in the second step to direct transcription of the downstream therapeutic or reporter gene. Adapted from Figueiredo et al. [135]. (B) The VISA system is comprised of the TSTA module plus WPRE. The combination of transcription amplification plus RNA stabilization enhances and prolongs expression of the target gene.

Figure 5.2 A simplified schematic representation of transcriptionally targeted gene expression by cancer-specific or cancer-targeting promoter. In the first step, genes that are highly expressed in specific cancer types are identified and considered candidates for cancer-specific or cancer-targeting promoters. Then, regulatory elements in the promoter region upstream of the transcriptional start site (TSS) are cloned and characterized. A region containing elements or a combination of regulatory elements carrying the highest transcriptional activities is cloned into a reporter expression vector to test for transcriptional activities in cancer cells. Expression of the reporter gene is limited to cancer cells by presence of specific transcription factors. Reporter gene expression is compared between cancer-specific/-targeting promoter alone, cancer-specific/-targeting promoter plus the VISA/TSTA module, and the CMV promoter. Ideally, to achieve the highest therapeutic window (++++), the promoter activity should be higher than the nonspecific CMV promoter (++++) and show little or no expression (+/−) in normal cells. CSP, cancer-specific promoter; CTP, cancer-targeting promoter.

Chapter 6

Figure 6.1 MiRNA biogenesis. MiRNAs are transcribed from the genome by RNA polymerase II (Pol II) into pri-miRNAs, which are then processed by Drosha into hairpin structures called pre-miRNAs. The pre-miRNAs are transported by exportin-5 from nucleus to the cytoplasm, where Dicer further processes them into miRNA duplex. The miRNA duplex unwinds and the mature miRNAs are incorporated onto the RISC. By base pairing with the target mRNAs, miRNA affects protein production either by interfering with protein translation or by changing the mRNA's stability.

Figure 6.2 Strategies of miRNA therapeutics. Generally, two strategies can be employed to modulate miRNA expression or function: using miRNA mimics or viral constructs to restore miRNA function, and using antisense oligonucleotides (including antagomirs and locked nucleic acids) or miRNA sponge constructs to block miRNA function.

Figure 6.3 The chemical structures of RNA and LNA. By forming a C3′-endo conformation by the introduction of a 2′-O,4′-C methylene bridge, the LNA modification greatly increases the stability and specificity of the antisense oligonucleotide.

Figure 6.4 The mechanism of miRNA sponges. MiRNA sponges are transcribed at high levels by using expression vectors in the nucleus. After release into the cytosol, these sponges compete with the miRNA-targeting mRNAs for the binding of miRNAs. As a result, the mRNAs are released from the inhibition by miRNAs, and consequently the expression levels of proteins translated by these mRNAs are upregulated.

Chapter 7

Figure 7.1 Producer line Master Cell Bank production and testing. Manufacturers are cautioned to contact the FDA to determine current testing requirements. QC, Quality Control; RCR, replication-competent retrovirus; USP, United States’ Pharmacopoeia.

Figure 7.2 Retrovirus producer working cell bank freezing and testing. Manufacturers are cautioned to contact the FDA to determine current testing requirements.* Indicates required for vaccine product. PERT, product-enhanced reverse transcriptase; QC, Quality Control; USP, United States’ Pharmacopoeia.

Figure 7.3 Major steps in the manufacture and testing of an adenoviral vector. Manufacturers are cautioned to contact the FDA to determine current testing requirements. In this schema part of the Master Virus Bank has been used as the final clinical product. TGB, Tris/glycerol buffer; USP, United States’ Pharmacopoeia; VP, viral particles.

Figure 7.4 Major steps in the manufacture and testing of a retroviral vector. Manufacturers are cautioned to contact the FDA to determine current testing requirements. RCR, replication-competent retrovirus; USP, United States’ Pharmacopoeia.

Figure 7.5 Floor plan of the cGMP facilities at the Baylor Center for Cell and Gene Therapy. The Vector Production Facility has unidirectional flow of staff, materials, and waste, whereas the Cell Processing Facility is a single-corridor design with multidirectional flow.

Figure 7.6 Vector manufacturing. Showing the interior of a Class 10,000 (ISO 7) manufacturing suite during retroviral vector production. Staff at the left-hand BSC are harvesting the supernatant. The staff member at the right-hand BSC is labeling the tubes for storage of the supernatant. The staff member in the foreground is performing in-production environmental monitoring.

Chapter 8

Figure 8.1 Flow chart for development and review of a gene therapy clinical trial. PI, Principal Investigator.

Figure 8.2 An example of the organizational chart developed to provide support for gene-transfer studies.

Guide

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Table of Contents

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TRANSLATIONAL ONCOLOGY

SERIES EDITORS

ROBERT C. BAST, MD

Vice President for Translational ResearchThe University of Texas MD Anderson Cancer CenterHouston, TX, USA

MAURIE MARKMAN, MD

Senior Vice President for Clinical AffairsCancer Treatment Centers of AmericaClinical Professor of MedicineDrexel University College of MedicinePhiladelphia, PA, USA

ERNEST HAWK, MD, MPH

Vice President, Division of OVP, Cancer Prevention and Population SciencesThe University of Texas MD Anderson Cancer CenterHouston, TX, USA

Cancer Gene Therapy by Viral and Non-viral Vectors

Edited by

Malcolm K. Brenner, MB, BChir, PhD

Director, Center for Cell and Gene Therapy

Baylor College of Medicine

Houston, TX, USA

Mien-Chie Hung, PhD

Chair, Department of Molecular and Cellular Oncology

The University of Texas MD Anderson Cancer Center

Houston, TX, USA

Copyright © 2014 by John Wiley & Sons, Inc. All rights reserved

Published by John Wiley & Sons, Inc., Hoboken, New JerseyPublished simultaneously in Canada

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Library of Congress Cataloging-in-Publication Data

Cancer gene therapy by viral and non-viral vectors / edited by Malcolm K. Brenner, Mien-Chie Hung.p. ; cm. – (Translational oncology ; 4)Includes bibliographical references and index.ISBN 978-1-118-50162-7 (hardback)I. Brenner, Malcolm K., 1951– editor of compilation. II. Hung, Mien-chie, editor of compilation.[DNLM: 1. Neoplasms–therapy. 2. Genetic Therapy. 3. Genetic Vectors–therapeutic use. 4. Oncolytic Virotherapy. QZ 266]RC268.4616.99′4042–dc23

2013049552

Cover image: Left hand image – iStock #18471119 / © David_Ahn. Top right hand image – iStock #17000305 / © Pgiam. Bottom right hand image – iStock #24015041 /© monkeybusinessimages.Cover design by Modern Alchemy

Printed in the United States of America

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List of Contributors

Malcolm K. Brenner, MB, BChir, PhDCenter for Cell and Gene TherapyBaylor College of MedicineHouston, TX, USA

John C. Burnett, PhDDepartment of Molecular and Cellular BiologyBeckman Research Institute of City of HopeDuarte, CA, USA

George A. Calin, MD, PhDDepartment of Experimental TherapeuticsThe University of Texas MD Anderson Cancer CenterHouston, TX, USA

Vincenzo Cerullo, PhDLaboratory of ImmunovirotherapyCentre for Drug Research (CDR) and Division ofPharmaceutical BioscienceFaculty of PharmacyUniversity of HelsinkiHelsinki, Finland

Laurence J.N. Cooper, MD, PhDDivision of PediatricsThe University of Texas MD Anderson Cancer CenterHouston, TX, USA

Yue Ding, MScDepartment of Molecular and Cellular BiologyIrell and Manella Graduate School of Biological SciencesBeckman Research Institute of City of HopeDuarte, CA, USA

Adrian P. Gee, PhDCenter for Cell and Gene TherapyBaylor College of MedicineHouston, TX, USA

Bambi Grilley, RPh, RAC, CIP, CCRC, CCRPCenter for Cell and Gene TherapyBaylor College of MedicineHouston, TX, USA

Kilian Guse, PharmD, PhDCancer Gene Therapy GroupHaartman InstituteUniversity of HelsinkiHelsinki, Finland

Akseli Hemminki MD, PhDCancer Gene Therapy GroupHaartman InstituteUniversity of HelsinkiHelsinki, Finland

Jennifer L. Hsu, PhDDepartment of Molecular and Cellular OncologyThe University of Texas MD Anderson Cancer CenterHouston, TX, USA

Yi-Hsin Hsu, PhDDepartment of Molecular and Cellular OncologyThe University of Texas MD Anderson Cancer CenterHouston, TX, USA

Leaf Huang, PhDDivision of Molecular PharmaceuticsEshelman School of PharmacyUNC-NCSU Joint Department of Biomedical EngineeringUniversity of North Carolina at Chapel HillChapel Hill, NC, USA

Mien-Chie Hung, PhDDepartment of Molecular and Cellular OncologyThe University of Texas MD Anderson Cancer CenterHouston, TX, USA

Longfei Huo, PhDDepartment of Molecular and Cellular OncologyThe University of Texas MD Anderson Cancer CenterHouston, TX, USA

Chia-Wei Li, PhDDepartment of Molecular and Cellular OncologyThe University of Texas MD Anderson Cancer CenterHouston, TX, USA

Hui Ling, MD, PhDDepartment of Experimental TherapeuticsThe University of Texas MD Anderson Cancer CenterHouston, TX, USA

Zhuyong Mei, MDCenter for Cell and Gene TherapyBaylor College of MedicineHouston, TX, USA

Judy S.E. Moyes, MB, BChirDivision of PediatricsThe University of Texas MD Anderson Cancer CenterHouston, TX, USA

Amer M. Najjar, PhDCancer Systems ImagingThe University of Texas MD Anderson Cancer CenterHouston, TX, USA

John Rossi, PhDDepartment of Molecular and Cellular BiologyBeckman Research Institute of City of HopeDuarte, CA, USA

Andrew B. Satterlee, BSDivision of Molecular PharmaceuticsEshelman School of PharmacyUNC-NCSU Joint Department of Biomedical EngineeringUniversity of North Carolina at Chapel HillChapel Hill, NC, USA

Markus Vähä-Koskela, PhDCancer Gene Therapy GroupHaartman InstituteUniversity of HelsinkiHelsinki, Finland

Yan Wang, PhDDepartment of Molecular and Cellular OncologyThe University of Texas MD Anderson Cancer CenterHouston, TX, USA

Jiehua Zhou, PhDDepartment of Molecular and Cellular BiologyBeckman Research Institute of City of HopeDuarte, CA, USA

Series Foreword

While our knowledge of cancer at a cellular and molecular level has increased exponentially over the last decades, progress in the clinic has been more gradual, largely depending upon empirical trials using combinations of individually active anticancer drugs to treat the average patient. The challenge for the immediate future is to accelerate the pace of progress in clinical cancer care by enhancing the bidirectional interaction between laboratory and clinic. Our new understanding of human cancer biology and the heterogeneity of cancers at a molecular level must be used to identify novel targets for therapy, prevention, and detection focused on each individual. Barriers must be removed to facilitate the flow of targeted agents and fresh approaches from the laboratory to the clinic, while returning relevant human specimens, images, and data from the clinic to the laboratory for further analysis.

In this title we will provide a brief overview of our current understanding of human cancer biology that is driving interests in targeted therapy and personalized management. Further development of molecular diagnostics should facilitate earlier detection, more precise prognostication and prediction of response across the spectrum of cancer development. Targeted therapy has already had a dramatic impact on several forms of cancer and strategies are being developed to identify small groups of patients who would benefit from novel targeted drugs in combination with each other or with more conventional surgery, radiotherapy or chemotherapy. Development of personalized interventions – whether preventive or therapeutic in nature – will require multidisciplinary teams of investigators and the infrastructure to match patient samples and agents in real time.

To accelerate translational cancer research, greater alignment will be required between academic institutions, the US National Cancer Institute, the US Food and Drug Administration, foundations, pharma, and community oncologists. Ultimately, new approaches to prevention, detection, and therapy must be sustainable. In the long run, translational research and personalized management can reduce the cost of cancer care which has escalated in recent years. More accurate and specific identification of at-risk members and risk stratification will be helpful to minimize the risks of overdiagnosis and overtreatment, while maximizing the benefits of screening, early detection, and preventive intervention. Patients who would benefit most can be identified and funds saved by avoiding treatment in those whose cancers would not respond. Participation and education of community oncologists will be required, as will modification of practice patterns. For progress in the clinic to occur at an optimal pace, leaders of translational teams must envision a clear path to bring new concepts and new agents from the laboratory to the clinic, to complete pharmaceutical or biological development, to obtain regulatory approval, and to bring new strategies for detection, prevention, and treatment to patients in the community.

In a series of additional volumes regarding translational cancer research, several topics are explored in greater depth, including Biomarkers, Targeted Therapy, Immunotherapy, and this volume concerning Cancer Gene Therapy by Viral and Non-Viral Vectors. The purpose of these books has been not only to describe different strategies for particular forms of cancer, but also to identify some of the barriers to translation using different reagents or different strategies around common therapeutic or diagnostic modalities. Potential barriers include not only the need for a deeper understanding of science, methods to overcome the challenge of tumor heterogeneity, the development of targeted therapies, the availability of patients with an appropriate phenotype and genotype within a research center with the investigators, research teams, and infrastructure required for clinical/translational research and the design of novel trials, but also adequate financial support, a viable connection to diagnostic and pharmaceutical development, and a strategy for regulatory approval, as well as for dissemination in the community.

Cancer Gene Therapy by Viral and Non-Viral Vectors considers many of these areas, including the strengths and limitations of the several types of viral and non-viral delivery systems, the potential importance of tumor-specific promoter systems, examples of where gene therapy has succeeded, the challenge of targeting all cancer cells, the advantages of targeting the tumor stroma and immunocytes, and the logistic barriers to preparation of materials required for clinical trials. The need for substantial antitumor activity and the importance of clinical responses in phase I–II trials are also highlighted. Overall, this volume provides substantial perspective regarding the translational potential of cancer gene therapy.

Robert C. BastMaurie MarkmanErnest Hawk

Preface

The idea of gene therapy was first proposed to correct errors associated with genetic disease by supplementing defective or missing genes. Advances in DNA technology and in understanding the basis of genetic diseases gave high hopes that gene therapy would be the next big breakthrough in medicine. However, the journey ahead was not without challenges and roadblocks. In 1999, a tragedy occurred when an 18-year-old gene therapy trial participant, Jessie Gelsinger, died 4 days after receiving adenoviral treatment for a genetic disorder from a massive immune response that led to multiple organ failure. This incident caused a major setback in the gene therapy field and the US Food and Drug Administration placed a hold on active gene therapy trials. Yet another event that followed brought more bad news to the field. In 2003, five patients who received CD34+ hematopoietic (bone marrow) stem cells transduced with a retrovirus carrying the interleukin-2 receptor γ chain gene to treat inherited X-linked severe combined immunodeficiency (SCID-XI) developed T-cell leukemia. One patient later died. Despite the dismissal of promising hopes of gene therapy in the early days as a result of these events, there is now optimism as more current research data have shown substantial progress in the clinical development of gene therapy after years of intense investigations to improve vector design and safety. Several successful gene therapy trials, including treatment of an inherited eye disease (Leber’s congential amaurosis), Parkinson’s disease, blood disorders, SCID-XI, adenosine deaminase-deficient SCID, and Siemerling–Creutzfeldt disease (X-linked adrenoleukodystrophy), have been reported in the last few years. Most recently, two studies published in July 2013 in Science reported clinical efficacy in lentivirial-mediated gene therapy to treat metachromatic leukodystrophy and Wiskott–Aldrich symdrome (see Chapter 2 for more details). In addition to human trials, studies conducted in canines showed that achromatopsia (an inherited form of total color blindness), diabetes, and Duchenne muscular dystrophy were successfully treated by gene therapy; these encouraging findings will undoubtedly continue to pave the way for conducting human clinical trials to develop new drugs to treat these diseases.

While no gene therapy has yet been approved in the USA, two have been approved for use in other parts of the world. The Chinese State Federal Drug Administration approved the world’s first gene therapy to treat head and neck cancer using Gencidine, an adenoviral vector expressing tumor suppressor p53. However, concerns about the therapeutic efficacy have been raised [1], and there are no further reported clinical outcomes after a decade of approval. In 2012, the European Commission approved the first gene therapy product (Glybera) in the Western world to treat lipoprotein lipase deficiency, a rare inherited disease of fat metabolism. The company uniQure is currently seeking regulatory approval in the USA, Canada, and other countries.

Cancer, cardiovascular, and infectious diseases, among many others, are also targets of gene therapy. Adenovirus remains the most popular type of vector used in gene therapy clinical trials worldwide, followed by retrovirus, naked/plasmid DNA, vaccinia virus, and lipofection in the top five. For viral vectors, the important parts of the virus required for gene delivery are kept and those that are not required are deleted, and the development of self-inactivating integrating viruses such as retrovirus and lentivirus eliminates the transactivation of neighboring genes after integration. Current investigations also continue to broaden the viral vectors’ cell host range. For non-viral vectors, improvements have focused on the delivery system for therapeutic agents, including plasmid DNA, RNA interference (RNAi), and microRNAs, by increasing cellular uptake, protecting against microphage digestion, and optimizing nucleic acid payload release.

In the USA, clinical studies must be reviewed by regulatory committees such as the Institutional Review Board (IRB), Food and Drug Administration (FDA), Institutional Biosafety Committee (IBC), and Recombinant DNA Advisory Committee (RAC). Moreover, manufacture must also comply with Good Manufacturing Practice (GMP) guidelines set out by the FDA. The development of sufficient manufacturing capacity to meet the clinical demands after gene therapy attains approval is another concern.

The discovery of monoclonal antibodies brought much excitement as a new treatment modality in early 1980s. However, the lack of efficacy and the rapid clearance of murine monoclonal antibodies due to the development of human antimouse antibodies in patients led to the failure of many clinical trials. Nonetheless, perseverance allowed the development of technological improvements resulting in the eventual clinical success of monoclonal antibodies, which are now a standard approach for producing therapeutics targeting cell surface receptors. In a similar way, further improvements in gene therapy may allow this approach to follow the successful journey of monoclonal antibodies.

To ensure that gene therapy can be successfully developed into new drugs following the fate of monoclonal antibodies, there are several areas needing critical improvement, including efficient delivery, specificity, and well-designed clinical trials. In this book, we have invited experts to discuss the current updates on cancer gene therapy. The opening chapter by Cerullo et al. describes various types of viral therapy, particularly DNA viruses (adenovirus, vaccinia virus, herpes virus, parvovirus) and provides examples of their use in clinical studies. The following chapter by Zhou et al. focuses on the principal types and evolution of lentiviruses in cancer and HIV therapy with special interest in gene silencing by RNAi. The next two chapters describe non-viral delivery systems. First, in Chapter 3 Najjar et al. review various methods of plasmid DNA delivery, optimization of gene expression, and their application for therapy including cancer. Satterlee and Huang then explain in Chapter 4 the design and challenges of nanoparticles to deliver therapeutic RNAi. In the second part, starting with Chapter 5, Hsu et al. provide an introduction to the clinical applications of tissue-specific and cancer-targeting promoters in cancer gene therapy. As aberrant microRNA expression has been implicated in promoting and initiating carcinogenesis, in Chapter 6 Ling and Calin present an overview of the role of microRNAs in cancer and other diseases and discuss examples of anti-microRNA therapeutics.

The last part of the book provides some insight on the regulatory compliance of gene therapy clinical trials focusing on manufacturing regulations of viral vectors by Gee and Mei in Chapter 7 and review processes and requirements prior to obtaining FDA approval by Grilley in Chapter 8. In the closing chapter, Brenner discusses the tasks that must be accomplished to make gene therapy drugs more broadly applicable and the improvements in clinical trial design, as the development pathway of cancer gene therapy is distinct from and more complex than the traditional pharmaceutical model. It is our hope that this book can facilitate the maturation of gene therapy for its clinical application.

Malcolm K. BrennerMien-Chie Hung

Reference

1 Guo J, Xin H. (2006) Splicing out the West? Science 314: 1232–1235.

Part 1Delivery Systems

Chapter 1Translational Cancer Research: Gene Therapy by Viral and Non-viral Vectors

Vincenzo Cerullo, Kilian Guse, Markus Vähä-Koskela, and Akseli Hemminki

University of Helsinki, Helsinki, Finland

Adenovirus

Adenovirus is among the most used vectors for gene therapy and gene transfer, and about 23% of all vector-based clinical trials have been performed with it (www.wiley.com//legacy/wileychi/genmed/clinical/). Adenovirus was first isolated in 1953 from human adenoids [1]. To date, 55 different human serotypes, subdivided into seven subgroups (A–G), have been characterized [2,3].

Adenovirus is a nonenveloped double-stranded DNA virus surrounded by an icosahedral protein capsid (Table 1.1). The capsid comprises penton and hexon proteins with knobbed fibers protruding from the vertices of the capsid [4]. Soon after its entry into the target cell viral DNA reaches the nucleus where starts its replication. Early genes, mainly involved in DNA replication, are transcribed first [5], followed by late genes mainly coding for structural proteins [4].

Table 1.1 The main characteristics of the viruses discussed in this chapter.

Adenovirus

Vaccinia virus

Herpes simplex virus

Parvovirus

Genome

Linear dsDNA, 36 kb

Linear dsDNA, 200 kb

Linear dsDNA, 150 kb

Linear ssDNA, 5.1 kb

Transgene capacity

Replication-competent vectors

≈3 kb

> 25 kb

>25 kb?

First-generation adenoviral vectors

FG-Ad: 7.8 kb

MVA, ΔD4R: > 50 kb

≈40 kb?

Helper-dependent adenoviral vectors

HD-Ad: ≈36 kb

HSV amplicon: ≈150 kb

<4.6 kb

Genetic targeting

Deletion of genes essential for replication in normal cells (E1A, E1B)

Tumor-specific promoters

MicroRNA targets in genome

Deletion of genes essential for replication in normal cells (VGF, TK)

MicroRNA targets in genome

Deletion of genes essential for replication in normal cells (TK, RR, γ34.5)

Tumor-specific promoters

MicroRNA targets in genome

Tumor-specific promoters

Particle retargeting

scAb-binding domain in knob

Cell-specific peptides in fiber/knob

Serotype fiber/knob exchange

scAb-binding domain in knob

Cell-specific peptides in fiber/knob

serotype fiber/knob exchange

scAb-binding domain in knob

Cell-specific peptides in fiber/knob

Serotype fiber/knob exchange

dsDNA, double-stranded DNA; HSV, herpes simplex viruse; MVA, modified vaccinia Ankara; ssDNA, single-stranded DNA; TK, thymidine kinase; VGF, vaccinia growth factor.

Adenoviruses tend to be species-specific with regard to permissivity to replication. However, there may be some exceptions to this general rule. It has been reported that adenovirus serotype 5 subgroup C (usually referred as Ad5, the most used gene therapy vector) can replicate to some degree in cotton rats [6,7], New Zealand rabbits [8], and Syrian hamsters [9]. This feature of Ad5 has been very important for scientists around the world because it has allowed them to use these animal models to develop new therapies for disease.

Historically adenovirus has been the most used vector for gene therapy and gene-transfer purposes. In 1970s F. Graham and colleagues discovered the importance of the E1 gene, that made possible the use of adenovirus as a viral vector for gene therapy [10]. In fact, as E1 gene products initiate the replication of the viral DNA, serotype 5 adenoviruses with E1 deleted are incapable of replicating and remain episomal. Taking advantage of this characteristic, scientists replaced E1 with different expression cassettes to avoid virus replication while promoting expression of the transgene inserted in place of E1. Later on, E1-deleted adenoviral vectors, also known as first-generation adenoviral vectors (FG-Ad), were developed into high-capacity adenoviral vectors or Helper-dependent adenoviral vectors (Hd-Ad). HD-Ad are devoid of all viral genes except the two inverted terminal repeats (ITRs) and the packaging signal (psi). They show a high cloning capacity (up to 36 kb) and reduced immunogenicity and toxicity [11] (Figure 1.1). Since then, it has been mainly used as vector for gene transfer for genetic diseases [12] or to treat cancer [13]. The immunogenicity of adenovirus may render it unsuitable for long-term gene expression but makes it attractive for treatment of cancer. Use of a replication-deficient adenovirus as a gene delivery vehicle is the classic approach, with some exciting clinical results [14,15,16,17], but no products have been approved outside of China. This approach has been reviewed recently [18]. In the past decade, many adenoviral gene therapists have focused on use of adenovirus as a replication-competent oncolytic virus and thus this will be focus of this chapter.

Figure 1.1 Schematic diagram representing the different kinds of adenovirus-derived vectors used for gene therapy. (A) Wild-type adenovirus is able to replicate and kill all permissive cells. (B) The E1 gene is replaced by the expression cassette; this vector can infect all permissive cells but they cannot replicate unless E1 is not transcomplemented by the packaging cell line. (C) All viral genes are deleted except ITRs and the packaging signal. These vectors can infect all permissive cells but they cannot replicate. (D) Oncolytic adenoviruses. These viruses have been engineered to selectively replicate in and kill cancer cells.

Oncolytic Adenoviruses for Treatment of Cancer

Oncolytic adenoviruses are specifically modified to selectively replicate in and destroy cancer cells. This selectivity is achieved by modifications of the genes involved in viral replication so that the life cycle of the virus can occur only in cells than can transcomplement the defect, including cancer cells, while the replication of the virus is arrested in normal cells (transcriptional targeting) (Figure 1.2). An alternative approach is to use tumor-specific promoters to “drive” E1 expression to allow selective replication of the virus in cancer cells [19] (Figure 1.2).

Figure 1.2 Transcriptional targeting. Simplified schematic illustrating the strategies used to achieve transcriptional targeting of tumor cells. (A) For example, a viral genome is modified to not be able to counteract the defense mechanisms that a normal cell turns on following a viral infection. (B) Tumor cells are defective of such mechanisms hence the virus can have its normal life cycle. (C) Tumor-specific promoter can initiate virus DNA replication, starting its life cycle.

Historically, the first adenoviruses used in patients were wild-type viruses [20]. The concept was revived with the first adenovirus proposed to have tumor selectivity, dl1520 (today known as ONYX-015) [21]. This adenovirus bears a naturally occurring variation that results in a nonfunctional E1B-55k product. E1B-55k is one of the proteins encoded by the early gene E1 and its normal function is to promote the degradation of p53 to avoid the infected cell undergoing apoptosis [22]. In infected normal cells p53 is not degraded by the mutated E1B-55k so that they can smoothly continue towards cell cycle arrest and apoptosis, which causes the arrest of the virus’s life cycle; on the other hand, in cancer cells, where the p53/p14ARF pathway is universally defective, the mutation is not needed to avoid apoptosis [21]. An issue with this type of virus is that E1B-55k is needed for late mRNA transport and its absence results in ineffective oncolysis, several orders of magnitude less than with the wild-type virus [23].

An alternative strategy used to generate adenoviruses selective for cancer cells is a 24 bp deletion of the E1A gene [23,24,25]. This deletion results in the inability of E1A to bind to retinoblastoma tumor-suppressor protein (Rb) and to release eukaryotic initiation factor E2F, which in the case of wild-type adenovirus would result in S-phase induction in normal cells. Therefore the “delta-24” viruses are unable to induce S-phase in host cells and no viral replication follows. In contrast to normal cells, most if not all cancer cells have a defective Rb/p16 pathway, rendering the Rb-binding property of E1A dispensable [26]. An important difference to dl1520 is that these types of viruses are not attenuated in comparison to wild-type adenovirus with regard to replication in cancer cells [24].

Another strategy to restrict virus replication to tumor tissue is to drive E1A gene expression with a tumor-specific promoter. The first example of this type of modification was an adenovirus with prostate-specific antigen promoter driving expression of E1A [27]. Since then a multitude of different tissue-specific promoters have been used, including α-fetoprotein for hepatic cancer [28], tyrosinase for melanoma [29], and carcinoembryonic antigen (CEA) for colorectal cancer [30]. Also, tissue-specific promoters that are activated in a variety of cancer types have been employed, including cyclo-oxygenase 2 promoter [31,32,33,34], L-plastin promoter [29,35], and human telomerase reverse transcriptase promoter [36,37]. The selectivity of dl1520 and delta-24 occurs after E1 expression, while tumor-specific promoters act prior to E1 expression. Therefore, an appealing approach is to combine both [33].

In addition to these strategies that restrict viral replication to tumor cells (transcriptional targeting), effort has also been put into modifying the adenovirus capsid to increase transduction of cancer cells (transductional targeting) (Figure 1.3). To this purpose different serotypes or chimeras have been tested to increase tumor transduction (recently reviewed by Cerullo and colleagues [13]). Particularly noteworthy has been the adenovirus 5/3 chimera. This modified adenovirus has been generated by placing the Ad3 fiber knob into the Ad5 backbone, resulting in an Ad5/3 chimera that displays the cell-binding properties of serotype 3 [38,39]. These chimeras also exhibit enhanced gene delivery and efficacy in preclinical animal models [39,40,41]. Recently this approach was taken a step further by developing the first fully serotype 3 (Ad3)-based oncolytic adenovirus, which has shown very encouraging results in animal models and human patients [42,43]. Transcriptional targeting is fully compatible with transductional targeting and an appealing concept is to combine them, as seen in many advanced-generation viruses [33].

Figure 1.3 Transductional targeting. Schematic diagram representing the strategies used for transductional targeting. (A) Tumor expressing a specific receptor can be infected and killed by an adenovirus that infects through the same receptor, e.g. adenovirus serotype 5 and Coxackie adenovirus receptor-expressing tumor. (B) Some tumors do not express Coxackie adenovirus receptors hence they are not susceptible to infection by Ad5. (C) Viruses bearing the knob from a different serotype (chimera viruses) are used to overcome the lack of Coxackie adenovirus receptors.

Importantly, oncolytic adenoviruses have also been used as delivery vehicles to produce molecules (such as antibodies, drugs and prodrugs, cytokines and chemokines, and so on) directly at the tumor site [13]. This approach has been particularly helpful because it allows, especially for molecules that have high systemic toxicity, a high local concentration associated with less systemic exposure.

For this purpose, a common way to insert foreign DNA into the adenovirus genome is by replacement of small proteins encoded by early or late genes. Transgenes can completely replace E3 [44] or just part of this gene [26,28]. Transgenes can also be inserted in the late genes and the expression level of the transgene could depend on the insertion site [45].

A multitude of different proteins have been investigated as “arming devices” for oncolytic adenoviruses. Tumor-suppressor genes such as p53 have been used to enhance oncolytic cell killing regardless of the p53 status of the cancer cell line [46]. Prodrug-converting-enzyme-based systems commonly employ either cytosine deaminase for 5-fluorocytosine conversion to 5-fluorouracil [35,47], HSV-tk for ganciclovir conversion to its active metabolite [48], or both [49]. Antiangiogenic molecules have also been used for arming [50], in addition to various other molecules such as human sodium iodide symporter, which has been used to concentrate radioiodine in target cells [51]. Furthermore, immunostimulatory cytokines such as granulocyte macrophage colony-stimulating factor (GM-CSF) [52,53,54,55] used to boost antitumoral immunity have been under active investigation as transgenes.

Another interesting approach recently explored for adenovirus has been the enrichment of its genome with TLR9-specific sequences to increase TLR9 stimulation and consequently to enhance the antitumor immunity [56]. Along the same line arming the adenovirus with ligand for CD40 has also shown enhanced antitumor immunity in animal models and in cancer patients as well [57,58]. Unfortunately oncolytic viruses – which need most of their genome to replicate – have a limited cargo size capacity. A larger payload can be achieved by FG-Ad or HD-Ad but these vectors are not capable of replicating in cancer cells. In fact it would be interesting to combine the cloning capacity of HD-Ad with the killing capacity of oncolytic viruses; these approaches are under investigation. This should be feasible as oncolytic viruses have already been utilized to amplify first-generation vectors to combine the merits of each [59].

Use of Adenovirus in the Clinic

The observation that wild-type adenoviruses can kill cancer cells has been acknowledged for a long time. In fact, the first in-human use of adenovirus was in the 1950s [20], when 10 different serotypes were used to treat 30 cervical cancer patients. The treatments were quite safe, which is remarkable considering that wild-type viruses were used. With regard to efficacy, two-thirds of the patients had a “marked to moderate local tumor response” [20] with necrosis and ulceration of the tumor. Although “response” was not defined, these numbers are not so far from what is seen with modern viruses [58,60].

Since then, a multitude of different oncolytic adenoviruses have been conceived and tested in human clinical trials for different tumor types such as pancreatic cancer [61], brain tumors [62], prostate cancer [63], bladder cancer [64], and ovarian cancer [65], among others.

The first oncolytic adenovirus used in clinical trials in modern times was ONYX-015. More than 300 cancer patients with different tumor types were treated in several clinical trials from phase I to phase II, but a phase III trial was never initiated in the West. Instead, in China a similar virus, H101, was rapidly taken through all phases and approved in 2006 as Oncorine [66]. The overall results from these clinical trials were that this virus is safe and selective for cancer [67], and has antitumor efficacy, especially when combined with chemotherapy. However, preclinical data suggest that the oncolytic potency is up to 100 times lower than, for example delta-24-type viruses [25]. Also, an unarmed virus might be at a disadvantage compared to armed viruses.

Recently, scientists have realized that the use of oncolytic adenoviruses for treatment of cancer is particularly intriguing given their ability to wake up the immune system, stimulating a response to the cancer [13,68]. Although a clear mechanism on how this happens still remains to be fully clarified, we believe that even in the tumor-immunosuppressive microenvironment adenoviral particles have the ability to (i) stimulate dendritic cells, predisposing them to crosspriming, (ii) promote antitumor immunity by enhancing the release of tumor-associated antigens in the presence of a danger signal [69], and (iii) break tolerance of the immunosuppresive tumor environment through interaction with pathogen-associated molecular pattern receptors [11,70,71,72,73]. These “natural” features can be further improved when adenoviruses are armed with immunostimulatory molecules.

We have been among the first laboratories to demonstrate the involvement of the immune system in human cancer patients. In our first study patients were treated with a GM-CSF-encoding serotype 5 virus (Ad5D24-GM-CSF) bearing a 24 bp deletion in E1A [68]. We assessed the tumor-specific immune response by ELISPOT and by flow cytometry. ELISPOT was performed on fresh peripheral blood mononuclear cells pulsed for 12 h with tumor-specific and adenovirus-specific pools of peptides. Tumor specificity was assessed using survivin as an example of a pan-carcinoma antigen commonly expressed by most tumors [74].

Similar immunological data were observed with a serotype chimera 5/3 (Ad5/3D24-GM-CSF) [60] and with an integrin-targeted virus [75]. In an interesting contrast, when an unarmed oncolytic adenovirus (Ad3hTERT) was used in humans, antiviral responses were equally emphatic but less evidence of antitumor response was seen [42]. It remains to be studied how important the immunostimulatory transgene is or if the serotype also plays a role.

Interesting results have also been reported by Li et al. [76]. They present the data of a phase I dose-escalating trial with an oncolytic adenovirus expressing the heat shock protein 70 (HSP70), emphasizing some aspects of the antitumor immune-mediated response. Specifically they observed elevation of the number of CD4+ and CD8+ T cells as well as natural killer (NK) cells in the blood of the patients after the administration of the virus [76].

Similar results were also reported in another phase I trial with an oncolytic adenovirus expressing GM-CSF [54]. Similarly an important involvement of the immune system was also demonstrated with CD40L-expressing oncolytic adenoviruses [57,58].

Vaccinia Virus

Vaccinia virus (VV) is best known for its use as an efficient vaccine against smallpox, which led to the worldwide eradication of the disease. Due to this important historical role, VV has the longest and most extensive history of use in humans of any virus and a wealth of basic, preclinical, and clinical data are now available. Different strains of VV exist, of which modified vaccinia Ankara (MVA), New York VV (NYVAC), Lister, Wyeth, and Western Reserve (WR) are the most commonly used. Some of these (MVA, NYVAC, Lister) are completely or partially replication-deficient and are therefore mostly used as gene-transfer vehicles and vaccines. Wyeth and WR instead are mostly employed as replicating viruses in experimental cancer therapies due to their strong oncolytic properties. VV has an approximately 200 kb double-stranded DNA genome, which encodes about 200 genes (Table 1.1). The large, enveloped, and brick-shaped virus particles are about 300 nm in the longest dimension. VV does not require specific cell-surface receptors for transduction of target cells. Rather, it enters cells through membrane fusion or macropinocytosis. Thus, VV is able to infect a wide range of cell types. Upon entering the cytosol, VV immediately starts transcribing a defined set of early mRNAs using the transcription enzymes that the virus brings with it. Subsequently ribosomes and other components of the host cell translation machinery are recruited into defined granular structures called virus factories. Here viral gene replication and viral protein production take place and new infectious viral particles are formed. The majority of the new particles are intracellular mature virions, which have a single lipid bilayer envelope and remain inside the cell until lysis. However, a small subset of the viral particles wrap themselves in an additional lipid bilayer derived from the trans-Golgi network before egressing from the cell as extracellular enveloped virions (EEVs). The EEV particles can spread efficiently through the system of the host and are largely shielded from the immune system; therefore, they are also known as stealth particles.

VV for Treatment of Cancer

VV has several unique features that make it an attractive cancer gene therapy vector. Firstly, VV has a wide host range and is able to efficiently transduce a broad range of mammalian cell types in vitro. However, in vivo after intravenous injection in mice VV exhibits a natural tropism to tumors, which has been suggested to be due to the leaky vasculature in cancer tissue that allows the large virus particles to enter. Another advantage is that VV can hold up to 25 kb of foreign DNA, allowing the insertion of large genes and/or multiple expression cassettes. Furthermore, for efficient expression of the genes of interest, a number of strong viral promoters exist. Another advantage is that VV is a highly immunogenic agent that triggers a strong antibody and T-cell response, making VV an efficient vaccine vector. Moreover, replication-competent VV strains are highly oncolytic due to their rapid and efficient replication cycle, resulting in strong antitumor efficacy. Lastly, VV does not enter the nucleus at any time during the infection; thus, there is no danger of insertional mutagenesis. These attributes make recombinant VVs attractive agents for the treatment of many diseases, especially cancer. For cancer therapy VV has mainly been used in three ways: (i) as a replication-deficient expression vector of therapeutic genes; (ii) as a replication-competent, oncolytic virus; and (iii) as a vaccine expressing cancer epitopes and/or immune-stimulatory molecules. Many VV constructs, some of which combine the above-mentioned mechanisms of action, have been generated and evaluated preclinically and some of them have entered clinical trials with exciting results.