Adenoviral vectors for gene therapy second edition

Adenoviral Vectors for Gene Therapy Second Edition Edited by David T Curiel AMSTERDAM • BOSTON • HEIDELBERG • LONDON NEW YORK • OXFORD • PARIS • SAN DIEGO SAN FRANCISCO • SINGAPORE• SYDNEY • TOKYO Academic Press is an imprint of Elsevier Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, UK 525 B Street, Suite 1800, San Diego, CA 92101-4495, USA 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, USA The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, UK Copyright © 2016 Elsevier Inc All rights reserved First Edition 2002 This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein) No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher Details on how to seek permission, further 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products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress ISBN: 978-0-12-800276-6 For information on all Academic Press publications visit our website at https://www.elsevier.com/ Acquisition Editor: Linda Versteeg-Buschman Editorial Project Manager: Halima Williams Production Project Manager: Karen East and Kirsty Halterman Designer: Alan Studholme Typeset by TNQ Books and Journals www.tnq.co.in List of Contributors Yadvinder S Ahi HIV Drug Resistance Program, National Cancer Institute, Frederick National Laboratory for Cancer Research, Frederick, MD, USA Steven M Albelda Thoracic Oncology Research Group, Pulmonary, Allergy, and Critical Care Division, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA Yasser A Aldhamen Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI, USA Ramon Alemany IDIBELL-Institut Català d’Oncologia, L’Hospitalet de Llobregat, Barcelona, Spain Marta M Alonso Department of Medical Oncology, Clínica Universidad de Navarra, University of Navarra, Pamplona, Spain P.M Alves iBET, Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal; Instituto de Tecnologia Qmica e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal Andrea Amalfitano Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI, USA; College of Osteopathic Medicine, Michigan State University, East Lansing, MI, USA Rachael Anatol Office of Cellular, Tissue, and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD, USA C.A Anderson Merck Research Laboratories, Merck & Co., Inc., West Point, PA, USA Svetlana Atasheva Lowance Center for Human Immunology, Departments of Pediatrics and Medicine, Emory University, Atlanta, GA, USA Michael A Barry Division of Infectious Diseases, Department of Internal Medicine, Mayo Clinic, Rochester, MN, USA; Department of Immunology, Mayo Clinic, Rochester, MN, USA; Department of Molecular Medicine, Mayo Clinic, Rochester, MN, USA Raj K Batra UCLA School of Medicine, Division of Pulmonary and Critical Care Medicine, GLA-VAHCS, Los Angeles, CA, USA; Jonsson Comprehensive Cancer Center, UCLA, Los Angeles, CA, USA xvi List of Contributors A.J Bett Merck Research Laboratories, Merck & Co., Inc., West Point, PA, USA A Bout Crucell NV, Leiden, The Netherlands K Brouwer Crucell NV, Leiden, The Netherlands Nicola Brunetti-Pierri Telethon Institute of Genetics and Medicine, Pozzuoli, Italy; Department of Translational Medicine, Federico II University, Naples, Italy Andrew P Byrnes Division of Cellular and Gene Therapies, FDA Center for Biologics Evaluation and Research, Silver Spring, MD, USA Shyambabu Chaurasiya Department of Oncology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada L Chen Merck Research Laboratories, Merck & Co., Inc., West Point, PA, USA A.S Coroadinha iBET, Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal; Instituto de Tecnologia Qmica e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal Igor P Dmitriev Department of Radiation Oncology, School of Medicine, Washington University, St Louis, MO, USA Hildegund C.J Ertl Wistar Institute, Philadelphia, PA, USA P Fernandes iBET, Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal; Instituto de Tecnologia Qmica e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal; Autolus, London, UK Juan Fueyo Department of Neuro-Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA; Department of Neurosurgery, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA S.M Galloway Merck Research Laboratories, Merck & Co., Inc., West Point, PA, USA Thomas A Gardner Department of Urology, Indiana University Medical Center, Indianapolis, IN, USA; Department of Microbiology and Immunology, Indiana University Medical Center, Indianapolis, IN, USA Candelaria Gomez-Manzano Department of Neuro-Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA; Department of Genetics, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA Urs F Greber Institute of Molecular Life Sciences, University of Zurich, Zurich, Switzerland Diana Guimet Department of Molecular Genetics and Microbiology, School of Medicine, Stony Brook University, Stony Brook, NY, USA List of Contributors xvii Michael Havert Office of Cellular, Tissue, and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD, USA Patrick Hearing Department of Molecular Genetics and Microbiology, School of Medicine, Stony Brook University, Stony Brook, NY, USA Masahisa Hemmi Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan R.B Hill Merck Research Laboratories, Merck & Co., Inc., West Point, PA, USA Mary M Hitt Department of Oncology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada Ying Huang Office of Cellular, Tissue, and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD, USA Ilan Irony Office of Cellular, Tissue, and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD, USA Hong Jiang Department of Neuro-Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA Sergey A Kaliberov Department of Radiation Oncology, School of Medicine, Washington University, St Louis, MO, USA Chinghai H Kao Department of Urology, Indiana University Medical Center, Indianapolis, IN, USA; Department of Microbiology and Immunology, Indiana University Medical Center, Indianapolis, IN, USA Dayananda Kasala Department of Bioengineering, College of Engineering, Hanyang University, Seongdong-gu, Seoul, Republic of Korea D Kaslow Merck Research Laboratories, Merck & Co., Inc., West Point, PA, USA Benjamin B Kasten Department of Radiology, The University of Alabama at Birmingham, Birmingham, AL, USA Johanna K Kaufmann German Cancer Research Center (DKFZ), Heidelberg, Germany Jay K Kolls Richard King Mellon Foundation Institute for Pediatric Research, Children’s Hospital of Pittsburgh of UPMC, Pittsburgh, PA, USA; Department of Pediatrics, School of Medicine, University of Pittsburgh, Pittsburgh, PA, USA Johanna P Laakkonen Department of Biotechnology and Molecular Medicine, A.I Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland xviii List of Contributors R Lardenoije Crucell NV, Leiden, The Netherlands J Lebron Merck Research Laboratories, Merck & Co., Inc., West Point, PA, USA B.J Ledwith Merck Research Laboratories, Merck & Co., Inc., West Point, PA, USA J Lewis Merck Research Laboratories, Merck & Co., Inc., West Point, PA, USA Erik Lubberts Department of Immunology, Erasmus MC, University Medical Center, Rotterdam, The Netherlands; Department of Rheumatology, Erasmus MC, University Medical Center, Rotterdam, The Netherlands Stefania Luisoni Institute of Molecular Life Sciences, University of Zurich, Zurich, Switzerland S.V Machotka Merck Research Laboratories, Merck & Co., Inc., West Point, PA, USA S Manam Merck Research Laboratories, Merck & Co., Inc., West Point, PA, USA D Martinez Merck Research Laboratories, Merck & Co., Inc., West Point, PA, USA Suresh K Mittal Department of Comparative Pathobiology, College of Veterinary Medicine and Purdue University Center for Cancer Research, Purdue University, West Lafayette, IN, USA Hiroyuki Mizuguchi Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan Edmund Moon Thoracic Oncology Research Group, Pulmonary, Allergy, and Critical Care Division, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA Stephen J Murphy Molecular Medicine Program, Mayo Clinic, Rochester, MN, USA Dirk M Nettelbeck German Cancer Research Center (DKFZ), Heidelberg, Germany Philip Ng Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA W.W Nichols Merck Research Laboratories, Merck & Co., Inc., West Point, PA, USA Raymond John Pickles Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA Sudhanshu P Raikwar Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, University of Missouri and Harry S Truman Veterans’ Memorial Hospital, Columbia, MO, USA List of Contributors xix Paul N Reynolds Department of Thoracic Medicine and Lung Research Laboratory, Royal Adelaide Hospital, Adelaide Jillian R Richter Department of Radiology, The University of Alabama at Birmingham, Birmingham, AL, USA Yisel Rivera-Molina Department of Neuro-Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA Qian Ruan PaxVax Inc., San Diego, CA, USA C Russo Merck Research Laboratories, Merck & Co., Inc., West Point, PA, USA Carl Scandella Carl Scandella Consulting, Bellevue, WA, USA Paul Shabram PaxVax Inc., San Diego, CA, USA Anurag Sharma Department of Pediatrics, Weill Cornell Medical College, New York, NY, USA Sherven Sharma UCLA/Wadsworth Pulmonary Immunology Laboratory, Division of Pulmonary and Critical Care Medicine, GLA-VAHCS, Los Angeles, CA, USA Dmitry M Shayakhmetov Lowance Center for Human Immunology, Departments of Pediatrics and Medicine, Emory University, Atlanta, GA, USA A.C Silva iBET, Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal; Instituto de Tecnologia Qmica e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal Phoebe L Stewart Department of Pharmacology and Cleveland Center for Membrane and Structural Biology, Case Western Reserve University, Cleveland, OH, USA Hideyo Ugai Cancer Biology Division, Department of Radiation Oncology, School of Medicine, Washington University, St Louis, MO, USA D Valerio Crucell NV, Leiden, The Netherlands M van der Kaaden Crucell NV, Leiden, The Netherlands Gary Vellekamp Vellekamp Consulting LLC, Montclair, NJ, USA Sai V Vemula Laboratory of Molecular Virology, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD, USA Richard G Vile Molecular Medicine Program, Mayo Clinic, Rochester, MN, USA R Vogels Crucell NV, Leiden, The Netherlands xx List of Contributors Stefan Worgall Department of Pediatrics, Weill Cornell Medical College, New York, NY, USA; Department of Genetic Medicine, Weill Cornell Medical College, New York, NY, USA Lily Wu Jonsson Comprehensive Cancer Center, UCLA, Los Angeles, CA, USA; Department of Urology, UCLA School of Medicine, Los Angeles, CA, USA; Department of Pediatrics, UCLA School of Medicine, Los Angeles, CA, USA Enric Xipell Department of Medical Oncology, Clínica Universidad de Navarra, University of Navarra, Pamplona, Spain Seppo Ylä-Herttuala Department of Biotechnology and Molecular Medicine, A.I Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland; Department of Medicine, University of Eastern Finland, Kuopio, Finland; Gene Therapy Unit, Kuopio University Hospital, Kuopio, Finland Chae-Ok Yun Department of Bioengineering, College of Engineering, Hanyang University, Seongdong-gu, Seoul, Republic of Korea Kurt R Zinn Department of Radiology, The University of Alabama at Birmingham, Birmingham, AL, USA D Zuidgeest Crucell NV, Leiden, The Netherlands Adenovirus Structure Phoebe L Stewart Department of Pharmacology and Cleveland Center for Membrane and Structural Biology, Case Western Reserve University, Cleveland, OH, USA 1.  Historical Perspective on Adenovirus Structure The structure of the adenovirus virion is quite complex and our understanding of it has been evolving from before 1965 Early negative stain electron micrographs of adenovirus revealed an icosahedral capsid with 252 capsomers and long fibers protruding from the vertices.1 Later these capsomers were identified as 240 hexons and 12 pentons, with the pentons at the fivefold vertices of the capsid The pentons each have five neighboring capsomers and the hexons each have six neighboring capsomers As the adenoviral molecular components were identified and their stoichoimetries characterized, it became apparent that the hexons and pentons were different proteins The hexons are trimeric proteins and the pentons are formed by two proteins, a pentameric penton base and a trimeric fiber.2 Subsequently, X-ray crystallography provided atomic resolution structures of hexon,3 penton base,4 fiber,5,6 and adenovirus protease,7 which is involved in virion maturation In addition to the three major protein components of the capsid (hexon, penton base, and fiber), there are four minor capsid proteins (proteins IIIa, VI, VIII, IX).8,9 The minor proteins are also referred to as cement proteins as they serve to stabilize the capsid They also play important roles in the assembly, disassembly, and cell entry of the virus Atomic resolution structures have not yet been determined for the minor proteins isolated from the adenovirus capsid However, cryo-electron microscopy (cryoEM) has provided moderate structural information on the density of the minor proteins in the context of the virion.10–13 In 2010, atomic resolution structures of adenovirus were determined by cryoEM and X-ray crystallography.14,15 Despite these two atomic, or near atomic, resolution (3.5–3.6 Å) structures, controversies remained regarding the structure and assignment of the minor capsid proteins In 2014, a refined crystal structure of adenovirus at 3.8 Å resolution revised the minor capsid protein structures and locations.16 The adenoviral genome is relatively large, with ∼30–40 kb.8 It is notable in that large deletions and insertions can be tolerated, a feature that contributes to the enduring popularity of adenovirus as a gene delivery vector.17 Within the core of the virion there are five proteins associated with the double-stranded DNA genome (proteins V, VII, mu, IVa2, and terminal binding protein).9 The structure of the genome and how it is packaged with its associated proteins in the core of the virion is not well understood Early negative stain EM and ion etching studies suggested that the core is organized as 12 large spherical nucleoprotein assemblies, termed adenosomes.18,19 However, cryoEM and crystallographic structures of adenovirus show that the core does not follow the strict icosahedral symmetry of the capsid.14–16 Adenoviral Vectors for Gene Therapy http://dx.doi.org/10.1016/B978-0-12-800276-6.00001-2 Copyright © 2016 Elsevier Inc All rights reserved Adenoviral Vectors for Gene Therapy Adenovirus was one of the first samples imaged during the development of the cryoEM technique20 and was among the first set of viruses to have its structure determined by the cryoEM single particle reconstruction method.21 Since then cryoEM structures have been determined for multiple types of adenovirus and adenovirus in complex with various host factors.10–12,14,22–29 Docking of crystal structures of capsid proteins into the cryoEM density and difference imaging have been useful approaches for dissecting the complex nature of the capsid An early example of difference imaging was applied in two dimensions to scanning transmission electron microscopy (STEM) images of the group-of-nine hexons and this work helped to elucidate the position of protein IX within the icosahedral facet.30 Difference imaging in three dimensions led to an early tentative assignment for the positions of the minor capsid proteins within the capsid based on copy number and approximate mass.13 As higher resolution cryoEM structures were determined, some of these initial assignments were revised.10–12 Visualization of α-helices was achieved with a 6 Å resolution cryoEM structure.12 This structure facilitated more accurate docking of hexon and penton base crystal structures and produced a clearer difference map and more detailed density for the minor capsid proteins Secondary structure prediction for the minor capsid proteins was used to tentatively assign density regions to minor capsid proteins Determination of an atomic resolution (3.6 Å) structure by cryoEM was facilitated by the use of a high-end FEI Titan Krios electron microscope.14 Micrographs for this dataset were collected on film and scanned for digital image processing The final dataset included 31,815 individual particle images The resolution was estimated by reference-based Fourier shell correlation coefficient and supported by observation of both α-helical and β-strand density Density was also observed for some of the side chains, particularly bulky amino acids The assignments for the minor capsid protein locations were assumed to be the same as interpreted from the 6 Å resolution cryoEM structure.12 Atomic models were produced for minor capsid proteins IIIa, VIII, and IX from the atomic resolution cryoEM density map using bulky amino acids as landmarks.14 Attempts to crystallize intact adenovirus began in 1999 and proceeded for more than 10 years before the first atomic resolution crystal structure was published.15,31 Several factors hampered early crystallization efforts, including the long protruding fiber, the instability of virions at certain pH values, the tendency of adenovirus particles to aggregate, and relatively low yields from standard virus preparations Use of a vector based on human adenovirus type (HAdV5), but with the short fiber from type 35 (Ad5.F35, also called Ad35F), helped to solve some of the production and crystallization difficulties This vector was also used for several moderate resolution cryoEM structural studies.11,12 Collection of diffraction data for atomic resolution structure determination spanned several years Even though crystals were flash-cooled in liquid nitrogen, they were still highly radiation sensitive and only 2–5% of the crystals diffracted to high resolution Diffraction data from nearly 900 crystals were collected but only a small subset of these data was used to generate the dataset The best crystals diffracted well to 4.5 Å resolution and weakly to 3.5 Å at synchrotron sources The initial phase information was derived from a pseudo-atomic capsid of adenovirus generated from fitting the crystallographic structures of hexon and penton base into a cryoEM structure of Ad5.F35 at 9 Å resolution.11 In 2010, partial atomic models were built for some of the minor capsid proteins.15 804 Adenoviral Vectors for Gene Therapy must be in compliance with the NIH Guidelines for research involving recombinant nucleic acids and register their clinical protocol with the NIH/Office of Biotechnology Activities.7,8 A review of oversight responsibilities for gene therapy products at the NIH and institutional level has been recently published and is beyond the scope of this chapter.9 This chapter provides OCTGT’s perspective on the regulation of adenovirus-based vectors The first section provides an overview of how these vectors are designed and implications for regulatory oversight Design considerations have a significant impact on how the product will be manufactured, tested, and used in a clinical study The remaining three sections describe more broadly how the scientific and medical disciplines in OCTGT review submissions for adenoviral vector-based products This chapter describes general manufacturing requirements for products under IND, preclinical supporting information needed for initiating clinical studies, and considerations for clinical trial design and for investigators involved in treating patients with adenovirus-based investigational products All of these requirements and recommendations have been published previously as FDA guidance or regulation, but here we present this information specifically in the context of developing adenovirus-based products 2.  Regulatory Considerations in the Design of Adenoviral Vector-Based Therapies The first step in the development of an adenoviral vector-based therapy is the molecular design of a product for further study In general, an understanding of the molecular biology of the virus, the transgene cassette, and its associated genetic elements influences the design and construction of adenovirus-vectored products Researchers have found that the virus can accommodate a number of design configurations from which clinical grade production should be readily achievable Ultimately, the design of these products is determined by the specific requirements of the user The FDA recommends that construction and testing of these products should be done in consultation with the Agency before IND submission in what is called a preIND interaction.10 For many investigators who are developing adenoviral vector-based products, the selection of a transgene(s) is the first and most significant design consideration Adenoviruses are very efficient at gene transfer and are capable of high-level expression of transgenes; however, the viral constructs are known to be immunogenic and usually have limited duration of gene expression in vivo Therefore these vectors are, for the most part, best suited for applications in which the transgenes require only transient gene expression Such applications would include expression of vaccine antigens, genes related to immune modulation, or cancer cell-specific cytotoxicity All of these applications are being explored (some in combination) for cancer treatments and, after demonstrating a level of tumor response, may accelerate quickly through clinical development.11 The size of the transgene cassette must be taken into consideration because of size restrictions for the virus Insertion capacity of the virus is limited by the amount of genomic DNA that can be efficiently packaged into the capsid.12,13 Adenovirus-based Adenoviral Vector-Based Therapies 805 vectors usually have at least one deletion to make space for the transgene cassette The most common deletion is in the early region (E1 region) of the virus In addition to providing space for the transgene, an E1 deletion removes the principal activating proteins of the virus, thus rendering the adenovirus replication deficient For so-called first generation constructs, the deletion of E1 significantly attenuates the virus and eliminates its ability to replicate This feature restricts viral replication to cells capable of trans-complementing the deletion during manufacture (e.g., HEK293 or PER.c6 cells) It also simplifies safety studies needed to establish a basis for conducting a clinical trial In most circumstances, the toxicity of a replication defective adenovirus can be evaluated in mouse model systems, even though the replication of human adenoviruses is greatly restricted in mice Because of their transient expression, E1-deleted vector designs also limit the duration of shedding studies and long-term follow-up data collection in the clinical trial Commercially available reagents may be used to generate these products, which also simplifies and standardizes the construction, and in turn enables faster initiation of clinical studies Incorporating additional deletions in second generation and high capacity (or gutless) vectors has allowed greater transgene sizes These deletions eliminate expression of viral gene products that may be immunogenic or cytotoxic, which in turn may increase the duration of transgene expression For example, high capacity vectors in which all the viral open reading frames have been removed are much less immunogenic and provide longer transgene expression times than first generation constructs (reviewed by Ventiri and Ng).14 The elimination of additional viral genes required for replication, such as E2 or E4, will require a more specialized production cell line In this situation, manufacturers must develop cells that are able to trans-complement the additional deletions, but many cell lines have already been established for this purpose (reviewed by Kovesdi and Hedley).15 These cells that complement multiple deletions also have the advantage of significantly reducing the likelihood of replication-competent adenovirus (RCA) contamination in the product by the fact that additional homologous recombination events are required to generate an RCA Another significant design consideration is the selection of transcriptional and targeting control elements for the transgene For most investigators, the selection of transcriptional control elements (i.e., promoter) is based on commercially available reagents Strong promoters that are active in a variety of cell types are usually assumed to allow for the greatest amount of protein production in vivo This is a reasonable starting point, although expression levels may vary from cell line to cell line and may not necessarily correlate with activity in vivo Promoter selection is a particularly important manufacturing consideration when expression of the transgene can interfere with virus replication Transgene interference may decrease yields and impose a negative selection pressure against expression, leading to genetic instability.16,17 In these situations, inducible promoters for transgene expression or promoters that are active in restricted cell types may be required for production Cell-restricted promoters may also improve the safety profile of a product For example, the safety profile of a virus expressing a cytotoxic gene for cancer gene therapy may be improved with the addition of a tumor-specific promoter In this case, establishing the safety profile for clinical use would include a demonstration of promoter selectivity 806 Adenoviral Vectors for Gene Therapy The designers of a new adenovirus-based product may also choose to incorporate a variety of capsid serotypes or capsid modifications that can direct cellular targeting of their product Many designers choose a serotype adenovirus capsid based on the convenience of commercially available reagents A large body of knowledge has been generated regarding the pharmacokinetics of serotype adenovirus after administration in humans via a number of routes and at a number of dose levels However, some investigators are exploring pseudotyping, retargeting, or use of novel serotypes as a way to improve target cell uptake These approaches are being rationally designed as we understand more about the biology of adenoviruses and their interaction with the human host Specific manufacturing concerns regarding these targeted adenoviruses include the efficiency of assembly of an altered capsid and potential selection against the intended modification during production and the stability of the modified virus capsid during manufacturing and storage, all of which will be taken into consideration during the development process for these products For investigators who are developing adenovirus vector-based products that are replication selective, the mutation or mechanism by which the virus is made to be replication selective is a significant design consideration that affects manufacturing, preclinical safety testing, and clinical trial design Most of these vectors are being developed as oncolytic products to allow limited or selective replication in tumor cells, but there has also been interest in developing replication-competent vectored vaccines.18–20 For replication-competent oncolytic products, tumor cell-specific replication is desirable Complete infection of all tumor cells may not be achievable using the standard serotype adenovirus vector Low levels of coxsackievirus and adenovirus receptor expression on tumor cells and preexisting serotype immunity limit transduction Low viral transduction limits the ability of the virus to replicate and spread in tumor cells.21–23 Advances are being made in the development of vectors that target and replicate in tumors Complete infection of all tumor cells may not be required because host immune responses to virus-infected cells also generate anticancer effects.24–26 The oncolytic methods currently being explored for adenovirus-based therapies include transcriptional targeting of E1 gene expression and removal of tumor suppressor binding domains in the E1 genes (reviewed in Kruyt and Curiel).27 In addition, replication-competent viruses from different serotypes with low seroprevalence are being investigated because they are unimpeded by preexisting immunity, as well as constructs selected for the ability to lyse cancer cell lines in vitro.28,29 The degree to which these viruses are replication selective is an important consideration to be established prior to use in human clinical studies.30 In addition, steps to assess shedding and the possible transmission from treated to untreated individuals may be required Recommendations regarding shedding studies have been published in FDA guidance (FDA, Draft Guidance for Industry: Design and Analysis of Shedding Studies for Virus or Bacteria-Based Gene Therapy and Oncolytic Products) and discussed publicly in a recent Cellular, Tissue, and Gene Therapies Advisory Committee (CTGTAC) meeting.31 Finally, another significant design consideration is the compatibility between the adenovirus vector and the delivery devices used to administer the product For some adenoviral products, delivery to a specific organ or tissue may require a specialized Adenoviral Vector-Based Therapies 807 delivery device Medical devices are cleared by the FDA for their intended use, which may include placement in a specific tissue, accessing a particular body compartment, or injecting/infusing an approved drug Adenovirus vectors have known stability problems when they come into contact with certain materials, including some materials that are commonly used in catheters.32–34 Contact surfaces or leachables from these surfaces may bind or inactivate the virus in as little as 10 min and may result in suboptimal delivery of the product Therefore delivery devices should be carefully considered and assessed for their biocompatibility with adenoviruses before use in clinical studies 3.  Chemistry, Manufacturing, and Control Requirements A summary of regulatory requirements and considerations for specific manufacturing steps for adenoviral vector-based products is presented in the following section This section describes measures taken to qualify cell substrates, viral banks, purification strategies, and reagents and raw materials used in production More specific information can be found in relevant guidance documents published by the Agency and referenced in this section 3.1  Establishing a Cell Bank Cells used for production are a critical reagent in the manufacturing of clinical grade adenoviral vector-based products The properties of the production cells and the potential adventitious contaminants harbored by them influence the quality of the viral product As with any biological product, cells used for production should be characterized with respect to history, in vitro growth characteristics, and presence of detectable microbial agents (21 CFR 610.18 (c)) There are limitations when selecting a production cell line based on the design requirements of the adenoviral vector For replication-defective viruses in which coding sequences needed for viral replication have been removed, viral proteins must be provided in trans by the production cell line Cells suitable for the generation of E1-deleted replication-defective adenovirus vectors contain and express the E1 region of the viral genome The two most common are cell lines derived from embryonic human kidney fibroblasts (HEK 293) or retinoblasts (PER.C6) In HEK 293 cells, the integrated E1 sequences contain enough flanking adenovirus sequence such that homologous recombination with the vector will occur, resulting in contamination of the product with E1-containing RCA.35 PER.C6 cells were engineered to contain minimal regions of homology to prevent recombination and reduce the occurrence of RCA.36 PER.C6 and other similar cells may be advantageous; however, there have also been reports of a rare RCA-like material known as helper-dependent E1-positive virus particle during production with PER.C6 cells.37,38 For more complicated vector designs with additional essential viral genes missing, manufacturers may have to develop custom cells that express additional viral genes15 and/or qualify additional reagents (i.e., a helper virus bank) to provide viral genes necessary for replication For production of adenovirus gene therapy products in which no complementation 808 Adenoviral Vectors for Gene Therapy is required (e.g., conditionally replicating vectors having modified E1 regions), a cell line should be chosen that does not contain adenovirus sequences in order to prevent homologous recombination Cell lines used for adenovirus vector production can be easily frozen and a cell banking system can be established to ensure consistency and control of production cells With an appropriate cell bank system, production with the same stock of cells can occur for the lifetime of the product The Agency has published guidance documents with recommendations for the qualification of cell banks.39,40 The recommendations include a description of the history of the cell line and the banking system used for storage This history should include a reference to the cell source and where the cells were first obtained For the derivation of new cell lines from primary human tissue the history should include whether the requirements for screening and testing of human donors were met (21 CFR 1271) Characterization tests are performed when the bank is developed, and include tests for phenotype, genotype, and cellular isoenzyme expression to establish identity In addition to consistency and control, a cell bank is tested to ensure safety of the production cell lines The Agency recommends that testing be performed to demonstrate that a master cell bank (MCB) is free from Mycoplasma, endotoxin, bacteria, fungi, and adventitious agents (through in vitro and in vivo assays) Depending on its species of origin, the MCB should be tested for species-specific pathogens For human adenoviruses made in human cells, this will include CMV, HIV1/2, HTLV1/2, EBV, B19, HBV, and HCV The Agency recommends that sponsors summarize the analytical testing in the regulatory file and establish acceptance criteria for these tests of the MCB Many cell lines used in the production of adenoviral gene therapies are known to be tumorigenic in animal models and therefore additional testing to verify a tumorigenic phenotype is not usually required for qualification of a cell bank.40 In these cases, the manufacturers will demonstrate in final product testing that the product is free of the transforming agent (e.g., host cell DNA) More on host cell DNA is presented later in this chapter The MCB is usually the first tier of a two-tier cell bank system; the second is the working cell bank (WCB) Usually, the purpose of the WCB is to extend the supply of the qualified MCB and it is created by expansion of one or more vials of the MCB The amount of information for characterizing a WCB is generally less extensive because it is generated from the fully qualified MCB, usually at the same facility, in a similar manner, and using the same, or similarly qualified, raw materials and ancillary agents Therefore, WCB qualification is typically limited to tests for sterility, Mycoplasma, adventitious agents (by the in vitro assay) and identity 3.2  Establishing a Virus Bank The generation of a master viral bank (MVB, sometimes referred to as the master virus seed) is the next stage in development after the adenovirus product has been designed and a cell bank has been established A banking system is needed to ensure the control and consistency of a product and, like the MCB, the MVB is expected to last for the lifetime of a product The MVB requires proper storage, identification, and appropriate record keeping (21 CFR 610.18) OCTGT has published guidance documents that Adenoviral Vector-Based Therapies 809 review recommendations for the qualification of an MVB under IND These recommendations include an assessment of the history of the virus stock and a description of the banking system used for storage.39 The MVB is an important control point that allows thorough characterization and qualification of virus stocks used for production Characterization and safety tests for qualification of the MVB involve some of the same testing recommended for the MCB and final product For most manufacturers, the history of the virus can be fully documented and viral stocks have only been in contact with qualified cells However, in situations where the initial virus construct was made using nonqualified cells or under poorly defined laboratory conditions, it would be appropriate to clone the virus by plaque purification or limiting dilution to remove any potential contaminants before being expanded into an MVB Because of the redundancy in testing for the MVB and final clinical product, investigators for most first-in-human trials sometimes combine testing to qualify a new master virus bank and the release of a final product for clinical use at the same time In these situations, after the first production run is completed, a fraction of the final vialed product is used for clinical release and the remaining vials are stored as the MVB Combining tests for the MVB and clinical lot has a cost advantage As an important control point in manufacturing, the MVB is used for full sequence analysis and RCA testing All adenovirus vectors for gene therapy should be sequenced prior to being used in clinical trials, based on recommendations in the November 2000 Advisory Committee and published in guidance.39,41 Most vectors have been derived from serotype and can be compared to the complete sequence in GenBank such as the adenovirus reference material (ARM) sequence (Accession Number AY339865) Some minor changes may be detected based on passage history and plasmids used during construction The virus has an error-checking polymerase and a relatively low mutation rate As such, evaluation of sequence stability of the transgene in the MVB and during production is not usually recommended for early-phase trials However, some vector constructs have been reported to be unstable; examples include those that have repetitive sequences, which might allow for recombination, or those with transgene deleterious to the virus.42 Testing the MVB for the presence of sequence changes in these situations may be recommended at early stages of development Another factor that may have a significant impact on the qualification of the virus bank is the presence of RCA RCA arises by homologous recombination during replication of E1-deleted constructs in a complementing cell line Recombination occurs between the left end of the vector and the adenovirus sequence in the cell, resulting in the acquisition of E1 by the vector The effect of RCA on production and control of the product can be significant If RCA-contaminated stocks are expanded, RCA levels can increase at the expense of the intended product after multiple passages.35 The presence of RCA can interfere with in vitro adventitious agents testing performed for lot release RCA-contaminated products in animals have been demonstrated to have reduced transgene expression and increased inflammatory responses.43 High levels of RCA in the final product are also a potential safety consideration that may necessitate additional toxicology studies or additional precautions taken in the design of the clinical trial In general RCA levels can be reduced by limiting the amount of homologous sequence in the vector and producer cell line.36,44–47 The Agency has recommended 810 Adenoviral Vectors for Gene Therapy that products contain no more than one RCA in 3 × 1010 vp.39 This number was chosen based on an estimation of the level of purity that typical manufacturers were able to achieve If a manufacturer is not able to meet this recommendation, a new production system with a lower chance of producing RCA should be considered 3.3  Production and Purification Strategies Most gene therapy applications require relatively high doses of a pure product High titers are usually needed to target sufficient cells for gene transfer, and impurities may limit transgene expression by inducing immune response or inflammation Adenovirus products are attractive candidate vector systems because of the ability to manufacture a high titer and high purity product High titers are possible because the virus is efficient at creating progeny One infected cell can result in 10,000 new virus particles and yields of about 1 × 1011 vp per milliliter of cell culture are commonly achieved.48 The typical purification process involves harvest of infected cells and a lysis step to release cell-associated virus This is followed by clarification of cellular debris, digestion of nonencapsidated DNA, separation step(s), and buffer exchange for final formulation The stable, nonenveloped nature of the capsid allows for robust enrichment methods, and the typical approaches include density gradient ultracentrifugation or anion exchange chromatography Both allow purification of virus particles from cellular components and adventitious agents Density gradient ultracentrifugation has the advantage of removing empty capsids based on the lower density of empty capsids, but this technique cannot be performed as a closed system and is difficult to scale-up In the past, many investigators started with purification by ultracentrifugation in early-phase studies, and then switched to chromatographic separation later in development The Agency advises IND sponsors to discuss major manufacturing changes, including scale-up, before implementation Prior to initiation of Phase studies the Agency will consider the potential impact manufacturing changes have on the safety profile of the product After Phase studies have been initiated, manufacturing changes are reviewed for potential impacts on the efficacy and safety of the product Product-related impurities include noninfectious particles, aggregates, free viral capsid proteins, and vector nucleic acid Process-related impurities include host cell protein, host cell DNA, residual culture media components, residual solvents, additives, antifoaming agents, or enzymes used in production The Agency recommends that manufacturers develop quantitative lot release assays for purity prior to initiating clinical studies (e.g., relative amount of infectious particles expressed as a ratio compared to viral particles).39 Other tests should be developed during early-phase studies and be in place by the start of Phase Probably the most significant product-related impurity at the production and purification stage is DNA The greatest by-product of replication is viral nucleic acid, and both viral and cellular DNA may be present as impurities in the final product Steps to remove DNA are needed for many reasons During manufacturing the presence of DNA can interfere with chromatography, and thus digestion of DNA can reduce fouling of columns DNA may also interfere with quantitation methods for calculating virus particle levels, and the presence of low Adenoviral Vector-Based Therapies 811 levels of DNA has also been associated with vector aggregation.49 For all these reasons, removal of DNA is an important process step during purification The presence of DNA from tumorigenic cell lines in the final product is also a safety concern For gene therapy vectors, it has been recommended that products contain not greater than 10 ng host cell DNA per dose.50 This is consistent with general guidelines for vaccines and other biologicals prepared in continuous cell lines.51 A size limit for host cell DNA is also recommended since it is likely that reduction of DNA fragment size reduces the risk from DNA, as the smaller the DNA fragments are, the lower the probability that intact oncogenes and other functional sequences would be present DNA fragments smaller than 200 bp will give substantial safety margins for products that meet the 10 ng per dose limit.52 Therefore host cell DNA is recommended to be between 100 and 200 bp Process-related impurities also include chemicals such as plasticizers and plastic additives that may leach from contact surfaces into the adenovirus product during production or storage Leachables may pose safety concerns or interfere with product performance, although they are usually present only at very low levels Another significant concern is that leachables can also interfere with analytical test methods Some contaminants have been identified that absorb strongly at 260 nm and can interfere with viral quantification methods, leading to inaccurate measurements of viral dose.53 Careful study of leachables is usually performed during late stages of development Prior to this stage, manufacturers should carefully qualify and control the plastic material used in production that comes into contact with the viral product 3.4  Qualification of Raw Materials and Reagents Used in Production Various raw materials are used in the production of adenoviruses Use of these materials may affect the quality, safety, and efficacy of the final product The quality of the culture media and raw materials should be documented in the regulatory file with respect to identity, purity, sterility, biologic activity, and absence of adventitious agents Typically trypsin and serum are the only animal-derived components commonly used in adenovirus gene therapy production For all animal-derived components, the manufacturer should provide information on the source organism, supplier/vendor, country of origin, infectious agent testing, and stage of manufacture in which the component is used This information is collected to mitigate potential concerns regarding transmission of bovine spongiform encephalopathy and transmissible spongiform encephalopathy and other infectious agents Trypsin and serum should be tested by vendors to ensure the absence of bovine viruses and porcine parvoviruses If these are not documented in the certificates of analysis, these tests will be required to qualify the MCB and/or MVB Animal-derived materials should be compliant with the requirements for the ingredients of animal origin used for the production of biologics (9 CFR 113.53) Serum and trypsin may also harbor infectious agents not detected by qualification tests Because these reagents may potentially introduce contaminants into the production process that may not be removed during purification and may not be detected during final product testing, 812 Adenoviral Vectors for Gene Therapy nonanimal-derived, alternative synthetic, or gamma-irradiated materials may be advantageous If proprietary serum-free medium is used, the sponsor must provide a letter of cross-reference for the material 4.  Manufacturing Control 4.1  Product Testing Testing on the adenoviral vector-based product should include the routine safety testing outlined in the general biological standards (21 CFR 610) and applicable guidance documents.39 These include tests for sterility, Mycoplasma, endotoxin, and adventitious agents (including RCA) In addition to these assays, additional customized assays will be required over the course of clinical development Common virological assays are used throughout development for quantification of the virus and these assays play an important role in the analytical characterization and release testing of adenoviral vector-based products Testing the final product relies on measures of both physical and biological properties of the viral particles Viral particles are assessed through detection of particle components such as DNA or capsid proteins The most common method for calculating virus particles is an absorbance measurement of lysed particles at OD 260.54,55 The data are then converted to a particle number based on the extinction coefficient for a 36 kb adenovirus, or based on an extinction coefficient specifically determined for the genome size of the adenovirus product The OD 260 method is commonly used to determine dose for clinical-grade preparations of adenovirus To facilitate the standardization of this method (as well as other methods noted below), an ARM was established in 2002 This material serves as a quantitative and qualitative reference of serotype adenovirus, and is commercially available from ATCC to be used by manufacturers in their qualification program.56 Another common assay for calculating adenovirus virus particles is anion exchange high-performance liquid chromatography (AEX-HPLC) AEX-HPLC is useful for analyzing both crude and pure samples.57,58 Virus particle quantification by AEX-HPLC relies on an absorbance measurement after elution from a positively charged matrix Pure adenovirus can be eluted at a nearly symmetrical peak and can be distinguished from residual DNA, hexon protein, and cellular debris The viral capsids remain intact during chromatography, and empty and full capsids are not separated Virus particle concentration is derived from a calibration curve that reports the known virus particle concentrations against the corresponding absorbance area of the viral peak using a reference standard This approach is more robust than the OD260 method because it can be used to measure virus particle concentration in samples of nonpurified cell lysate.57 This method can also measure the overall purity of a preparation by integrating all of the absorbance peaks and determining the percentage of the viral peak Physical particle counts alone are not sufficient to measure the activity of a virus preparation because they are unable to distinguish functional from nonfunctional particles In order to assess the infectivity of a preparation (or number of infectious particles), cell culture infectivity assays are performed These types of assays report the infectivity of the virus by assessing the ability of the virus particles to be taken Adenoviral Vector-Based Therapies 813 up by cells and execute one or more steps in the virus life cycle A large number of assays can be used to measure infectivity The most common infectivity assays are based on cell killing or CPE of the infected cell and these include plaque assays and limiting dilution assays The theory behind the limiting dilution assay is that, at limiting dilution, each CPE-positive well will represent a single infectious particle, distributed according to the Poisson distribution This method is more standardizable than the plaque assay, but as with any biological assay the results are highly variable Under typical conditions, only a small percentage of virions actually infect the cell monolayer, and therefore the results are often corrected for the theoretical diffusion rate of the virus This calculation is often referred to as the NAS (normalized and adjusted standard) method.59 NAS titers can correct for differences in incubation time and incubation volume that may otherwise affect the results of this assay A number of other infectious titer assays have been developed, including focus-forming assays and polymerase chain reaction (PCR)- or flow cytometry-based assays (reviewed in Dormond et al.).60 All of these are acceptable approaches for measuring infectious virus A well-characterized reference standard is an important control to include in these assays to understand assay variation The ARM has been useful in qualifying a manufacturer’s in-house reference standard and standardizing infectious titer results between manufacturers.56 The Agency has developed recommendations to ensure that preparations of adenovirus have a minimum activity in terms of infectivity per physical particle.39 This ratio was designed to be a measure of the specific activity of a preparation and is expressed as a ratio of particles to infectious units The current recommendation is that the particle to infectious unit ratio be no greater than 30:1 The previous recommendation of 100:1 plaque-forming units was developed shortly after the first adenovirus gene therapy trials were initiated, and remained in effect until 2000.50 After review of data received in response to the March 6, 2000, Gene Therapy letter, it was apparent that almost all vector lots have a ratio of less than 30:1 particle to infectious units.61 However this ratio can be highly variable, depending on the assay used to measure infectivity Using the NAS titer can help overcome some of the variability of different infectivity assays However, it may not be possible to manufacture some viral constructs, including those from different serotypes, at this level of specific activity This recommendation was developed based on experience with the serotype viruses and is applied generally as a target level for early development Ultimately, manufacturers will have to set a specification for the particle–infectious unit ratio based on manufacturing experience Investigators will also have to develop specific custom assays for a product over the course of clinical development These include the development of methods for assessment of potency, identity, and purity The development of specifications for each of these parameters is an important part of product development and characterization Potency assays are probably the most challenging custom assays to develop Tests for potency should consist of either in vitro or in vivo tests (or both) that measure a biological activity(ies) linked to how the product functions.62 Acceptability will be determined on a case by case basis, but these tests must (1) measure the biological activity of active components, (2) be available for lot release, (3) provide a quantitative 814 Adenoviral Vectors for Gene Therapy readout, (4) meet predefined acceptance/rejection criteria, (5) include appropriate reference controls, (6) be stability indicating, and (7) be validated before licensure Identity assays are also specific for each product Before initiation of Phase studies, an identity assay is recommended that will adequately identify the product and distinguish it from any other product being processed in the same facility.39 For most adenoviruses, this would include restriction enzyme digestion patterns and PCR An appropriately designed PCR should be specific enough to satisfy this recommendation, but may need to be updated if other similar products are made in the same facility over time Tests for purity may vary based on the purification methods used in the manufacturing process For example, process-related impurities such as CsCl, detergents, or Benzonase® are necessary only when these substances are used during the purification Not all tests necessarily need to be developed into lot release tests with specifications Rather some may be used mainly in situations when more extensive testing is required, such as process validation and comparability testing Custom tests may be designed with a specific understanding of the product and manufacturing process For example, an improved manufacturing process to remove human SET and Nucleolin was able to provide higher purity vector preparations.63 Tests to measure specific protein impurities can be very sensitive, and these tests may be helpful for supplementing more general methods to assess viral purity Normal adenovirus replication results in the production of a small proportion of empty capsids.64 Empty capsids may copurify with viral particles using anion exchange chromatography based on a similar capsid structure and charge.65 Empty capsids are immature viral particles that contain no DNA when measured by the OD 260 assay, and therefore the extinction coefficient for empty capsids is very different than that of intact capsids It can be difficult to accurately measure the percentage of empty capsids through direct measurements Measurements of empty capsids are not required for initiation of Phase studies, but may be included as a part of product characterization later in development Direct measurements of empty capsids usually involve analytical ultracentrifugation or transmission electron microscopy However, Vellekamp et al have identified a unique approach for measuring a marker of empty capsids, a capsid precursor protein (pVIII).65 Thus quantification of pVIII by SDS-PAGE or RP-HPLC may help simplify empty capsid measurements for some vectors Like other highly purified, highly concentrated protein preparations, adenovirus vectors may aggregate under certain circumstances Aggregation might be visible to the naked eye as cloudiness or a white precipitate, but aggregation can occur that is not visible A simple spectrophotometer-based static light scattering assay has been reported and is suitable for routine lot release; however, it is somewhat qualitative and not very sensitive More sophisticated assays use disc centrifuge or dynamic light scattering, which can measure particle diameter and dispersity.66,67 Finally, analytical ultracentrifugation can provide detailed information about particle sizes.68 Development tests for aggregation may be more sensitive in detecting loss of infectious particles than infectivity assays, and therefore perhaps may serve as a sensitive indicator of batch-to-batch activity Adenoviral Vector-Based Therapies 815 4.2  Quality and Current Good Manufacturing Practices A goal of the Agency’s current good manufacturing practices (cGMPs) is to ensure quality through control and regulation at each step of a manufacturing process The cGMPs are directed at the commercial manufacturer for large, repetitive commercial batch production Therefore, full application of cGMPs may not be achievable for the manufacturing of most investigational drugs used for Phase clinical trials In 2008, the FDA amended the cGMP regulations under 21 CFR part 211 to exempt most products made for use in Phase clinical trials and published the Guidance for Industry “CGMP for Phase I Investigational Drugs” The Agency provides recommendations in this guidance document that manufacturers of Phase investigational drugs can use to comply with cGMP requirements of section 501(a)(2)(B) of the Food, Drug and Cosmetics Act The adherence to cGMP during the manufacture of Phase investigational drugs occurs through having well-defined, written procedures, and having adequately controlled equipment and an adequately controlled manufacturing environment Consistent with the cGMPs, the Phase manufacturing program should include accurate and systematically recorded data from manufacturing (including process and final product testing) In addition, a quality control (QC) program, which is separate from manufacturing, should be in place at the earliest phase of product manufacture This QC unit should be responsible for ensuring the quality of the product and for product release for clinical use The role of the QC unit is described in Part 211 of Section 21 of the CFR 5.  Preclinical Evaluation of Adenoviral Vector-Based Therapies Advances in science and technology have resulted in the development of many types of adenovirus vector-based therapies, intended to treat a diversity of medical diseases and conditions However, prior to administration of such products in humans, “adequate information about pharmacological and toxicological studies…on the basis of which the sponsor has concluded that it is reasonably safe to conduct the proposed clinical investigations” is needed (21 CFR 312.23 (a)(8)) The preclinical program for each adenovirus-based therapy should thus be designed to evaluate the benefit of product administration in relation to the risk of administration in the identified patient population This section will provide an overview of preclinical evaluation considerations for adenoviral vector-based products to support administration in clinical trials For a more comprehensive discussion, refer to the FDA/CBER guidance titled, Guidance for Industry: Preclinical Assessment of Investigational Cellular and Gene Therapy Products (herein referred to as the “preclinical guidance”).69 5.1   Proof-of-Concept Studies The proof-of-concept (POC) studies, the initial translational step from the discovery stage, help establish the feasibility of use of a specific investigational product in a 816 Adenoviral Vectors for Gene Therapy particular disease/condition The data generated for the adenoviral-based gene therapy can (1) help define a pharmacologically active dose range, (2) determine a potentially optimal route and anatomic location for product administration, and (3) inform a possible dosing regimen for the clinical trial In addition, a preliminary biodistribution (BD) profile of the administered vector can help delineate potential target and nontarget tissues The POC program can consist of both in vitro and in vivo studies In vitro studies provide additional insight into the biological activity and mechanism of action of the gene therapy product and help support the biological relevancy of animal species/ model used, or may be used for POC if there is not an applicable animal model The use of animal models of disease/injury allows for potential further insight and understanding of the pharmacological profile of the administered product For example, antitumor activity may be evaluated in syngeneic murine tumor models, murine:human hybrid tumor xenografts, transgenic models, and animals with spontaneously occurring cancers Considerations when selecting an appropriate animal species can include (1) assessment of the animal’s pharmacological response to the clinical transgene delivered by the adenoviral vector or to the ex vivo adenoviral transduced cells, (2) permissiveness/ susceptibility of the species to vector transduction or to virus replication, and (3) the comparative physiology and pathophysiology of the animal species to the proposed human population.70 These considerations also affect the use of a particular model of disease/injury to study the adenoviral vector-based therapy 5.2  Safety Testing The overall goal of the toxicology studies is to further understand the benefit:risk ratio for use of a specific investigational product in a defined clinical trial Characterizing the vector-based product proposed for clinical use (e.g., vector backbone, replication status, expressed transgene) and knowing the administration route/anatomic location for product delivery are considerations in the overall paradigm for safety assessment Additional factors are the BD, viral replication, and potential persistence of the vector in nontarget tissues and the potential for inappropriate immune activation in the host Another important element is the expressed transgene, which can link to risks such as undesired immunogenicity and toxicity from overexpression in target as well as nontarget tissues The design of studies to evaluate the safety of gene therapies, including adenoviral vector-based products, should consider the biological relevancy and disease status of the animal species, the route of administration, and other factors, as discussed in the preclinical guidance 5.2.1  Selection of Animal Species Appropriate scientific justification should accompany the selection of the relevant animal species/models used in the safety assessment of the adenoviral vector-based therapy Consideration of anatomy, physiology, age, clinical delivery system and Adenoviral Vector-Based Therapies 817 procedure, and the anatomic site of product delivery is important The inclusion of multiple animal species (e.g., rodent and nonrodent) for safety testing of gene therapy products is not a default position In addition, selection of a particular animal species/strain may affect the interpretation of resulting study data For example, intravenous injection of adenovirus vectors in rats resulted in induction of shock mediated by platelet-activating factor.71 Adenovirus interaction with a variety of host proteins, in particular coagulation Factor X, has also been identified as part of the inherent effects on liver tropism, which, combined with macrophage scavenging, may impact viral activity and induce potential adverse effects.72,73 In many instances, toxicities related to the vector itself (e.g., inflammatory reaction to adenovirus capsid proteins) can occur In some cases, the expressed human transgene may be biologically active only in humans and in nonhuman primates In such cases, consideration is given to other animal species, such as (1) rodents responsive to adenovirus that are subsequently “humanized” to express the human target receptor(s) and (2) to vector expressing an analogous animal transgene The use of large, nonrodent animal species may be appropriate in certain instances, such as for the evaluation of a new delivery system and novel anatomic site proposed for the clinical trial; however, providing appropriate rationale for the species is important If feasible, safety endpoints should be added to POC studies to facilitate assessment of the disease-related pathology to any toxicity observed However, when analyzing the resulting data, the underlying pathology associated with the disease state may be a possible confounder 5.2.2  Route of Administration and Dosing Regimen It is important that the route of administration, delivery device, and the delivery procedure for the adenoviral vector-based therapy reflect the clinical plan to the extent possible Although the dosing regimen should mimic the intended clinical trial regimen, this may be difficult to achieve in rodents A modified regimen may be substituted if it reflects a worst case scenario in terms of the frequency of exposure and total exposure levels that are equivalent in humans The adequacy of the modified regimen should be based on the data derived from BD studies 5.2.3  Dose Level Selection Knowing the pharmacologically effective dose level range obtained from POC studies is an important element when designing the toxicology studies It is important that dose levels bracketing this range be administered in the safety studies in order to determine a possible threshold at which toxicity is observed The highest dose level administered may be restricted due to animal size, route of administration, tissue volume/size, and/or product manufacturing capacity The dose level at which no biologically or statistically significant increase in the severity or frequency of adverse findings in safety parameters such as clinical observations, clinical pathology, histopathology, or other observation parameters selected with the specific product in 818 Adenoviral Vectors for Gene Therapy evaluation, as compared to appropriate concurrent controls, is observed is termed a “no-observed-adverse effect dose level” (NOAEL) The extrapolation of the NOAEL in animals to the starting clinical dose level for most gene therapy products can be determined based on body weight, if the product is delivered systemically or results in systemic exposure, organ mass or volume if the product is delivered locally, or other factors It is important to justify the extrapolation method used In all preclinical studies appropriate concurrent controls, such as animals that are untreated, given vehicle alone, or administered null vector, should also be included 5.3  BD Assessment Studies evaluating the BD of the in vivo administered vector in “expected” target tissues, “unexpected” nontarget tissues, and biological fluids (e.g., blood, semen) help inform the design of the toxicology studies (e.g., dose levels, dosing regimen, route of administration) Adenovirus vectors can remain in the host tissues indefinitely following administration Thus, the data generated from analysis of blood samples obtained at various time points are not necessarily indicators of the virus level in tissues; therefore, multiple sacrifice intervals are usually needed in order to obtain a comprehensive BD profile The BD profile for the adenoviral vector-based therapy is characterized prior to initiation of the initial clinical trial For subsequent clinical trials, additional BD studies may be needed Examples include a change in product (e.g., formulation), route of administration, or dosing schedule Although the risk of inadvertent gene transfer to germ cells or vertical transmission of the foreign gene is not significant for adenoviral vectors, if the BD data indicate high levels of vector DNA in the reproductive tissues and germ cells, then reproductive/developmental toxicity concerns may need to be assessed prior to administration of the product to humans Further discussion on this aspect can be found in the preclinical guidance In addition, the guidance document titled, Guidance for Industry: Gene Therapy Clinical Trials—Observing Subjects for Delayed Adverse Events contains recommendations regarding tissues to be collected and analyzed via a quantitative PCR assay Depending on the route of administration used, additional tissues may need to be collected and analyzed Vector presence in tissues or biological fluids may trigger further analysis to determine transgene levels 5.4  Good Laboratory Practice Preclinical toxicology studies should be conducted in compliance with Good Laboratory Practice (GLP) as set forth in 21 CFR Part 58, in order to ensure the quality and integrity of the safety data that are generated However, some toxicology studies not fully comply with GLP In such cases, an explanation for this noncompliance is expected in the final study report and aspects of the study that deviate from the protocol and the potential impact of these deviations on study integrity should be also ... capsid.14–16 Adenoviral Vectors for Gene Therapy http://dx.doi.org/10.1016/B978-0-12-800276-6.00001-2 Copyright © 2016 Elsevier Inc All rights reserved 2 Adenoviral Vectors for Gene Therapy Adenovirus... contain exposed Adenoviral Vectors for Gene Therapy http://dx.doi.org/10.1016/B978-0-12-800276-6.00002-4 Copyright © 2016 Elsevier Inc All rights reserved 28 Adenoviral Vectors for Gene Therapy 3RODULVHGHSLWKHOLDOFHOO... apparent for several large side chains, including arginines and lysines, which aligned with the atomic model for the C-terminal domain of protein IX 12 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