Contributors Numbers in parentheses indicate the page numbers on which the authors' contributions begin. C.A.Anderson (129) Merck Research Laboratories, Merck & Company, Inc., West Point, Pennsylvania 19486 Raj K. Batra (533) Division of Pulmonary and Critical Care Medicine, Veterans Administration Greater Los Angeles Health Care System, and University of California, Los Angeles, School of Medicine and Jonsson Comprehensive Center, Los Angeles, California 90073 Steven R. Bauer (615) Division of Cellular and Gene Therapies, CBER Food and Drug Administration, Rockville, Maryland 20852 A. J. Bett (129) Merck Research Laboratories, Merck & Company, Inc., West Point, Pennsylvania 19486 Gerald W. Both (447) Molecular Science, CSIRO, North Ryde, Nev^ South Wales 1670, AustraHa A. Bout (129) Crucell NV, 2301 CA Leiden, The Netherlands K. Brouv^er (129) Crucell NV, 2301 CA Leiden, The Netherlands C. Chartier^ (105) Department of Genetic Therapy, Transgene, 67082 Stras- bourg Cedex, France Tandra R. Chaudhuri {6SS) University of Alabama at Birmingham, Birming- ham, Alabama 35294 L. Chen (129) Merck Research Laboratories, Merck & Company, Inc., West Point, Pennsylvania 19486 ^ Present address: Children's Hospital, Boston, Massachusetts xxiii X X I V Contributors Paula R. Clemens (429) Department of Neurology, University of Pittsburgh, Pittsburgh, Pennsylvania E. Degryse^ (105) Department of Genetic Therapy, Transgene, 67082 Stras- bourg Cedex, France Joanne T. Douglas (205) Division of Human Gene Therapy, Departments of Medicine, Pathology, and Surgery, and the Gene Therapy Center, University of Alabama at Birmingham, Birmingham, Alabama 35294 Jared D. Evans (39) Department of Molecular Genetics and Microbiology, State University of New York, School of Medicine, Stony Brook, New York 11794 S. M. Galloway (129) Merck Research Laboratories, Merck &c Company, Inc., West Point, Pennsylvania 19486 Thomas A. Gardner (247) Urology Research Laboratory, Indiana University Medical Center, Indianapohs, Indiana 46202 Frank L. Graham (71) Departments of Biology, Pathology, and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada L8S 4K1 Patrick Hearing (39) Department of Molecular Genetics and Microbiology, State University of New York, School of Medicine, Stony Brook, New York 11794 Daniel R. Henderson (287) Calydon, Incorporated, Sunnyvale, California 94089 R. B. Hill (129) Merck Research Laboratories, Merck & Company, Inc., West Point, Pennsylvania 19486 Hui-Chen Hsu (409) Department of Medicine, Division of Clinical Immunol- ogy and Rheumatology, University of Alabama at Birmingham, Birming- ham, Alabama 35294 Chinghai H. Kao (247) Urology Research Laboratory, Indiana University Medical Center, Indianapolis, Indiana 46202 D. Kaslow (129) Merck Research Laboratories, Merck &c Company, Inc., West Point, Pennsylvania 19486 David Kirn (329) Program for Viral and Genetic Therapy of Cancer, Imperial Cancer Research Fund, Hammersmith Hospital, Imperial College School of Medicine, London, W12 ONN, United Kingdom Stefan Kochanek (429) Center for Molecular Medicine, University of Cologne, D-50931 Cologne, Germany Jay K. KoUs {595) Department of Medicine and Pediatrics, Louisanna State University, Health Sciences Center, New Orleans, Louisianna 70112 Victor Krasnykh (205) Division of Human Gene Therapy, Departments of Medicine, Pathology, and Surgery, and the Gene Therapy Center, Uni- versity of Alabama at Birmingham, and VectorLogics, Inc., Birmingham, Alabama 35294 R. Lardenoije (129) Crucell NV, 2301 CA Leiden, The Netherlands ^ Present address: Laboratoire Microbiologie, Pernod-Ricard, Creteil Cedex, France. Contributors X X V J. Lebron (129) Merck Research Laboratories, Merck & Company, Inc., West Point, Pennsylvania 19486 B. J. Ledwith (129) Merck Research Laboratories, Merck &c Company, Inc., West Point, Pennsylvania 19486 J. Lew^is (129) Merck Research Laboratories, Merck & Company, Inc., West Point, Pennsylvania 19486 Erik Lubberts (595) University Medical Center St. Radboud, Nijmegen Cen- ter for Molecular Life Science, 6500 HB Nijmegen, The Netherlands M. Lusky (105) Department of Genetic Therapy, Transgene, 67082 Stras- bourg Cedex, France S. V. Machotka (129) Merck Research Laboratories, Merck & Company, Inc., West Point, Pennsylvania 19486 S. Manam (129) Merck Research Laboratories, Merck & Company, Inc., West Point, Pennsylvania 19486 D. Martinez (129) Merck Research Laboratories, Merck & Company, Inc., West Point, Pennsylvania 19486 M. Mehtali^ (105) Department of Genetic Therapy, Transgene, 67082 Stras- bourg Cedex, France John D. Mountz (409) Department of Medicine, Division of Clinical Immunol- ogy and Rheumatology, University of Alabama at Birmingham, and Birmingham Veterans Administration Medical Center, Birmingham, Alabama 35294 Stephen J. Murphy (481) Molecular Medicine Program, Mayo Clinic and Foundation, Rochester, Minnesota 55905 Glen R. Nemerov^ (19) Department of Immunology, The Scripps Research Institute, La JoUa, California 92037 Philip Ng"^ (71) Department of Biology, McMaster University, Hamilton, Ontario, Canada L8S 4K1 W. W. Nichols (129) Merck Research Laboratories, Merck & Company, Inc., West Point, Pennsylvania 19486 Catherine O'Riordan (375) Genzyme Corporation, Framingham, Massa- chusetts 01701 Raymond John Pickles (565) Cystic Fibrosis/Pulmonary Research and Treat- ment Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 Anne M. Pilaro (615) Division of Clinical Trial Design and Analysis, CBER Food and Drug Administration, Rockville, Maryland 20852 Sudhanshu P. Raikwar (247) Urology Research Laboratory, Indiana Univer- sity Medical Center, Indianapolis, Indiana 46202 ^ Present address: Deltagen, Illkirch, France. "^Present address: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030. X X V I Contributors C. Russo (129) Merck Research Laboratories, Merck &: Company, Inc., West Point, Pennsylvania 19486 Carl Scandella (167) Carl Scandella Consulting, Bellevue, Washington Gudrun Schiedner (429) Center for Molecular Medicine, University of Cologne, D-50931 Cologne, Germany Paul Shabram (167) Canji Inc., San Diego, California 92121 Thomas P. Shanley (349) Divisions of Pulmonary Biology and Critical Care Medicine, Children's Hospital Medical Center, Cincinnati, Ohio 45229 Sherven Sharma (533) Division of Pulmonary and Critical Care Medicine, Veterans Administration Greater Los Angeles Health Care System, and Wadsworth Pulmonary Immunology Laboratory, University of Califor- nia, Los Angeles, Los Angeles, California 90073 Phoebe L. Stewart (1) Department of Molecular and Medical Pharmacology, Crump Institute for Molecular Imaging, University of California, Los Angeles, School of Medicine, Los Angeles, California 90095 Bruce C. Trapnell (349) Divisions of Pulmonary Biology and Critical Care Medicine, Children's Hospital Medical Center, Cincinnati, Ohio 45229 D. Valerio (129) Crucell NV, 2301 CA Leiden, The Netherlands M. van der Kaaden (129) Crucell NV, 2301 CA Leiden, The Netherlands Gary Vellekamp (167) Shering-Plough Research Institute, Kenilworth, New Jersey Richard G. Vile (481) Molecular Medicine Program, Mayo Clinic and Foun- dation, Rochester, Minnesota 55905 R. Vogels (129) Crucell NV, 2301 CA Leiden, The Netherlands Christoph Volpers (429) Center for Molecular Medicine, University of Cologne, D-50931 Cologne, Germany Karen D. Weiss (615) Division of Clinical Trial Design and Analysis, CBER Food and Drug Administration, Rockville, Maryland 20852 Lily Wu (533) Departments of Urology and Pediatrics, University of Cal- ifornia, Los Angeles, School of Medicine and Jonsson Comprehensive Center, Los Angeles, California 90073 De-Chao Yu (287) Cell Genesys, Incorporated, Foster City, California 94404 Huang-Ge Zhang (409) Department of Medicine, Division of Clinical Immunology and Rheumatology, University of Alabama at Birmingham, and Birmingham Veterans Administration Medical Center, Birmingham, Alabama 35294 Kurt R. Zinn (655) University of Alabama at Birmingham, Birmingham, Alabama 35294 D. Zuidgeest (129) Crucell NV, 2301 CA Leiden, The Netherlands Freface The number of human gene therapy cHnical trials employing adenoviral vectors is expanding at an unprecedented rate. This increased use of adenoviral vectors has both fueled, and has in turn been fueled by, a parallel explosion in our knov^ledge of the biology of adenoviruses and their vectors. Moreover, there have been concomitant advances in associated technologies. It is therefore timely to reviev^ both basic and applied aspects of adenoviruses and adenoviral vectors in a single, comprehensive, multi-author volume. The first few chapters focus on basic virology—the structure of aden- oviruses and the biology of adenoviral infection and replication. Advances in our understanding of the parental virus have facilitated the rational design of adenoviral vectors for gene therapy. The construction, propagation, and purifi- cation of adenoviral vectors have benefited from a number of technological advances, as discussed in the next series of chapters. In addition to the underlying biological features that favor their use for gene therapy, it is recognized that adenoviral vectors have suffered from a number of limitations. These limitations, together w^ith strategies by which they might be overcome, are considered. Thus, separate contributions discuss approaches to target adenoviral vectors to specific cell types, as well as strategies to circumvent the host immune response. Replication-competent adenoviruses, which are increasingly being used as oncolytic agents for the treatment of cancer, are described. Other vectorological advances covered in this section include high capacity adenoviral vectors, xenogenic adenoviral vectors, and xxvii XXVIII Preface hybrid adenoviral vectors, which combine the advantages of adenoviral vectors with beneficial features derived from other vector systems. The next group of contributors describes the use of adenoviral vectors in animal models of human disease — cancer, genetic disease, and acquired diseases. These chapters discuss the lessons that have been learned from these model systems and their implications for the employment of adenoviral vectors in humans. Specific approval from the regulatory bodies must be obtained prior to the implementation of human trials, as detailed in the following chapter. Finally, the recognition of the need for noninvasive methods to monitor adenovirus-mediated gene transfer in human patients has predicated the development of novel imaging technologies. In the aggregate, we have provided herein a comprehensive overview of adenoviral technology, both classical and novel. This update should provide an entree into the field for the neophyte as well as a reference source for the practitioner. David T. Curiel Joanne T. Douglas C H A P T E R Adenovirus Structure Phoebe L. Stewart Department of Molecular and Medical Pharmacology Crump Institute for Molecular Imaging University of California, Los Angeles School of Medicine Los Angeles, California I. introduction The world got its first look at the icosahedral symmetry of adenovirus (Ad) in 1959 with published electron micrographs of negatively stained Ad5 [1]. In this classic work, Home et al. were able to resolve the basic subunits and thus determine that the adenovirus capsid is composed of 252 subunits, 12 of which have five neighbors (pentons) and 240 of which have six neighbors (hexons). A few years later, Valentine and Pereira [2] published a striking electron micrograph of a single Ad5 particle, revealing the long protruding fibers that are characteristic of adenovirus. In analogy to what was known at the time about the role of phage tails, the authors correctly deduced that the adenovirus fiber might be involved in adsorption to the host cell surface. Since then electron microscopy has continued to play a role in our understanding of the structure of adenovirus and its interaction with av integrins [3, 4]. In recent years X-ray crystallography has contributed atomic structures for the capsid proteins hexon [5, 6], fiber knob [7-9], and shaft [10], the fiber knob complexed with a receptor domain[ll], and the virally encoded protease [12]. Our growing knowledge of adenovirus structure has already contributed to the field of vector design [13]. For example, initial attempts at modifying the C-terminal end of the fiber protein gave suboptimal results for gene delivery [14], while subsequent efforts utilizing knowledge of the fiber knob structure produced vectors with enhanced performance [15, 16]. Strategies for improving adenoviral vectors by making genetic modifications to capsid proteins and by designing hybrid vectors are discussed in later chapters. An understanding of adenovirus structure will be essential for these endeavors. ADENOVIRAL VECTORS FOR GENE THERAPY | Copyright 2002, Elsevier Science (USA). All rights reserved. Phoebe L. Stewart II. Molecular Composition The approximately 50 known human adenovirus serotypes are classified into six subgroups, A-F, and all share a similar structure and genomic organi- zation [17]. Adenovirus is an nonenveloped virus of ~150 MDa, composed of multiple copies of 11 different structural proteins, 7 of which form the icosa- hedral capsid (II, III, Ilia, IV, VI, VIII, IX) and 4 of which are packaged with the linear double-stranded DNA in the core of the particle (V, VII, mu, and terminal protein). For clarification of the nomenclature, note that most of the Ad polypeptides were named based on their position on a polyacrylamide gel. The highest molecular mass protein band turned out to be a complex of com- ponents, and consequently there is no polypeptide I in adenovirus. Also note that polypeptide Ilia was not originally resolved as a separate band; however, it is a distinct structural protein. In addition to the capsid and core components, approximately 10 copies of the adenovirus protease are incorporated into each virion [18]. For many icosahedral viruses, determination of a crystal structure has resolved outstanding molecular composition issues. In the case of adenovirus, there is as yet no atomic structure for the intact virion. In 1985, a prelimi- nary X-ray crystallographic density map of the Ad2 hexon showed that the capsomer was a trimer of polypeptide II with a triangular top and a pseu- dohexagonal base [19]. Together with the early electron microscopy of the intact virion [1], the crystallographically observed hexon symmetry fixed the copy number of polypeptide II at 720 in the Ad virion. The stoichiometry of eight other structural proteins (III, Ilia, IV, V, VI, VII, VIII, and IX) was inferred by careful sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of radiolabeled virions ([^^S] methionine) using hexon as the standard [20]. After adenovirus protease cleavage sites were found in the sequences of polypeptides Ilia, VI, and VIII [18], changing the number of methionines in the mature proteins, their predicted copy numbers were revised [21]. The molecular stoichiometry indicated that there is symmetry mismatch in the Ad penton [20]. Symmetry mismatches are not unheard of in icosahedral viruses. One example is SV40, which has pentamers of VPl at sites of both local fivefold and sixfold symmetry in the crystal structure [22]. The conformationally flexible C-termini of VPl are able to adapt to the position of the pentamer within the SV40 capsid. In the case of adenovirus, three copies of polypeptide IV form the fiber and five copies of polypeptide III form the penton base. The fiber and penton base together compose the penton, which sits at the fivefold symmetry axes of the icosahedral capsid. Microheterogeneity in the Ad penton base has been offered as an explanation for the symmetry mismatch [20]. 1. Adenovirus Structure More recently a reversed-phase high-performance Hquid chromato- graphic (RP-HPLC) assay was developed in order to more fully characterize the Ad5 proteome [23]. N-terminal protein sequencing and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectroscopy were used to identify each component protein contributing at least 2% to the total protein mass of the virus. Peaks for the fiber protein, which contributes only 1.8% of the total protein mass, as well as the terminal protein and the protease, were not identified. The mass of the remaining structural proteins was determined to within ±0.1%. Their copy numbers were estimated using hexon as the standard and with the exception of the copy number for the core polypeptide VII, which was significantly reduced, the new copy numbers are in good agreement with the SDS-PAGE numbers [20, 21]. The precise mass measurements confirmed the proteolytic processing of polypeptides Ilia, VI, VII, VIII, and mu and interestingly cleaved precursor products of all but polypeptide Ilia were found to be present in the purified Ad5 virions. III. Structure of the Intact Virion In 1991 the first structure of an intact Ad particle was determined by cryo- electron microscopy (cryo-EM) and three-dimensional image reconstruction methods [24]. The technique of cryo-EM was developed in the mid-1980s by Dubochet and colleagues [25] for imaging viruses and other macromolecular assemblies in a native-like, frozen-hydrated state. Since then it has proven to be a powerful approach for studying icosahedral viruses and it has been applied to numerous members of over 20 different viral families [26]. The method involves placing a droplet of concentrated virus on an EM grid layered with a holey carbon film (carbon with holes 1-10 ixm in diameter), blotting with a piece of filter paper to leave a thin (~1000 A) layer of water and sample stretched across the holes of the grid, and then plunge freezing into a cryogen such as ethane slush chilled by liquid nitrogen. This rapid freezing causes formation of vitreous (amorphous) ice rather than crystalline ice. Formation of normal crystalline (hexagonal) ice would be harmful to the biological sample because of its expansion relative to liquid water. After cryo-freezing the sample grids are maintained at liquid nitrogen temperature to preserve the vitreous state. Transmission electron micrographs are collected using a low dose of electrons to avoid significant radiation damage to the frozen, unstained sample. The real power of the technique lies in the fact that many particle images can be computationally combined to generate a three-dimensional density map [26-28]. In the early 35-A-resolution reconstruction of Ad2, the features of the icosahedral protein capsid were clear and its dimensions without the fiber were measured as 914, 884, and 870 A along the five-, two-, and threefold symmetry Phoebe L. Stev^art axes, respectively [24]. The reconstruction showed the trimeric shape of the hexon, the pentameric shape of the penton base, and a short portion (^^88 A) of the fiber shaft. The full-length fiber, ~300 A long including the knob at the distal end, was occasionally visible in cryo-electron micrographs. Comparison of these particle images with projections of modeled full-length fibers indicated that the knobs were not positioned as would be expected if the fibers were straight. This suggested that the Ad fibers in the intact Ad2 particle are bent or flexible. Electron micrographs of negatively stained Ad2 fibers show a bend close to the N-terminal end, which binds the penton base [29]. A pseudo repeat of 15 residues was noted in the central section of the Ad2 fiber sequence [30] and later analysis of the fiber sequences from a variety of Ad serotypes revealed a range of 6-23 pseudorepeats in the shaft [31]. A long, nonconsensus repeat at motif 3 was proposed to induce a bend in the shaft of many Ad serotypes [31]. The idea that the fiber is bent for many Ad serotypes is consistent with both negative-stain electron micrographs [29] and the fact that only a short rigid portion of the Ad2 fiber shaft was reconstructed [24]. A more recent cryo-EM reconstruction of Ad2 [3] is shown in Fig. 1 (see color insert) with modeled full-length fibers. Reconstructions have now been pubHshed of Ad2 at 17-A resolution [32], Ad5 [33], Adl2 [3], Ad2 complexed with a Fab fragment from a monoclonal antibody directed against the integrin-binding region of the penton base [34], both Ad2 and Adl2 complexed with a soluble form of avp5 integrin, the internalization receptor for many Ad serotypes [3], and a fiberless Ad5 vector [33]. The capsids of these Ad serotypes appear quite similar, with only subtle differences observed in the size and flexibility of the surface protrusions of the hexon and penton base [3]. IV. Structure of the Capsid Components A. Hexon, Polypeptide II Crystal structures have been published for hexon of serotype Ad2 [5] and Ad5 [6], two members of subgroup C. The sequences of these hexons (967 amino acids for Ad2, 951 for Ad5) are closely related with 86% amino acid identity. Both structures show that the monomer has two eight-stranded P-barrels at the base and long loops that intertwine in the trimer to form a triangularly shaped top (Fig. 2). The high degree of interlocking observed between the monomers might explain why an adenovirus-encoded 100-kDa protein is required for trimer assembly [35]. In the trimer the six ^-barrels, two from each monomer, form a ring with pseudohexagonal symmetry that allows for close packing with six neighboring capsomers in the icosahedral capsid. Regions of the electron density for the Ad2 hexon, refined to 2.9-A resolution.