Adenoviral Vectors for Gene Therapy

David T. Curiel

• Publisher: Academic Press
• Year: 2016
• eBook ISBN: 9780128005101
• Edition: 2
• Topic: Scienze biologiche
• Subtopic: Genetica e genomica

Adenoviral Vectors for Gene Therapy, Second Edition provides detailed, comprehensive coverage of the gene delivery vehicles that are based on the adenovirus that is emerging as an important tool in gene therapy. These exciting new therapeutic agents have great potential for the treatment of disease, making gene therapy a fast-growing field for research.This book presents topics ranging from the basic biology of adenoviruses, through the construction and purification of adenoviral vectors, cutting-edge vectorology, and the use of adenoviral vectors in preclinical animal models, with final consideration of the regulatory issues surrounding human clinical gene therapy trials. This broad scope of information provides a solid overview of the field, allowing the reader to gain a complete understanding of the development and use of adenoviral vectors.- Provides complete coverage of the basic biology of adenoviruses, as well as their construction, propagation, and purification of adenoviral vectors- Introduces common strategies for the development of adenoviral vectors, along with cutting-edge methods for their improvement- Demonstrates noninvasive imaging of adenovirus-mediated gene transfer- Discusses utility of adenoviral vectors in animal disease models- Considers Federal Drug Administration regulations for human clinical trials

1

Adenovirus Structure

Phoebe L. Stewart Department of Pharmacology and Cleveland Center for Membrane and Structural Biology, Case Western Reserve University, Cleveland, OH, USA

Abstract

This chapter presents a synopsis of our current understanding of adenovirus structure. Structures of the isolated forms of the major capsid proteins, hexon, penton base, fiber, and adenovirus protease are reviewed. Information derived from cryo-electron microscopy and X-ray crystallography on the molecular architecture of the intact adenovirus virion is summarized including alternate assignments for the four-helix coiled coil at the facet edges. In the most recent refined X-ray structure of adenovirus, the four-helix bundle is assigned to protein IIIa. The N-terminal domain of protein IX forms triskelions between hexons on the exterior of capsid and density is not resolved for the C-terminal domain of protein IX. On the inner capsid surface, a portion of core protein V is found to interact with proteins VI and VIII below the peripentonal hexons. Additional copies of proteins VI and VIII interact below the hexons at the icosahedral twofold axis.

Keywords

Adenovirus; CryoEM; Fiber; Hexon; Penton base; Protease; Structure; X-ray crystallography

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

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 5 (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

After collection of more diffraction data and additional refinement a refined crystal structure was published with more complete models for minor capsid proteins IIIa, VI, VIII, and IX and surprisingly for a portion of the core protein V.¹⁶ To compensate for the relatively modest resolution (3.8 Å) of the structure, a method was devised to evaluate the reliability of assigned amino acid sequences to the experimental electron density. This gives credence to the latest assignments for the locations of the minor capsid proteins within the capsid. It is important to recognize that adenovirus is one of the largest biomolecular assemblies with an atomic resolution structure determined by X-ray crystallography (>98,000 nonhydrogen atoms used in refinement of the asymmetric unit). With an assembly of this size and complexity and with less than ideal resolution data, assigning the locations of the minor capsid proteins is quite a challenging task.

There are over 60 HAdV types categorized in seven species (human adenovirus A–G). Species D adenoviruses are the most numerous, many of which were identified during the AIDS e...

Table of contents

• Cover image
• Title page
• Table of Contents
• Copyright
• List of Contributors
• 1. Adenovirus Structure
• 2. Biology of Adenovirus Cell Entry: Receptors, Pathways, Mechanisms
• 3. Adenovirus Replication
• 4. Adenoviral Vector Construction I: Mammalian Systems
• 5. Adenoviral Vector Construction II: Bacterial Systems
• 6. Upstream Bioprocess for Adenovirus Vectors
• 7. Propagation of Adenoviral Vectors: Use of PER.C6™ Cells
• 8. Purification of Adenovirus
• 9. Targeted Adenoviral Vectors I: Transductional Targeting
• 10. Targeted Adenoviral Vectors III: Transcriptional Targeting
• 11. Adenoviral Vector Targeting via Mitigation of Liver Sequestration
• 12. Molecular Design of Oncolytic Adenoviruses
• 13. Conditionally Replicative Adenoviruses—Clinical Trials
• 14. Innate Immune Response to Adenovirus Vector Administration In Vivo
• 15. Antibodies against Adenoviruses
• 16. Methods to Mitigate Immune Responses to Adenoviral Vectors
• 17. Helper-Dependent Adenoviral Vectors
• 18. Hybrid Adenoviral Vectors
• 19. Xenogenic Adenoviral Vectors
• 20. Engineering Chimeric Adenoviruses: Exploiting Virus Diversity for Improved Vectors, Vaccines, and Oncolytics
• 21. Adenoviral Vector Vaccines Antigen Transgene
• 22. Adenoviral Vectors Vaccine: Capsid Incorporation of Antigen
• 23. Utility of Adenoviral Vectors in Animal Models of Human Disease I: Cancer
• 24. In Situ Vaccination with Adenoviral Vectors to Treat Cancer
• 25. Utility of Adenoviral Vectors in Animal Models of Human Disease II: Genetic Disease
• 26. Adenoviral Vectors for Pulmonary Disease (Pulmonary Vascular Disease)
• 27. Utility of Adenoviral Vectors in Animal Models of Human Disease III: Acquired Diseases
• 28. Animal Models of Gene Therapy for Cardiovascular Disease
• 29. Polymer-Anchored Adenovirus as a Therapeutic Agent for Cancer Gene Therapy
• 30. Adenoviral Vectors for RNAi Delivery
• 31. Imaging and Adenoviral Gene Therapy
• 32. Regulation of Adenoviral Vector-Based Therapies: An FDA Perspective
• Index