Adeno-associated virus (AAV) is a prime candidate vector for human gene therapy. The long term objective is an understanding of the structural and mechanistic bases of capsid-host interactions and viral assembly. This is needed to engineer modifications of the capsid to maximize AAV's potential to deliver therapeutic DNA to targeted cells afflicted with cancer or an inherited disorder. Mapping the footprint of the cellular receptor binding site is critical to modifying tissue tropism, while mapping of the neutralizing antigenic determinants is needed to engineer viruses that can reach target cells in patients previously exposed to AAV. Similar challenges will be faced with other viral vectors, for which this work will be a paradigm. The research will build upon our recent 3 Angstrom resolution crystallographic structure of AAV serotype 2 (AAV-2), and its continuing refinement and analysis. Interactions with the cellular receptor, heparan sulfate proteoglycan, will be characterized through crystallographic analysis of AAV-2 complexed with small heparan fragments, through cryo-electron microscopy (EM) of complexes with larger fragments (in collaboration with Ken Taylor), and through site-directed mutagenesis of the binding site. AAV-antibody interactions will be studied using panels of monoclonal antibodies (MAb) prepared by Barrie Carter and Jurgen Kleinschmidt. Functional epitopes will be mapped through the sequencing of mutants to be selected by viral propagation in the presence of antibodies. Physical epitopes for representative MAb will be mapped by cryo-EM-imaging, interpreted at molecular resolution using the known AAV-2 structure. Structural studies of at least one of the other 4 serotypes will be initiated. Comparative analysis will show the extent of conservation of receptor-binding regions, and reveal the extent of variability of regions most subject to immune surveillance. All of the proposed structural studies will complement and accelerate extensive efforts elsewhere to develop AAV-based therapies, by providing a structural rationale and set of limiting constraints for modifications that currently are being made by enlightened trial-and-error.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM066875-03
Application #
6849337
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Basavappa, Ravi
Project Start
2003-02-01
Project End
2007-01-31
Budget Start
2005-02-01
Budget End
2006-01-31
Support Year
3
Fiscal Year
2005
Total Cost
$259,539
Indirect Cost
Name
Florida State University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
790877419
City
Tallahassee
State
FL
Country
United States
Zip Code
32306
Pillay, Sirika; Zou, Wei; Cheng, Fang et al. (2017) AAV serotypes have distinctive interactions with domains of the cellular receptor AAVR. J Virol :
Xie, Qing; Spear, John M; Noble, Alex J et al. (2017) The 2.8 Å Electron Microscopy Structure of Adeno-Associated Virus-DJ Bound by a Heparinoid Pentasaccharide. Mol Ther Methods Clin Dev 5:1-12
Earley, Lauriel F; Powers, John M; Adachi, Kei et al. (2017) Adeno-associated Virus (AAV) Assembly-Activating Protein Is Not an Essential Requirement for Capsid Assembly of AAV Serotypes 4, 5, and 11. J Virol 91:
Pillay, S; Meyer, N L; Puschnik, A S et al. (2016) An essential receptor for adeno-associated virus infection. Nature 530:108-12
Spear, John M; Noble, Alex J; Xie, Qing et al. (2015) The influence of frame alignment with dose compensation on the quality of single particle reconstructions. J Struct Biol 192:196-203
Stagg, Scott M; Noble, Alex J; Spilman, Michael et al. (2014) ResLog plots as an empirical metric of the quality of cryo-EM reconstructions. J Struct Biol 185:418-26
Xie, Qing; Spilman, Michael; Meyer, Nancy L et al. (2013) Electron microscopy analysis of a disaccharide analog complex reveals receptor interactions of adeno-associated virus. J Struct Biol 184:129-35
Zhang, Fuming; Aguilera, Javier; Beaudet, Julie M et al. (2013) Characterization of interactions between heparin/glycosaminoglycan and adeno-associated virus. Biochemistry 52:6275-85
Chapman, Michael S; Trzynka, Andrew; Chapman, Brynmor K (2013) Atomic modeling of cryo-electron microscopy reconstructions--joint refinement of model and imaging parameters. J Struct Biol 182:10-21
Lerch, Thomas F; O'Donnell, Jason K; Meyer, Nancy L et al. (2012) Structure of AAV-DJ, a retargeted gene therapy vector: cryo-electron microscopy at 4.5 A resolution. Structure 20:1310-20

Showing the most recent 10 out of 26 publications