The Adeno-associated viruses (AAVs) are not associated with any diseases and their ability to package non-genomic DNA and to transduce different cell/tissue populations for corrective gene delivery has generated significant interest in understanding their basic biology. This includes their capsid structure, cellular tropism and interactions for entry, uncoating, replication, DNA packaging, capsid assembly, and antibody neutralization. The goal is to improve their specificity and efficacy as vectors. However, while the majority of the characterization of the AAVs has been directed at serotype 2 (AAV2), studies on some of the more recently identified antigenically distinct human and primate viruses show enhanced transduction properties for particular cell types compared to AAV2. This property is mediated by their capsid sequence. Thus while providing the gene therapy community with a more expansive choice of potential AAV vectors for development, specific tissue/organ targeting for improving safety profiles and efficacy as well as engineering a faster onset of transduction would be greatly aided by identifying the capsid features of the other AAVs that correlates with their distinct tissue tropism and transduction phenotypes as well as their antigenic reactivities. The overall objective of this proposal is a structure-function analysis of the AAV capsid to identify features that (I) determine differential cell tropism; (II) affect transduction efficiency, (III) and are utilized for cell receptor recognition. The analysis will also provide information on capsid features that are conserved and thus could be important for the fidelity of viral capsid assembly interactions, and on capsid regions that dictate the distinct antigenicity of the AAV clade groups. Genetically manipulating these features could give rise to a new generation of corrective viral gene delivery vectors with synergistic improvements in tissue tropism and specificity, transduction efficiencies, and the ability to evade existing host immune responses. To achieve our objectives, we will determine the capsid structures for representative members of the AAV clade groups, alone and in complex with identified carbohydrate receptors, by X-ray crystallography, and functionally annotate AAV capsid regions involved in the receptor interactions using mutagenesis, cell binding and transduction assays, and biophysical measurements of binding affinity. We have developed a scaleable baculovirus system for the expression of wild type and mutant virus capsids for these studies. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM082946-02
Application #
7496948
Study Section
Virology - A Study Section (VIRA)
Program Officer
Marino, Pamela
Project Start
2007-09-17
Project End
2012-07-31
Budget Start
2008-08-01
Budget End
2009-07-31
Support Year
2
Fiscal Year
2008
Total Cost
$275,626
Indirect Cost
Name
University of Florida
Department
Biochemistry
Type
Schools of Medicine
DUNS #
969663814
City
Gainesville
State
FL
Country
United States
Zip Code
32611
Bennett, Antonette D; Wong, Kristine; Lewis, Jordyn et al. (2018) AAV6 K531 serves a dual function in selective receptor and antibody ADK6 recognition. Virology 518:369-376
Ilyas, Maria; Mietzsch, Mario; Kailasan, Shweta et al. (2018) Atomic Resolution Structures of Human Bufaviruses Determined by Cryo-Electron Microscopy. Viruses 10:
Marr, Matthew; D'Abramo, Anthony; Pittman, Nikea et al. (2018) Optimizing the Targeting of Mouse Parvovirus 1 to Murine Melanoma Selects for Recombinant Genomes and Novel Mutations in the Viral Capsid Gene. Viruses 10:
Wang, Dan; Li, Shaoyong; Gessler, Dominic J et al. (2018) A Rationally Engineered Capsid Variant of AAV9 for Systemic CNS-Directed and Peripheral Tissue-Detargeted Gene Delivery in Neonates. Mol Ther Methods Clin Dev 9:234-246
Pittman, Nikéa; Misseldine, Adam; Geilen, Lorena et al. (2017) Atomic Resolution Structure of the Oncolytic Parvovirus LuIII by Electron Microscopy and 3D Image Reconstruction. Viruses 9:
Bennett, Antonette; Patel, Saajan; Mietzsch, Mario et al. (2017) Thermal Stability as a Determinant of AAV Serotype Identity. Mol Ther Methods Clin Dev 6:171-182
Mietzsch, Mario; Hering, Henrik; Hammer, Eva-Maria et al. (2017) OneBac 2.0: Sf9 Cell Lines for Production of AAV1, AAV2, and AAV8 Vectors with Minimal Encapsidation of Foreign DNA. Hum Gene Ther Methods 28:15-22
Tse, Longping Victor; Klinc, Kelli A; Madigan, Victoria J et al. (2017) Structure-guided evolution of antigenically distinct adeno-associated virus variants for immune evasion. Proc Natl Acad Sci U S A 114:E4812-E4821
Kondratov, Oleksandr; Marsic, Damien; Crosson, Sean M et al. (2017) Direct Head-to-Head Evaluation of Recombinant Adeno-associated Viral Vectors Manufactured in Human versus Insect Cells. Mol Ther 25:2661-2675
Mietzsch, Mario; Kailasan, Shweta; Garrison, Jamie et al. (2017) Structural Insights into Human Bocaparvoviruses. J Virol 91:

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