Type of Document	Dissertation
Author	Mitchell, Daniel Anthony
URN	etd-04222008-155053
Title	Methods for Epitope Characterization of Adeno-Associated Virus Type-2 Through Neutralization Escape Mutants
Degree	Doctor of Philosophy
Department	Molecular Biophysics, Institute of
Advisory Committee	Advisor Name Title Hong Li Committee Chair Hengli Tang Committee Member Kenneth Roux Committee Member Michael Chapman Committee Member Robert Reeves Committee Member
Keywords	Mutants Parvovirus Escape Epitope Antibody Aav
Date of Defense	2008-04-17
Availability	unrestricted
Abstract The Adeno-Associated Virus has moved to the forefront as a vector for human gene therapy. Vectors have been constructed with AAV to repair many genetic deficiencies such as cystic fibrosis and hemophilia and have demonstrated remarkable success. For the application of a gene therapy vector, the particle must be afforded maximal capability to deliver the therapeutic gene without immunological elimination. Unfortunately, AAV is endemic in the human population and as a result, a large proportion of individuals harbor immunity to AAV leading to rapid elimination upon subsequent exposure. One of the major obstacles for the development of AAV as a vector is the absence of robust epitope data. If such data was in hand, gene therapy vectors could be constructed with modifications to these highly immunogenic sites on the viral particle. Given the non-cytopathic nature of AAV, the virus does not lend itself well to traditional plaque assays. Without such assays in hand, the ability to isolate and recover viable viral clones is severely limited. We set out to map the viral epitope to monoclonal antibody A20 by generating monoclonal antibody neutralization escape mutants in cell culture. The lack of plaque assays had prompted us to focus on the development of methods that would facilitate these selection experiments. Having successfully developed a robust and reliable method for plaquing AAV we were capable of applying it to the preliminary selection experiments. The initial application of these methods did not yield the desired mutant. Presented here are the methods developed, their preliminary applications, analysis of the results and future prospective for escape mutant production.
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