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. / A Dissertation Submitted to the Institute of Molecular Biophysics in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy. / Summer Semester, 2008. / April 17, 2008. / Mutants, Parvovirus, Escape, Epitope, Antibody, Aav / Includes bibliographical references. / Hengli Tang, Professor Co-Directing Dissertation; Michael Chapman, Professor Co-Directing Dissertation; Hong Li, Committee Member; Kenneth Roux, Committee Member; Robert Reeves, Outside Committee Member.
| Identifer | oai:union.ndltd.org:fsu.edu/oai:fsu.digital.flvc.org:fsu_180589 |
| Contributors | Mitchell, Daniel Anthony (authoraut), Tang, Hengli (professor co-directing dissertation), Chapman, Michael (professor co-directing dissertation), Li, Hong (committee member), Roux, Kenneth (committee member), Reeves, Robert (outside committee member), Program in Molecular Biophysics (degree granting department), Florida State University (degree granting institution) |
| Publisher | Florida State University, Florida State University |
| Source Sets | Florida State University |
| Language | English, English |
| Detected Language | English |
| Type | Text, text |
| Format | 1 online resource, computer, application/pdf |
| Rights | This Item is protected by copyright and/or related rights. You are free to use this Item in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s). The copyright in theses and dissertations completed at Florida State University is held by the students who author them. |
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