LEADER 03292ctm a22002777 4500
008 110630s2010 xx a b 000 0 eng d
z| 9781124517438 q| (electronic)
a| PU c| PU
a| Mays, Lauren E.
a| The impact of capsid structure on the immune response to adeno-associated viral vectors / c| Lauren E. Mays.
a| xiii, 237 p. : b| ill. (some col.) ; c| 29 cm.
a| Adviser: James M. Wilson.
a| Thesis (Ph.D. in Cell and Molecular Biology) -- University of Pennsylvania, 2010.
a| Includes bibliographical references.
a| Penn dissertations x| Cell and molecular biology.
a| Cell and molecular biology x| Penn dissertations.
a| Cell and Molecular Biology.
a| Dissertations, Academic.
a| Wilson, James M., e| advisor.
a| University of Pennsylvania.
a| g121 a| Cell and molecular biology.
a| Adeno-associated virus shows great promise as a gene delivery vehicle. However, understanding the immune response following AAV gene transfer is critical to its safe and efficacious use. While the earliest studies using AAV resulted in stable beta-gal expression in the absence of adaptive responses, studies using alternative transgenes, doses, or routes of administration have shown that it is possible for AAV to generate immunity in the mouse. Here, we have begun by showing that the threshold for immune activation to AAV is even lower in nonhuman primates (NHP), which more readily activate cytotoxic T lymphocytes to vector encoded transgenes. Secondly, we have observed that even in cases where T cell responses are elicited in mice, those CD8+ T cells are often non-functional, limiting our ability to study mechanisms of immune activation within a small animal model. By screening the expanded family of AAVs, we were able to identify a capsid variant which elicits strong capsid-specific T cells in mice, that are not only present but capable of secreting IFNgamma in response to antigenic stimulation. This novel variant, AAVrh32.33, also elicits a robust, poly-functional transgene-specific T cell response, capable of eliminating transduced cells, and properly modeling the immune activation observed with AAV in higher order species. In contrast, AAV8 expressing an identical transgene results in minimal T cell activation allowing for stable expression in the muscle, representative of what is typically seen with AAV in mice. A functional comparison of AAV8 and AAVrh32.33 in the mouse has allowed us to study the mechanism of differential immune activation to AAV vectors within a small animal model. A structural comparison was then used to inform the generation of hybrid capsids between AAVB and AAVrh32.33, which has allowed us to map the structural domain responsible for the generation of adaptive immune responses and the loss of transgene expression over time. This work has enhanced our understanding of the mechanisms of immune activation to AAV encoded transgenes and our knowledge of the structural determinants responsible for dictating immunophenotypes which are critical to the safety and efficacy of future gene transfer applications.