Adeno-associated virus (AAV) vector has been successfully applied in phase I clinical trials in hemophilia B patients with liver targeting. However, these studies have suggested that AAV capsid specific cytotoxic T lymphocytes (CTL) have the potential to eliminate AAV transduced hepatocytes and result in the therapeutic failure. Our prior studies have demonstrated that AAV capsid antigen presentation is dose-dependent and requires capsid ubiquitination for proteasome mediated degradation. The contamination of empty virions in AAV preparation inhibits transduction from full particles of AAV vectors and potentially increases the risk of virus capsid antigen load. In this proposal we will investigate capsid antige presentation from AAV empty virions and the effect of empty particles on antigen presentation from full virus transduction (Aim 1). To decrease antigen presentation on AAV transduced cells for avoiding capsid specific CTL-mediated elimination, it has been proposed to modify the AAV capsid surface or apply proteasome inhibitors to enhance AAV transduction while lowering the effective dose or to escape capsid ubiquitination. We will study the effect of AAV mutants and proteasome inhibitors on AAV capsid antigen presentation (Aim 2). It is well-known that the transduction of AAV vectors in mouse models does not always translate into the human. Finally, we will explore the directed evolution approach combined with a rational design strategy to isolate AAV vectors with human hepatocyte specific tropism and the ability to evade a capsid specific CTL response in humanized mice (Aim 3). Elucidation of AAV empty capsid antigen presentation in vivo and the development of an AAV vector with enhanced human liver transduction and CTL immune-evasion will allow us to design safer and more effective strategies that address the current clinical complications for human liver gene therapy using AAV.

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Having demonstrated that AAV capsid antigen presentation is dose-dependent and requires proteasome mediated degradation, and modification of the AAV capsid surface induces enhanced AAV transduction while lowering the effective dose or decreases capsid antigen presentation, we will explore to develop AAV mutants with the ability to evade capsid specific CTL mediated elimination and with human hepatocyte tropism. This study will allow us to design safer and more effective strategies for human liver gene therapy using AAV.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Gene and Drug Delivery Systems Study Section (GDD)
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Gondre-Lewis, Timothy A
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University of North Carolina Chapel Hill
Schools of Medicine
Chapel Hill
United States
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