The overall goal of Project 1 is to understand the nature of the immune response to AAV vectors in humans. This line of investigation is based on a clinical trial of AAV-Factor IX (F.IX) administered to the hepatic artery in men with severe hemophilia B, in which we documented the simultaneous occurrence of loss of F.IX expression, and transient liver transaminase elevation, beginning 3-4 weeks after vector injection. In the previous funding period we documented: 1) that the rise and fall in liver enzymes was accompanied by expansion and contraction of a population of capsid-specific CD8+ T cells, but no T cell response to F.IX;2) that a substantial proportion of normal human subjects harbor AAV capsid-specific T cells as documented by IFN-7 ELIspot;3) that human hepatocytes can process and present preformed capsid antigen on the surface ofthe transduced cell;4) in preliminary studies, that the clinically approved proteasome inhibitor bortezomib reduces capsid antigen presentation on the surface ofthe transduced cell;5) in non-human primates, and one human subject thus far, that the immunosuppressive regimen MMF/rapamycin can be safely coadministered with AAV vector delivered to the hepatic artery. In the next funding period, we propose three aims to build on these observations by;1) monitoring and characterizing the immune response to the AAV capsid in human subjects undergoing AAV-mediated gene transfer in seven different AAV trials. We will use ELISpot for screening and polyfunctional T cell analysis for more comprehensive assessment of T cell responses;we will also obtain a complete serum cytokine expression profile over time in the subjects studied;2) determining the levels and the biological significance of capsid antigen presentation of alternate AAV serotypes and capsid variants carrying mutations in surface-exposed tyrosine residues;these experiments will be carried out in a fully-humanized in vitro system;and 3) investigating new strategies to block or reduce antigen presentation through a pharmacologic treatment with bortezomib or exploiting the molecular mechanisms of viral immune evasion. Regulatory T cells will also be tested as modulators of capsid T cells.
Aims 1 and 2 will involve extensive interaction with Project 2, and Aim 3 with Project 3.

Public Health Relevance

Gene therapy represents a new method of treatment for previously untreatable genetic diseases. Immune responses to gene transfer vectors represent one of the last barriers to successful gene therapy. In this proposal we will define the nature of these responses in human subjects undergoing AAV-mediated gene transfer. We will also analyze capsid antigen presentation and test novel strategies to block or modulate recognition of AAV vectors by the immune system.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Program Projects (P01)
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Heart, Lung, and Blood Initial Review Group (HLBP)
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Wistar Institute
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