Enhancing immune regulation in gene therapy for hemophilia

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PROJECT SUMMARY/ABSTRACT Adeno-associated viral (AAV) vectors are widely used in human gene therapy: e.g. two products have been FDA approved and three vectors, which correct the X-linked bleeding disorder hemophilia by hepatic gene transfer, are in Phase III clinical trials. Undesired and sometimes unexpected immune responses to vector or transgene product can impede long-lasting therapy, including CD8+ T cell and antibody responses to transgene product and viral capsid, which at high systemic vector doses occasionally induce severe immune toxicities. Consequently, short-term immune suppression, for example by corticosteroids has now been incorporated into many protocols and B cell depletion is being explored. This proposal seeks to elucidate the molecular and cellular underpinnings of innate and adaptive immune responses to AAV-transduced mouse and human hepatocytes. The experiments are designed to test the central hypothesis that a targeted blockade of innate immune responses and specific cytokine signaling pathways combined with an empowered immune regulation are requisite for a successful hepatic AAV-directed gene transfer. This concept is based upon the outcomes of studies, which establish that: 1. The liver is an excellent target for immune tolerance induction to transgene products owing to its microenvironment that ultimately empowers regulatory T cells (Treg) to control antibody and T cell responses. 2. Immune modulatory protocols using rapamycin by itself or in combination with other drugs result in the desired immune regulation. 3. Cross-priming of capsid-specific CD8+ T cells requires cooperation between different subsets of dendritic cells, which critically involves TLR9-MyD88 signaling and IFN I. 4. For TLR9-independent activation of transgene product-specific CD8+ T cells in the liver, IL-1R is requisite. 5. State-of-the art in vitro culture techniques and ability to in vivo expand both human hepatocytes and human innate immune cells in immunodeficient mice have been developed. 6. Formation of neutralizing antibodies directed against coagulation factor VIII and capsid upon hepatic AAV gene transfer can be prevented and sustained therapy is achieved hemophilia A mice by optimal immune modulation. Based on these findings, the proposal seeks to i) define the innate immune response of human hepatocytes and liver environment in AAV gene transfer; ii) define the mechanism by which the IL-1R-MyD88 signaling pathway promotes CD8+ T cell responses in hepatic AAV gene transfer; and iii) develop optimal transient immune modulatory regimens that prevent B and T cell responses to the transgene product and the vector and thereby assure sustained therapy.