PROJECT SUMMARY ABSTRACT Gene therapy for the X-linked bleeding disorder hemophilia holds much promise to accomplish a lasting cure. Four clinical trials utilizing adeno-associated viral (AAV) gene transfer to the livers of males with severe hemophilia are currently being investigated in multiple Phase III clinical trials. Hemophilia A (deficiency in factor VIII, FVIII), the more common form of the disease (~80% of patients), has traditionally been more difficult to treat by gene therapy because FVIII is a large molecule and not efficiently expressed and secreted. Nonetheless, initial results demonstrated complete correction of the disease. However, FVIII levels declined substantially over time, raising worrying questions about durability, and patients also experienced prolonged mild hepatotoxicity despite steroid drug treatment during the first year of gene therapy. Multiple recent observations raise serious questions about the safety of hepatic gene therapy for hemophilia A. These urgently need to be addressed so that this promising approach can be safely applied to patients and to achieve sustained correction. For instance, the reasons for hepatotoxicity and for the decline in FVIII expression are unclear, highlighting critical gaps in our knowledge of the interactions between the vector and hepatocytes and between the FVIII expression and hepatocytes, as well as the role of the immune system in long-term outcome. There is also renewed concern about insertional mutagenesis. We will address these basic and mechanistic questions related to the biology of AAV and FVIII. The central hypothesis of this proposal is that multiple interconnected features of AAV and FVIII biology limit durability of therapeutic expression and pose serious safety concerns. Further, we postulate that unraveling these mechanisms will allow for design of vectors and protocols that minimize these problems, thus resulting in lasting therapy and enhanced safety. The program combines expertise in FVIII biology, cellular stress responses, immunology, and AAV vector biology and is structured into 3 scientific Projects, an administrative Core and 2 scientific Cores. Project 1 (Kaufman) seeks to overcome FVIII protein misfolding and cell toxicity. Project 2 (Xiao) will uncover the mechanisms that lead to formation of subgenomic AAV vector particles that form during vector production through nuclease and recombination activities. Project 3 (Herzog) will define the mechanisms of innate and adaptive immune responses to AAV-FVIII gene transfer. The objectives of the three projects will be supported by an administrative core (Core A), a core that provides human hepatocytes for in vitro and in vivo studies (Core B), and a core that performs development and molecular analysis of AAV vectors (Core C). Overall, this project applies the expertise of the individual investigators towards addressing major unanswered questions in FVIII biology, gene therapy for hemophilia, liver-directed gene transfer, and molecular and immunobiology of AAV vectors.

PROJECT NARRATIVE Recent clinical studies have shown that the bleeding disorder hemophilia, which occurs in 1 of 5,000 male births worldwide, can be cured by gene therapy. Surprisingly however, the gene therapy has not been stable. Basic scientific studies are needed to fill in critical knowledge gaps, so that a safe and lasting cure of the disease can be achieved.