AAV mediated muscle directed gene therapy for Hemophilia B

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Hemophilia B is a severe bleeding diathesis caused by the absence of blood coagulation factor IX. The disease is currently treated by infusion of clotting factor concentrates either prophylactically or in response to bleeds. This approach to treatment is sub-optimally for a number of reasons, owing partly to the fact that the protein has a relatively short half-life in the circulation. The ability to treat hemophilia by a gene therapy approach will represent a considerable advance in the treatment of the disease, since it will insure that patients maintain continuously a level of clotting factor adequate to prevent most spontaneous bleeds. The underlying hypothesis of this application is that AAV-mediate, muscle- directed gene transfer of blood coagulation Factor IX can achieve this therapeutic goal. Previous work in mice and in hemophilic dogs supports this hypothesis although the levels of expression achieved in dogs were at the lower limit of the therapeutic range. Thus, successful translation of these promising pre-clinical results to a large clinical population in a safe and efficacious manner will require a more detailed understanding of the biology of the vector and of the biosynthesis of Factor IX in muscle. The specific aims outlined in this application build on the work accomplished in the previous funding period , and include 1) to identify those post- translational modifications that are limiting for expression of biologically active F.IX in muscle, and to determine whether F.IX expression in muscle can be enhanced by supplying the limiting enzyme(s) in trans; 2) to determine whether vector DNA introduced into muscle persists as a high molecular weight episome or is integrated into chromosomal DNA; and 3) to determine whether introduction of muscle-specific promoter/enhancer elements can result in higher levels of expression of F.IX following intramuscular injection of a recombinant AAV vector. Project #1 will make use of standard molecular biology techniques, of small and large animal models of hemophilia B, and of measurements of clotting factors through the Core. Project #1 will have extensive collaborations with Project #4 in that discoveries that enhance expression of F.IX will be translated to the bedside as feasible. Project #1 also has ongoing collaborations with Project #2 in the area of skin-directed gene transfer for hemophilia B, and development of a novel re transposon vector.