The goal of Project 1 is to determine the feasibility of expression of FVIIa as the transgene in a gene therapy approach for hemophilia. Work with recombinant proteins has established that hemophilia, caused by nutations in genes encoding Factors VIII or IX in the intrinsic pathway of coagulation, can be treated by infusion of activated Factor VII, an enzyme of the extrinsic pathway. In the previous funding period, we treated an engineered FVIIa variant, expressed it in a recombinant AAV vector, introduced the vector into the livers of hemophilic mice, and demonstrated long-term expression of activated FVII and amelioration of the hemophilic phenotype. We propose to build on this proof-of-concept by 1) defining precisely the minimum level of VIla required for improvement in hemostasis, and the maximum safe tolerated levels. This will be accomplished by studying hemostatic endpoints in both vector-treated and transgenic VIIa-expressing mice. We will use state-of-the-art methods to examine kinetics of clot formation, clot stability, and clot composition as a function of circulating levels of FVIIa in hemophilic mice. 2) In the second aim, we will extend this work to the large animal model of hemophilia. In these experiments we will infuse an AAV vector expressing activated canine FVII into the liver of dogs with severe hemophilia, and determine levels that are safely tolerated and that result in improved hemostasis. 3) Finally, we will capitalize on a novel vector delivery method that we have developed during the previous funding period. This route of administration exploits the extensive capillary network of skeletal muscle to effect transduction of large areas of skeletal muscle. We will determine whether regional intravascular delivery allows us to achieve adequate levels of FVIIa expression using a target organ (skeletal muscle) that is accessible for nearly all hemophilia patients, even those with severe liver changes due to viral hepatitis. Successful development of a VIla-based gene transfer strategy would be applicable for both FVIII and FIX deficiency, would avoid problems of immune response to the transgene product (FVIII or FIX) identified in preclinical gene transfer studies, by using a transgene to which the recipient is fully tolerant, and would circumvent problems of short half-life and need for IV infusion that characterize therapy with the recombinant VIla protein.
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