The goal of Project 2 is to investigate the safety and efficacy of regional intravascular approaches for the delivery of an AAV vector encoding Factor IX (F.IX) to skeletal muscle. Two major safety issues to be examined include immune response to the transgene product, Factor IX, and the risk of germline transmission of vector DMA using this delivery approach. In the previous funding period, we explored the safety and efficacy of direct intramscular injection of AAV-F.IX in animals and patients with severe hemophilia B. Although this approach proved safe at all doses tested in humans (up to 2 x 10[12] vg/kg), the large number of injections required to reach a therapeutic dose (1 x 10[13] vg/kg) made continuation of the study impractical. We therefore explored in a large animal model of hemophilia B alternative methods for delivering large doses of vector to skeletal muscle. We have shown long-term expression at levels of 4-14% in hemophilia B dogs using a technique involving intra-arterial delivery of vector via the femoral artery, and have also shown efficacy using a lacZ transgene for a second technique based on vector delivery to a distal vein. However, transient immunosuppression with cyclophosphamide was required to prevent inhibitor formation with the first technique. In this application, we will determine whether transient immunosuppression is also required with the second intravascular delivery technique and will characterize the effect on the immune response to Factor IX of the following parameters: route of administration of vector (isolated limb perfusion vs. anterograde perfusion);presence or absence of immunosuppression at time of vector delivery;AAV serotype;vector dose;and underlying mutation in the Factor IX gene, which determines the degree of immunologic tolerance to the transgene product. These studies must be carried out in an animal genetically deficient in F.IX, but cannot be done in mice, as they are too small for the delivery procedure. Recent advances in canine immunology now enable detailed immunologic studies in this species.
A second aim will be focused on examining the risk of germline transmission as a function of serotype, dose, and delivery method. In the third aim we will take advantage of the high levels of F.IX expression in dog muscle to determine the upper limit of fully functional Factor IX protein that can be synthesized in skeletal muscle and to characterize muscle-syntheszied F.IX biochemically.
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