The major morbidity of congenital factor Vlll and IX deficiencies (hemophilia A and B) is the progressive crippling arthropathy caused by repeated joint bleeding. The institution early in childhood of prophylactic intravenous factor Vlll or IX replacement throughout the circulation can prevent bleeding-induced joint damage. Apparent clinical success in some subjects receiving a liver-directed factor IX adeno-associated virus (AAV) gene therapy vector raises hope. Nevertheless, failed expression in other subjects receiving identical doses suggests the promise of this strategy may be limited to a select group of individuals, specifically, those without preexisting neutralizing antibodies against AAV; in addition, individuals having neutralizing antibodies against the clotting factor transgene and those with significant liver disease are excluded. The proposed investigations exploit the finding that neutralizing antibody titers appear lower in the hemophilic joint than in the circulation, and the studies explore the potential for local clotting factor gene delivery in the joint to decrease morbidity for hemophilic individuals not adequately treated by available prophylactic protein or emerging gene therapies. The studies compare phylogenetically distinct AAV serotypes for localized gene expression in joint synovial fibroblasts versus chondrocytes in nonnal and blood-damaged joints. Optimization of factor IX vectors having wild type or gain of function genes will he pursued in hemophilic mice, as well as evaluation of comparative immunogenicity of these variants in novel hemophilia B mouse models carrying both human hemophilia gene sequences and human MHC II haplotype. Intraarticular gene delivery following passive transfer of/^AV-neutralizing antibodies from AAV- immunized mice and dogs will be studied in these small and large hemophilia models. The FIX studies will inform evaluation of wild-type and mutant factor Vlll vectors delivered with adjuvant proteasome inhibitors for joint expression in hemophilia A mice in the setting of induced factor Vlll inhibitors or AAV-neutralizing antibodies. The broad objective of the studies is to advance tools for the study of blood-induced joint damage and examine therapies to circumvent major limitations imposed by systemic humoral immunity.