Phenotype Correction of Hemophilia A Mice with Adeno-Associated Virus (AAV) Vectors Carrying the B Domain Deleted Canine Factor VIII Gene.

Hemophilia A is a life-threatening bleeding disorder caused by mutations in the factor VIII (FVIII) gene that lead to deficiency of FVIII. Gene therapy is respected to provide an alternative to current FVIII supplemental therapy. Among a variety of vectors, AAV vectors are thought to be ideal for transfer of therapeutic genes since they are derived from non-pathogenic virus and have been demonstrated to provide sustained transgene expression in non-dividing cells with little toxicity, though, delivery of the FVIII gene using AAV vectors are limited by its small packaging capacity. To overcome the packaging capacity limit, we developed AAV1 vectors and AAV8 vectors carrying the B domain deleted canine FVIII gene (BDD cFVIII) utilizing a minimum promoter (150b) and expressed canine FVIII in hemophilia A mice. Previous reports suggested AAV1 serotype is suitable for transduction of skeletal muscles and AAV8 serotype is superior to other AAV serotypes for transduction of the liver, thus, AAV1 vectors carrying the BDD cFVIII gene (AAV1 cFVIII) were injected to the skeletal muscles of hemophilia A mice. Since the liver could be transduced with intravenously injected AAV8 vectors carrying the Lac Z gene as efficiently as intraportally injected vectors, AAV8 vectors carrying the BDD cFVIII gene (AAV8 cFVIII) were injected to hemophilia A mice intravenously. FVIII clotting activities measured by the APTT method increased in mice injected with AAV1cFVIII in a dose dependent manner. The FVIII activity level in peripheral blood increased to 2.9±1.0% in hemophilia A mice with the AAV1cFVIII dose at 1x10¹² gc/body, suggesting partial correction of phenotype with AAV1cFVIII vectors. The FVIII clotting activity levels in hemophilia A mice injected with AAV8cFVIII increased also in a dose dependent manner, achieving supernormal FVIII levels (436±219 %) in hemophilia A mice with the AAV8 cFVIII dose at 1x10¹² gc/body. It is apparent that transduction of the liver with AAV8cFVIII is superior to transduction of skeletal muscles with AAV1cFVIII regarding FVIII production, however, AAV1 vectors have advantages of removing the transgenes in case of inconvenience and of less systemic distribution of vectors. These data suggested that both AAV1 and AAV8 vectors carrying the FVIII gene utilizing a minimum promoter have a potential for hemophilia A gene therapy.