Adeno-associated virus-mediated gene therapy for hemophilia B in an inhibitor-prone canine model

Hemophilia B is an X-linked coagulopathy caused by a mutation in the factor IX (FIX) gene that results in the absence of functional FIX clotting protein. In this study, we report data obtained from muscle-directed as well as liver-directed AAV-mediated gene therapy for canine hemophilia B. The dogs used in this study have a null mutation resulting in an early stop codon and an unstable mRNA transcript. Previous gene and protein replacement therapies in these dogs resulted in rapid inhibitor formation and subsequent neutralization of FIX activity. In this study, 1 dog was treated by IV infusion of recombinant canine FIX (cFIX), 3 dogs were treated by muscle injection of an AAV-cFIX vector construct with a CMV promoter and 3 dogs were treated by mesenteric vein injection of an AAV-cFIX vector construct with a liver-specific promoter and enhancer element. The dog treated by IV infusion with recombinant cFIX developed a cRX-specific inhibitor within 10 days verifying previous observations that dogs of the Auburn hemophilia B colony are prone to inhibitor formation following treatment with a high concentration of cFIX. Two of the 3 dogs treated by muscle injection of the cFIX-encoding vector construct developed inhibitors to cFIX; however, a third dog transiently treated with cyclophosphamide prior to and for six weeks following gene therapy did not produce inhibitors and has continued to express cFIX sufficient to partially correct the hemophilia B phenotype for over a year. Also, 2 of the 3 dogs treated by mesenteric vein injection have continued to produce cFIX at therapeutic levels for over a year. The third dog developed inhibitors 5 weeks after gene therapy. This dog also had pyruvate kinase deficiency, an erythrocyte metabolism disorder that causes chronic hemolytic anemia and secondary hepatic fibrosis and cirrhosis, and may have caused a heightened immune sensitivity contributing to inhibitor formation. We conclude that either liver-directed gene therapy utilizing an AAV vector construct with a liver-specific promoter or the less invasive combination of AAV-mediated muscle-directed gene therapy and transient immune suppression can be used to establish stable gene transfer and sustained expression of cFIX in inhibitor-prone hemophilia B dogs.