Risk and prevention of anti-factor IX formation in AAV-mediated gene transfer in the context of a large deletion of F9

The safety of several gene therapy approaches for treatment of the severe, X-linked bleeding disorder hemophilia is currently being evaluated in early phase clinical trials. One strategy seeks to correct deficiency of functio nal coagulation factor IX (hemophilia B) by intramuscular (IM) administrati on of an adeno-associated viral (AAV) vector. A potentially serious complic ation of any treatment for hemophilia is formation of inhibitory antibodies against the coagulation factor protein, a risk that increases in the setti ng of null mutations in the factor IX gene (F9). Here, we describe hemophil ia B mice with a large F9 deletion that form inhibitors within 1 to 2 month s after IM administration of an AAV vector expressing mouse F9 or after rep eated intravenous infusion of mouse F9 concentrate. In both cases, inhibito rs are primarily IgG1 immunoglobulins representing a Th2-driven humoral imm une response. We further demonstrate that anti-mouse F9 antibody formation in the gene-based approach can be reduced by transient immune modulation at the time of vector administration. Moreover, this maneuver resulted in com plete absence of anti-mouse F9 and sustained expression of functional mouse F9 in some hemophilia B mice, particularly in those animals treated with t he immunosuppressive drug cyclophosphamide. These data have direct relevanc e for design of clinical trials and strategies aimed at avoiding immune res ponses against a secreted transgene product.