Efficient Induction of Tolerance to FIX by Direct Intramuscular Delivery of AAV1.

Hemophilia B is an X-linked recessive genetic disease resulting from deficiency in coagulation factor IX (FIX). The current therapy for hemophilia B is life-long replacement of FIX through recombinant FIX or blood products in response to bleeding events. However, this replacement therapy is non-prophylactic, costly, and can be complicated by formation of inhibitory anti-FIX antibodies in up to 5% of patients. While somatic gene therapy is expected to provide a final |[ldquo]|cure|[rdquo]| for hemophilia B, it may also cause high incidence of FIX antibodies formation and other adverse immune responses following gene delivery. Direct intramuscular injection of adeno- associated virus (AAV) is a safe and promising procedure for hemophilia B gene therapy. This treatment, however, elicits anti-FIX antibodies in immune competent animal models. We have previously reported that intramuscular injection of AAV1 expressed high levels of canine FIX and induced FIX tolerance in a mouse model of hemophilia B, but AAV2 elicited anti-FIX antibodies. Here, we report efficient induction of human FIX (hFIX) tolerance in na|[iuml]|ve as well as FIX-pre-immunized animals by direct intramuscular injection of AAV1 vectors. Following injection of 1x1011 of AAV1 expressing hFIX per mouse in hemostatically-normal and FIX knock out mice, we detected higher than 500ng/ml of hFIX antigen by ELISA as early as 2 weeks post AAV injection (n=5), while no significant level of anti-FIX antibodies could be detected in these mice, by either ELISA or modified Bethesda inhibitor assay. However, anti-FIX antibodies, but not hFIX antigen, could be measured in the mice injected with the same doses of AAV2 (n=5). Subsequent injection of AAV1 vector into the skeletal muscle of these AAV2-injected mice resulted in the disappearance of anti-FIX antibodies and emergence of FIX antigen at similar levels to AAV1- injected na|[iuml]|ve mice in the circulation of these mice as early as 2 weeks post AAV1 injection. In addition, direct intramuscular injection of AAV1 also induced FIX tolerance in mice that developed anti-FIX antibodies after exposure to recombinant FIX proteins (n=5). Investigation of FIX tolerance by AAV1 in mice with different genetic and MHC backgrounds is ongoing. We hypothesize that the immediate expression of high levels of FIX from the non-pathogenic AAV1 induces FIX tolerance. We previously showed a linear relation between canine FIX antigen levels and the AAV1 doses injected to skeletal muscle of mice. We are currently conducting a viral dose curve to determine the lowest titer of AAV1 (as well as the minimal levels of hFIX) necessary to induce tolerance to FIX. To elucidate the mechanism of the different immune responses to FIX following intramuscular injection of AAV1 and AAV2, we are examining variations in antigen presentation, interaction between antigen presenting cells and antigen-specific T cells, and fate of antigen-specific T cells following intramuscular injection of AAV1 and AAV2 vectors. In summary, our results demonstrate efficient induction of FIX following direct intramuscular injection of AAV1 vectors. Investigations to elucidate the underlying mechanism are ongoing in our lab.