Efficient Induction of Tolerance to FIX by Direct Intramuscular Delivery of AAV1.
Authors: Cohn Ellen F. , Kelly Meagan E. , Zhuo Jiacai , Chao Hengjun 
Published: October 2019

Abstract
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 purified 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 cure 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 naive as well as FIX-pre-immunized animals by direct intramuscular injection of AAV1 vectors. Following injection of 1×1011 of AAV1 expressing hFIX per mouse in hemostatically-normal and FIX knock out mice, we detected close to 1000ng/ml of hFIX antigen by ELISA 8 weeks post AAV injection (n=5). No significant level of anti-FIX antibodies could be detected in these mice, by either ELISA or modified Bethesda inhibitor assay. In addition, subsequent challenge with recombinant hFIX in complete Freund’s adjuvant did not cause anti-FIX antibodies to be produced and the level of hFIX in the blood remained constant. However, anti-FIX antibodies, but not hFIX antigen, were measured in the mice injected with the same dose of AAV2 (n=7). 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 naive mice in the circulation of these mice. 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=6). Similar experiments in mice with different genetic and MHC backgrounds have also demonstrated efficient induction of tolerance to FIX, implying that AAV1-hFIX can induce tolerance regardless of MHC haplotype. We hypothesize that the immediate expression of high levels of FIX from the non-pathogenic AAV1 induces FIX tolerance. To elucidate the mechanism of 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.