Major role of local immune responses in antibody formation to factor IX in AAV gene transfer

The risk of an immune response to the coagulation factor IX ( F. IX) transgene product is a concern in gene therapy for the X-linked bleeding disorder hemophilia B. In order to investigate the mechanism of F. IX-specific lymphocyte activation in the context of adeno-associated viral (AAV) gene transfer to skeletal muscle, we injected AAV-2 vector expressing human F. IX (hF. IX) into outbred immune-competent mice. Systemic hF. IX levels were transiently detected in the circulation, but diminished concomitant with activation of CD4(+) T and B cells. ELISPOT assays documented robust responses to hF. IX in the draining lymph nodes of injected muscle by day 14. Formation of inhibitory antibodies to hF. IX was observed over a wide range of vector doses, with increased doses causing stronger immune responses. A prolonged inflammatory reaction in muscle started at 1.5 - 2 months, but ultimately failed to eliminate transgene expression. By 1.5 months, hF.IX antigen re-emerged in circulation in similar to 70% of animals injected with high vector dose. Hepatic gene transfer elicited only infrequent and weaker immune responses, with higher vector doses causing a reduction in T-cell responses to hF.IX. In summary, the data document substantial influence of target tissue, local antigen presentation, and antigen levels on lymphocyte responses to F.IX.