1002. Immune Responses to AAV and to Factor IX in a Phase I Study of AAV-Mediated, Liver-Directed Gene Transfer for Hemophilia B

Studies in mice, hemophilic dogs, and non-human primates have shown long-term expression of Factor IX (FIX) after administration of a recombinant AAV-2 vector expressing FIX into the portal vein or hepatic artery. Specifically, infusion of ~1×1012 vg/kg AAV vector expressing canine FIX in hemophilic dogs showed sustained (>3 years) circulating levels of FIX in the range of 4-12% of normal (Blood 99:2670). In a Phase I dose escalation study of hepatic artery infusion of AAV-2-F.IX in humans with severe hemophilia B, two subjects receiving a dose of 2×1012 vg/kg (titered against a supercoiled standard) showed expression of detectable levels of FIX, but expression was transient, with peak levels of 3% in one subject and 11.8% in the other. FIX levels had returned to baseline by 4 weeks and 10 weeks post vector infusion respectively. In the latter subject, the decline in circulating FIX levels was accompanied by a transient rise in serum transaminase levels. This subject had a low baseline neutralizing antibody titer (NAB) to AAV-2 (1:2), which rose to >1:1000 two weeks later. The other subject experienced no change in serum transaminases; his pre-treatment NAB to AAV-2 was 1:17, and increased to 1:96,000 two weeks later. To further assess the role of the immune response, PBMCs from these two subjects were incubated with peptides derived from the AAV-2 capsid sequence and from the wild-type hF.IX sequence. IFN-γ secretion was measured in ELISpot assays, which showed, in the latter subject, a 10-fold elevation in IFN-γ compared to media control at the 4 wk time point following incubation with one of the AAV peptide pools and with the F.IX peptide pool. When normalized to levels of maximal IFN-γ secretion in response to a positive control, IFN-γ response was >10-fold that of normal controls for the F.IX peptide pool, and >2-fold that of normal controls for the reactive AAV-2 peptide pool. We conclude that: 1) AAV-F.IX can transduce human hepatocytes in vivo, resulting in therapeutic circulating factor levels; 2) the vector dose required to achieve a therapeutic factor level was accurately predicted by animal models; 3) the transient nature of expression seen in subjects treated at 2×1012 vg/kg was not predicted by earlier studies in animal models; 4) T cell responses to AAV-2 and to F.IX may determine the outcome of AAV-2-mediated gene transfer in human subjects. More recently the clinical study has resumed with a planned target dose of 1.2×1012 vg/kg. The goal is to determine whether subjects can achieve therapeutic levels of expression at this dose, and whether specific immune responses to either AAV or F.IX will limit duration of expression in humans.