Factor IX gene therapy for the treatment of hemophilia a complicated with inhibitory antibodies

The treatment of patients with inhibitory antibodies remains a major challenge in hemophilia therapy. Here, we pursue a gene transfer strategy using FIX variants which do not require FVIII. In initial variant screening experiments, we combined three to five amino acid substitutions which led to the generation of FIX variants with 17% clotting activity (V181I/K265T/ I383V) in absence of FVIII (ca. 100-fold increase compared to wild type FIX). FVIII inhibitor bypassing activity was confirmed in plasma of patients with high titers of inhibitory antibodies. Further, three different variants were expressed in FVIII knockout mice using non-viral gene transfer. At FIX expression levels ranging from 7500 to 19000 ng/ml partial normalization of the aPTT (from 70 to < 50 sec.) and of blood loss following tail clip assay (1.5 and 3 mm) were observed in all three variant groups (n = 5-9 mice/group, p < 0.05-0.005), while wild type FIX expressing mice did not differ from untreated animals. Similar results were obtained in FVIII knockout mice with high titers of anti-FVIII antibodies. Further, the efficacy of the FIX variants with FVIII bypassing activity was confirmed following laser induced injury of a cremaster arteriole by in vivo imaging technology. While no formation of a stable clot could be observed in FVIII knockout mice treated with wild-type FIX, a stable clot formed at 8 out of 8 injury sites using the V181I/K265T/I383V variant. The described FIX variants therefore offer a new FVIII inhibitor bypassing strategy. The use of these not previously activated proteases could presumably allow prophylactic treatment or even gene therapy in inhibitor patients.