684. Enhanced FVIII AAV Vector Cassette Produces Improved Virus Yields and Supraphysiological FVIII Levels In Vivo

Hemophilia A is the most common inherited bleeding disorder in humans and is caused by a deficiency of blood coagulation factor VIII (FVIII). This disease is an ideal candidate for liver-directed gene therapy, as even modest increases in FVIII activity (u003e1% of normal) can ameliorate the severe bleeding phenotype. Adeno-associated viral (AAV) vectors have shown great promise in both preclinical and clinical trials to efficiently deliver therapeutic transgenes to the liver. Suboptimal virus yields of AAV comprising human FVIII (hFVIII) for use in human therapy have hampered clinical scale manufacturing, both from mammalian (HEK293) and insect cells (Baculovirus system). We optimized an hFVIII AAV vector cassette to improve both virus yields and liver-specific hFVIII expression. Compared to historical hFVIII AAV vector cassettes that routinely produce 20% of standard, non-hFVIII containing AAV2/6 virus preparations, the improved hFVIII AAV vector cassette produced yields 100% of standard AAV2/6, both from HEK293 cells at the cell factory (CF) scale, but also in the Baculovirus system at large scale. Improved yields were also observed using other AAV serotypes including hFVIII AAV2/8 and AAV2/9. Using virus produced from the improved hFVIII AAV cassette we transduced mice at 6E+10 vg/mouse (~2E+12 vg/kg). Peak levels of hFVIII protein were achieved in mouse plasma at day 14 and represented supraphysiological levels of normal hFVIII in humans with serotypes AAV2/8 and AAV2/9; up to 337% and 516% of normal hFVIII plasma levels respectively (1U = 200 ng/mL = 100%). Transducing mice with serotype AAV2/6, known to be inefficient at transducing mouse liver, at 6E+10 vg/mouse (~2E+12 vg/kg) we achieved a mean peak value of 91.9 % +/- 15.5 SEM (n=6) of normal hFVIII plasma levels in humans. At a higher dose representing ~6E+12 vg/kg we achieved a mean peak value of hFVIII plasma over six independent in vivo mouse studies of 169.2 % +/- 10.1 SEM (n = 36). Supraphysiological hFVIII plasma levels were also achieved in non-human primates at a dose of 2E+12 vg/kg using serotype AAV2/6, representing up to 840% of normal hFVIII levels (8.4U). The robust production of hFVIII from the enhanced hFVIII AAV vector could significantly reduce the dose required to achieve therapeutic levels in human subjects. Additionally the improved hFVIII AAV vector cassette will enable clinical scale manufacturing.