Therapeutic levels of FVIII following a single peripheral vein administration of rAAV vector encoding a novel human factor VIII variant

Adeno-associated virus (rAAV) vectors encoding human factor VIII (hFVIII) were systematically evaluated for hemophilia A gene therapy. A 5.7kb rAAV-expression cassette (rAAV-HLP-codop-hFVIII-N6) containing a codon optimized hFVIII cDNA in which a 226 amino-acid B-domain spacer replaced the entire B-domain and a novel liver-specific promoter (HLP) mediated 10-fold higher hFVIII levels in mice compared to non-codon optimized variants. A further two fold improvement in potency was achieved by replacing the 226 amino-acid N6 spacer with a novel 17 amino-acid peptide (V3) in which six glycosylation triplets from the B-domain were juxtaposed. The resulting 5.2kb rAAV-HLP-codop-hFVIII-V3 cassette was more efficiently packaged within AAV virions and mediated supraphysiologic hFVIII expression (732±162% of normal) in HA-knock-out mice following administration of 2x1012 vector genomes/kg, a vector dose shown to be safe in subjects with hemophilia B. Stable hFVIII expression at 15±4% of normal was observed at this dose in a nonhuman primate. Human FVIII expression above 100% was observed in three macaques that received a higher dose of either this vector or the N6 variant. These animals developed neutralizing anti-FVIII antibodies that were abrogated with transient immunosuppression. Therefore, rAAV-HLP-codop-hFVIII-V3 substantially improves the prospects of effective HA gene therapy.