Adeno-Associated Viral Vector Delivery of Optimized Human Factor VIII Achieves Therapeutic Factor VIII Levels in Non-Human Primates

Clinical studies of adeno-associated viral (AAV)-mediated gene transfer of factor IX for hemophilia B have demonstrated long term expression of therapeutic levels of factor IX but revealed that the AAV vector dose may be limiting due to anti-AAV immune responses (Nathwani, 2011). While there is significant interest in moving this approach forward for hemophilia A, it is challenging to express high levels of human factor VIII (hFVIII) due to its intrinsic properties that result in lower expression levels compared to similarly sized proteins (Lynch, 1993). Approaches using codon optimization and variants of hFVIII with enhanced function (increased activity, stability and/or secretion) may provide strategies to increase hFVIII expression to support AAV clinical studies for hemophilia A. For example, we previously developed a codon-optimized hFVIII (CO3) that expressed 5-8-fold higher protein levels than wild type hFVIII after AAV delivery in the context of an optimized expression cassette utilizing a modified transthyretin (TTRm) promoter. Introduction of a PACE-furin (P/F) variant (Siner, 2013) that deletes residues 1645-47 (Δ3) or 1645-48 (Δ4) of the PACE-furin recognition site in CO3 resulted in hFVIII expression after AAV delivery that was 18 (Δ3) or 12-fold (Δ4) better than wild type hFVIII. To date, only one published study has reported clinically relevant levels of human FVIII following AAV treatment in a large animal model. This study used a hFVIII variant that contained a 17 amino acid synthetic sequence flanked by 14-amino acid SQ residues from the N- and C-terminal ends of the B domain (McIntosh, 2013). While the presence of the synthetic spacer allowed for an increase in circulating hFVIII levels, the use of a non-wild-type FVIII sequence in hemophilia A patients may increase the risk of development of neutralizing antibodies to FVIII due to its potential neo-antigenicity. Our goal in this study was to generate an AAV-hFVIII vector capable of expressing therapeutic doses of FVIII at a clinically relevant vector dose without adding any neoantigens to the protein. To this end, we generated 26 codon-optimized hFVIII-SQ constructs under the control of the TTRm promoter. Hydrodynamic delivery of the pAAV-TTRm-hFVIII plasmids identified 11 candidates that expressed FVIII 2-7 fold higher than CO3. Nine of these FVIII expression constructs were made into AAV vectors and delivered to hemophilia A/CD4 KO mice (1x10¹¹vg/mouse) using a novel capsid, AAV-Spark100. At 4 weeks post vector administration, 2/9 constructs were similar to CO3, 5/9 were 3-4 fold higher than CO3 and 2/9 (SPK-8003 and SPK-8005) were 4-6 fold higher than CO3. To determine if the deletion of the PACE-furin site would result in higher FVIII expression, the Δ4 P/F deletion was introduced into SPK-8003. The levels of FVIII expression after AAV-TTRm-SPK-8003-Δ4 P/F delivery were 2-fold higher than AAV-TTRm-SPK-8003. In order to evaluate the potency of these novel cassettes in a large animal model, SPK-8005 was administered as a single dose via intravenous infusion to male cynomolgus macaques and followed for 8 weeks of observation. At two weeks after gene transfer, NHPs transduced with 2x10¹² vg/kg of SPK-8005 expressed hFVIII antigen levels of 12.7 ± 2.1% (average ± standard error of the mean, n=3). Average FVIII expression after treatment with 5x10¹² vg/kg was 22.6 ± 0.8% (n=2). Finally, at the highest tested dose of 1x10¹³ vg/kg, hFVIII antigen levels of 54.1 ± 15.6% were observed two weeks after AAV infusion (n=3). As anticipated, hFVIII expression declined in approximately one third of the animals around week 4, concomitant with the appearance of inhibitory antibodies to human factor VIII in these macaques. In summary, these data using highly active, novel codon-optimized FVIII constructs devoid of potential neoantigens demonstrate the feasibility of lowering the AAV capsid load for a gene-based therapeutic approach for hemophilia A to a dosage level that appears to be efficacious and safe in the treatment of hemophilia B.