Engineered coagulation factor VIII with enhanced secretion and coagulation potential for hemophilia A gene therapy Open Access

• Engineered FVIII, based on amino acid sequence alignments among animal FVIIIs, had high FVIII activity, efficient secretion, and a low UPR.

• Engineered FVIII will enable treatment with low AAV vector doses and lead to the development of effective gene therapy.

The major challenges of gene therapy for hemophilia A using adeno-associated virus (AAV) vectors are reducing vector doses and the long-term maintenance of stable factor VIII (FVIII). Here, we developed engineered human B-domain-deleted FVIIIs (FVIIISQs) with enhanced secretion and coagulation potential. Intracellular accumulation was markedly reduced in some engineered FVIIISQs, resulting in reduced unfolded protein responses. The administration of AAV vectors carrying engineered FVIIISQ to hemophilia A mice resulted in approximately eight-fold higher FVIII activity and four-fold higher FVIII antigen levels compared with wild-type FVIIISQ administration. The specific FVIII activity of the engineered FVIIISQ was 3.6 times higher than that of the wild-type FVIIISQ, and its binding to activated coagulation factor IX was significantly enhanced, which is supported by the structural analysis. In macaques, the administration of AAV5 vector carrying the engineered FVIIISQ without CpG sequences resulted in a supra-physiological increase in plasma FVIII activity at a dose one-thirtieth that of valoctocogene roxaparvovec (2 × 1012 vg/kg). The engineered FVIIISQ may thus provide stable, long-term therapeutic efficacy in AAV-mediated hemophilia A gene therapy even at low doses.