Identification of Activity Enhancement Residue of Human FVIII and Novel Factor VIII Variant for Human Gene Therapy

Gene therapy for Hemophilia A using the recombinant Adeno-associated virus (rAAV) offers an alternative to FVIII protein infusions; However, due to limitations associated with rAAV and the FVIII protein itself, the end result is transgene expression below therapeutic limits.  One approach to improving the therapeutic value of rAAV gene therapy for HA is to engineer a more active FVIII protein through genetic modifications.  Preliminary testing revealed that canine FVIII Light Chain (kLC) enhances human FVIII coagulation activity.  Through the process of engineering, evaluation, positive and negative selection of kLC, key amino acids in canine factor VIII were identified. These amino acids were incorporated into human factor VIII and a new version of human factor VIII, hFVIII.JF12, was engineered.

The hFVIII.JF12 is a human FVIII B-domain deleted construct containing 12 amino acid changes in the light chain that work together to enhance coagulation activity. In vitro, hFVIII.JF12 resulted in a 4.3 fold increase in clotting activity, but no increase in protein production. CD4KO/HA mice injected with rAAV vector carrying the hFVIII.JF12 gene produced an average 4.6 fold increase in clotting activity compared to those injected with hFVIIIBDD.  An ELISA revealed no significant difference in protein production between these two groups of injected mice.  In order to determine the mechanism of enhancement, the hFVIIIBDD and hFVIII.JF12 proteins were purified and functional properties analyzed.  Results demonstrated that the hFVIII.JF12 protein produced a specific activity of 39,153.69 Units/m.  This is a 6.28 fold increase over hFVIIIBDD specific activity, which was 6,237.92 Units/mg. Measurement of conversion from FX to FXa revealed that the hFVIII.JF12 protein generated a higher amount of FXa at a quicker rate. 

The hFVIII.JF12 construct is novel because it enhances FVIII activity both in vitro and in vivo through modifications to the light chain based on the kLC.   This will be beneficial in the context of both gene and protein therapy because the protein is more specifically active.  This research is also innovative because it demonstrates a novel method of enhancing transgene expression of FVIII delivered by an AAV vector through modifications to the gene itself.