Enhanced Synthesis of Functional Recombinant Factors IX and VII by BHK Cells Engineered to Overexpress VKORC1 Combined with siRNA Silencing of the γ-Carboxylation Inhibitor Calumenin.

Because of the importance and demand for recombinant factors IX, VII and protein C in prophylactic medicine, our laboratory is focused on engineering eukaryotic cells with enhanced capacity to produce fully γ-carboxylated functional forms of these recombinant vitamin K-dependent proteins. Here we report our work on recombinant human factor IX (r-hFIX) and recombinant human factor VII (r-hFVII). Successful engineering was accomplished by overexpressing the vitamin K cofactor producing enzyme VKORC1 of the vitamin K cycle in BHK cells stably overexpressing r-hFIX and r-hFVII respectively. VKORC1 provides the γ-carboxylase with reduced vitamin K₁ (vit.K₁H₂) cofactor and is the rate limiting step in vitamin K-dependent post translational γ-carboxylation of proteins. We have identified the ER chaperone protein calumenin as an inhibitor of vitamin K-dependent γ-carboxylation. Here we show that siRNA silencing of calumenin in BHK cells engineered to overexpress VKORC1 and r-hFIX and r-hFVII respectively significantly increases production of both coagulation proteins as functional fully γ-carboxylated proteins. Engineering of the γ-carboxylation system and silencing of calumenin do not affect total synthesis of r-hFIX and r-hFVII by BHK cells. However the fraction of functional r-hFIX and r-hFVII isolated from the total pool of each recombinant coagulation factor pool was found to be 80 and 85% compared to 12 and 14 % from the pools isolated from non engineered BHK cells stably overexpressing r-hFIX and r-hFVII respectively. This new recombinant technology enforced on the vitamin K-dependent γ-carboxylation system demonstrates that r-hFIX and r-hFVII used as pharmaceuticals can be produced at a much higher yield than currently is accomplished from r-hFIX and r-hFVII overexpressing cells not modified by the new technology.