Propeptide-deleted von Willebrand Factor Improves Secretion of Protein Spliced L303E/F309S Mutated FVIII

Abstract: We recently demonstrated that L303E and F309S mutation in the A1 domain of heavy chain of coagulation factor VIII (FVIII) could improve secretion of spliced FVIII in protein-splicing based dual-vector delivery of FVIII gene. In this study， we further investigated the effect of a propeptide-deleted form of the von Willebrand factor (vWF-ΔPro)， a functional FVIII carrier co-transfection on secretion of protein spliced FVIII with L303E/F309S mutation. By co-transfection of HEK293 cell with both heavy and light chain genes fused to intein， a protein splicing element and vWF-ΔPro gene， an ELISA was performed to determine secreted spliced FVIII and Coatest was used to measure secreted bioactivity. The data demonstrated that vWF-ΔPro co-expressed cell displayed a much higher levels of secretion of spliced FVIII (196±27) ng/mL， compared to control cell (116±24) ng/mL. The secreted bioactivity by vWF-ΔPro co-expressed cell (1.39±0.31) IU/mL was also greater than that of control cell (0.91±0.18) IU/mL. Therefore， vWF-ΔPro may further improve efficacy of dual-vector delivery of FVIII gene by enhancing secretion of spliced L303E/F309S mutated FVIII encouraging our ongoing in vivo investigation for improvement of two-vector FVIII transgene.

CSTR: 32200.14.cjcb.2012.10.0005