Acidic region-3 fused heavy chain ameliorates intein-based dual-vector delivery of the B-domain deleted F VIII gene

It was previously demonstrated that an intein-catalyzed splicing of B-domain deleted coagulation factor VIII (BDD FVIII) heavy and light chains could improve the secretion of heavy chain in cis and the splicing can occur independently of cellular entities exhibiting a splicing activity in and out of the cell In order to improve the efficacy of intein-based dual-vector delivery of the BDD-F VIII gene, here an additional acidic region-3 (AR-3) between Pro(1640) and Ser(1690) of FVIII proven to be helpful for the secretion of heavy chain was incorporated into BDD-F VIII heavy chain to examine its effect on secretion and bioactivity of an intein-spliced BDD-F VIII protein By co-transfection of the cultured HEK293 cells with genes of the ar-3 incorporated heavy chain and light chain with fused intein (HCAR3IntN and IntCLC), an ELISA and Coatest assay were performed to determine the amount of spliced BDD-F VIII protein and coagulation activity secreted in the culture supernatant, and the intracellular BDD-F VIII splicing was observed by Western blotting The data showed that the amount of spliced BDD-F VIII protein (173 +/- 26) mu g/L and coagulation activity (1 31 +/- 0 15) U/ml of supernatant from gene co-transfected cells were greater than supernatant from intein-fused ar-3-free heavy and light chain genes (HCIntN and IntCLC) co-transfected cells (102 +/- 12) mu,g/L and (0 79 +/- 0 09) U/ml indicating the additional AR-3 in the heavy chain could dramatically improve the secretion and activity of intein spliced BDD-F VIII A spliced BDD-F VIE protein ((35 +/- 7) mu g/L) and coagulation activity ((0 28 +/- 0 08) U/ml) was also detected in the culture supernatant of combined cells separately transfected with the HCAR3IntN and IntCLC genes implying the cellular entities independent BDD-F VIII splicing of the intein The total protein from gene co-transfected cells displayed an obvious protein band of the spliced BDD-F VIII detected by a F VIII polyclone antibody indicating the intracellular BDD-F VIII splicing It paved a way for ongoing study on protein trans-splicing based dual-adeno-associated virus (AAV) delivery of the split BDD-FVIII gene in gene therapy for hemophilia A animals