Protein trans-splicing based dual-vector factor VIII(FVIII) gene delivery is adversely affected by less efficiency of protein splicing.We sought to increase the amount of spliced FVIII protein and plasma coagulation activity in dual-vector FVIII transgene in mice by means of strengthening the interaction of inteins,protein splicing elements,thereby facilitating protein trans-splicing.Dual-vector delivery of the FVIII gene in cultured cells showed that replacement of Met226 in the heavy chain and Asp1828 in the light chain with Cys residues could facilitate inter-chain disulfide linking and improve protein trans-splicing,increasing the levels of spliced FVIII protein.In this study,C57BL/6 mice were coadministered dual vectors of intein-fused human FVIII heavy chain and light chain with Cys mutations via portal vein injection into the liver.Forty-eight hours post-injection,plasma was collected and analyzed for FVIII antigen concentration and coagulation activity.These mice showed increased circulating FVIII heavy chain polypeptide(442±151 ng mL-1 vs.305±103 ng mL-1) and coagulation activity(1.46±0.37 IU mL-1 vs.0.85±0.23 IU mL-1) compared with control mice co-administered dual vectors expressing the heavy and light chains of wild-type FVIII.Moreover,coagulation activity was similar to that of mice receiving a single vector expressing FVIII(1.79±0.59 IU mL-1).These findings indicate that improving protein trans-splicing by inter-chain disulfide bonding is a promising approach for increasing the efficacy of dual-vector based FVIII gene transfer. Protein trans-splicing based dual-vector factor VIII (FVIII) gene delivery is adversely affected by less efficiency of protein splicing. We sought to increase the amount of spliced ​​FVIII protein and plasma coagulation activity in dual-vector FVIII transgene in mice by means of strengthening the interaction of inteins, protein splicing elements, facilitating treatment of trans-splicing. Dual-vector delivery of the FVIII gene in cultured cells showed that replacement of Met226 in the heavy chain and Asp1828 in the light chain with Cys residues could facilitate inter- chain disulfide linking and improve protein trans-splicing, increasing the levels of spliced ​​FVIII protein. In this study, C57BL / 6 mice were coadministered dual vectors of intein-fused human FVIII heavy chain and light chain with Cys mutations via portal vein injection into the liver. Forty-eight hours post-injection, plasma was collected and analyzed for FVIII antigen concentration and coagulation activity. ed circulating FVIII heavy chain polypeptide (442 ± 151 ng mL-1 vs. 305 ± 103 ng mL-1) and coagulation activity (1.46 ± 0.37 IU mL-1 vs.0.85 ± 0.23 IU mL-1) -administered dual vectors expressing the heavy and light chains of wild-type FVIII. Moreover, coagulation activity was similar to that of mice receiving a single vector expressing FVIII (1.79 ± 0.59 IU mL-1). These findings indicate that improving protein trans- splicing by inter-chain disulfide bonding is a promising approach for increasing the efficacy of dual-vector based FVIII gene transfer.