Substitution of A1 Domain Enhances Secretion of Human BDD-FVIII Heavy Chain and Relieves Chain Imbalance Secreted by the Heavy and Light Chain Co-transgenic Cells

Abstract: Dual-vector delivery of coagulation factor VIII (FVIII) gene is an alternative strategy to overcome package limitation of adeno-associated virus (AAV) vectors， but the main drawback is chain imbalance due to the inefficient secretion of the heavy chain. It demonstrated that the inefficient heavy chain secretion is caused by binding of its A1 domain to chaperon protein of ER. Our recent work showed that protein splicing could be used for a dual-vector co-delivery of a B-domain-deleted FVIII (BDD-FVIII) heavy and light chain genes. Currently， we observed the secretion of A1 domain substituted human BDD-FVIII heavy chain by A1-domain of porcine FVIII and its effect on secretion of spliced BDD-FVIII and corresponding activity by co-transfection of an intein-fused heavy and light chain genes. The results showed that the secretion of variant heavy chain by itself displays an obvious enhancement in terms of secretion compared to the wild-type heavy chain (89±12 ng/ml vs 25±9 ng/ml). The amount of spliced variant BDD-FVIII and activity in the culture supernatants of heavy and light chain co-transgenic cells were 219±51 ng/ml and 1.47±0.22 U/ml， respectively， higher than those of human BDDFVIII heavy and light chain co-transgenic cells (116±32 ng/ml and 0.8±0.11 U/ml). The spliced variant BDD-FVIII and activity were also detected in the culture supernatant of combined cells separately transfected with variant heavy and light chain genes (38±7 ng/ml and 0.22±0.05 U/ml) indicating the cellular mechanism-independent protein splicing. It suggests that enhanced secretion of the A1-domain substituted heavy chain could improve secretion of spliced BDD-FVIII and activity and relieve the chain imbalance secreted by the heavy and light chain co-transgenic cells with a protein splicing-based dual-vector. It provided evidence for ongoing research for an in vivo BDD-FVIII heavy and light genes co-transfer by a dual-AAV vector in animal models.

CSTR: 32200.14.cjcb.2010.06.0007