Dual-vector Delivery of Thr664Cys and Thr1826Cys Mutated BDD-FVIII Gene

Abstract: Dual-vector co-transfer of coagulation factor VIII(FVIII) has been used as an alternative strategy to overcome packaging limitation of adeno-associated virus (AAV) vectors in hemophilia A gene therapy， but leading to a chain imbalance problem for an inefficient heavy chain secretion. To improve heavy chain secretion， here we aimed to develop a strategy to enhance the interaction of heavy and light chains by introducing a disulfide linking between both chains. A pair of vectors was expressing Tyr664 to Cys mutated heavy chain and Thr1826 to Cys mutated light chain and co-transfected into cultured HEK293 cells to investigate the gene expression， heavy chain and bioactivity secreted in the culture medium. A disufide-crosslinked heavy and light chains dimer was observed from total cellular protein by Western blot under non-reduced condition. An ELISA for the heavy chain demonstrated high levels of heavy chain (125±29) ng/mL in the medium， greater than that secreted by wild-type heavy and light chains co-transfected cells (75±23) ng/mL. The bioactivity in the medium was determined by Coatest chromogenic assay showing as (0.78±0.29) U/mL higher than (0.34±0.12) U/mL in medium of wild-type heavy and light chains cotransfected cells. Thus， it suggests that inter-chain disulfide linking could improve efficacy of dual-vector delivery of FVIII gene providing a feasible approach for an in vivo study using dual-AAV vectors to transfer FVIII gene.

CSTR: 32200.14.cjcb.2012.01.0003