双载体转重链Tyr664Cys和轻链Thr1286Cys突变体BDD-FVⅢ基因

摘要用双载体转运凝血VⅢ因子基因在甲型血友病基因治疗研究中可克服AAV毒载体容量限制，但存在重链分泌低效和链不均衡性问题。为探索重、轻链间二硫键形成对重链分泌的促进作用，该丈用双载体转B结构域大部缺失型FVⅢ（BDD-FVⅢ）的重链和轻链基因，将重链的Tyr664和轻链Thr1826突变为Cys，研究了HEK293细胞共转基因后的基因表达、分泌至培养上清的重链量和凝血生物活性。用Western blot检测细胞裂解液结果显示，非还原条件下有明显的二硫键交联的重、轻链蛋白；链特异性ELISA定量检测细胞分泌的重链为（125＋29）ng／mL，明显高于共转野生型重链和轻链基因细胞的（75＋23）ng／mL；Coatest法显示细胞分泌的凝血活性为（0．784±0．29）U／mL．也明显高于共转野生型重链和轻链基因细胞（0．34＋0．12）U／mL。结果表明，重、轻链间的二硫键形成可提高双载体转FVⅢ基因的功效，为进一步在动物体内转基因提供了实验依据。

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

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 lead-ing 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 highlevels of heavy chain （125±29） ng/mL in the medium, greater than that secreted by wild-type heavy and light chainsco-transfected cells （75±23） ng/mL. The bioactivity in the medium was determined by Coatest chromogenic assayshowing as （0.784±0.29） U/mL higher than （0.344±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 deliveryof FVⅢ gene providing a feasible approach for an in vivo study using dual-AAV vectors to transfer FVⅢ gene.