一种分泌和活性改善的新型人凝血因子VIII变构体

甲型血友病基因治疗疗效受凝血因子VIII(FVIII)表达水平低下的制约。FVIII分泌低效,且活化产物的A2区易于自发脱落使其容易失活。犬属FVIII(cFVIII)的胞内Furin水解酶裂解位点变异,使其主要以单链多肽形式分泌,而人源FVIII(hFVIII)受Furin水解,主要以重链(HC)和轻链(LC)非共价二聚体形式分泌,这种差异使cFVIII分泌性和比活性均高于hFVIII。我们前期的工作在人源B结构域缺失型FVIII(hBDD-FVIII)HC的A2与轻链LC的A3区引入链间二硫键表明,HC和LC二聚体的分泌性得到改善,活化产物稳定性和血浆凝血活性提高。本文构建一种新型hBDD-FVIII变构体F8C-RH,包含链间二硫键突变M226C/D1828C和Furin水解位点突变R1645H。培养的CHO和BHK细胞转基因表明,F8C-RH的分泌性和活性有明显改善。小鼠门静脉注射肝靶向转基因,血浆F8C-RH浓度和凝血活性均明显升高。为进一步腺相关病毒(AAV)载体转F8C-RH的甲型血友病基因治疗研究提供了依据。

A Novel Variant of Human Coagulation Factor VIII with Improved Secretion and Activity

Gene therapy for hemophilia A is limited by the low expression levels of coagulation factor VIII(FVIII).Secretion of FVIII is inefficient and spontaneous dissociation of A2 domain from its active products leads to lower bioavailability.The secretion and activity of canine FVIII(cFVIII)are higher than that of human FVIII(hFVIII).Mutation of the intracellular Furin site the cFVIII results in its secretion mainly as single-chain polypeptides,while the secretion of hFVIII forms heterodimer after Furin hydrolysis.We previously demonstrated that a human B-domain-deleted FVIII(hBDD-FVIII)with an engineered disulfide bond between A2 domain in heavy chain and A3 domain in light chain could increase functional hBDD-FVIII plasma levels by promoting their intracellular assembly of the co-expressed heavy and light chains.In this study,we constructed a novel hBDD-FVIII variant F8C-RH,which includes an assumed inter-chain disulfide point mutations M226C/D1828C and an amino acid change at its Furin cleavage site(position R1645H)as the same as cFVIII sequence.Secretion and activity were improved by analysis of expression products of F8C-RH from cultured CHO and BHK cells.F8C-RH exhibited an increased plasma levels and biological activity after liver-targeted gene transfer via the portal vein injection in mice.It provided evidence for ongoing study of gene therapy using AAV vectors delivering the F8C-RH gene.