Life Science College of Ludong University, Yantai 264025, China.
Sci China Life Sci. 2012 Jun;55(6):521-6. doi: 10.1007/s11427-012-4333-8. Epub 2012 Jun 29.
Hemophilia A is caused by a genetic mutation in coagulation factor VIII (FVIII) gene and gene therapy is considered to be a promising strategy for its treatment. We recently demonstrated that co-delivery of two vectors expressing M662C mutated heavy and D1828C mutated light chain genes of B-domain-deleted coagulation factor VIII (BDD-FVIII) leads to inter-chain disulfide cross-linking and improved heavy chain secretion in vitro. In this study, co-injection of both M662C and D1828C mutated BDD-FVIII gene expression vectors into mice resulted in increased heavy chain secretion and coagulation activity in plasma in vivo. Approximately (239 ± 56) ng mL(-1) above endogenous levels of transgenic FVIII heavy chain was found in mouse plasma using a chain-specific ELISA. For FVIII coagulation activity, approximately (1.09 ± 0.25) IU mL(-1) above endogenous levels were detected in co-injected transgenic mouse plasma using a chromogenic assay. These data demonstrate that inter-chain disulfide bonds likely increase heavy chain secretion and coagulation activity in the plasma of transgenic mice with an improved efficacy of a dual-vector delivery of BDD-FVIII gene. These findings support our ongoing efforts to develop a gene therapy for hemophilia A treatment using dual-AAV vectors.
血友病 A 是由凝血因子 VIII (FVIII) 基因突变引起的，基因治疗被认为是其治疗的一种有前途的策略。我们最近证明，共递送两种载体，表达 B 结构域缺失凝血因子 VIII (BDD-FVIII) 的 M662C 突变重链和 D1828C 突变轻链基因，可导致链间二硫键交联，并提高体外重链分泌。在这项研究中，将两种 M662C 和 D1828C 突变的 BDD-FVIII 基因表达载体共注射到小鼠体内，导致体内血浆中重链分泌和凝血活性增加。使用链特异性 ELISA 在小鼠血浆中检测到转基因 FVIII 重链的水平比内源性水平高出约 (239 ± 56) ng mL(-1)。使用显色测定法，在共注射的转基因小鼠血浆中检测到 FVIII 凝血活性比内源性水平高出约 (1.09 ± 0.25) IU mL(-1)。这些数据表明，链间二硫键可能会增加转基因小鼠血浆中的重链分泌和凝血活性，从而提高 BDD-FVIII 基因的双 AAV 载体传递的疗效。这些发现支持我们正在努力开发用于治疗血友病 A 的基因治疗方法，使用双 AAV 载体。
