Shanghai Institute of Medical Genetics, Children's Hospital of Shanghai, Shanghai Jiao Tong University School of Medicine, Shanghai 200040, China.
Shanghai Institute of Medical Genetics, Children's Hospital of Shanghai, Shanghai Jiao Tong University School of Medicine, Shanghai 200040, China; Key Laboratory of Embryo Molecular Biology, Ministry of Health & Shanghai Key Laboratory of Embryo and Reproduction Engineering, Shanghai 200040, China.
J Genet Genomics. 2013 Dec 20;40(12):617-28. doi: 10.1016/j.jgg.2013.11.002. Epub 2013 Nov 16.
Hemophilia A (HA) is an inherited X-linked recessive bleeding disorder caused by coagulant factor VIII (FVIII) deficiency. Previous studies showed that introduction of mesenchymal stem cells (MSCs) modified by FVIII-expressing retrovirus may result in phenotypic correction of HA animals. This study aimed at the investigation of an alternative gene therapy strategy that may lead to sustained FVIII transgene expression in HA mice. B-domain-deleted human FVIII (hFVIIIBD) vector was microinjected into single-cell embryos of wild-type mice to generate a transgenic mouse line, from which hFVIIIBD-MSCs were isolated, followed by transplantation into HA mice. RT-PCR and real-time PCR analysis demonstrated the expression of hFVIIIBD in multi-organs of recipient HA mice. Immunohistochemistry showed the presence of hFVIIIBD positive staining in multi-organs of recipient HA mice. ELISA indicated that plasma hFVIIIBD level in recipient mice reached its peak (77 ng/mL) at the 3rd week after implantation, and achieved sustained expression during the 5-week observation period. Plasma FVIII activities of recipient HA mice increased from 0% to 32% after hFVIIIBD-MSCs transplantation. APTT (activated partial thromboplastin time) value decreased in hFVIIIBD-MSCs transplanted HA mice compared with untreated HA mice (45.5 s vs. 91.3 s). Our study demonstrated an effective phenotypic correction in HA mice using genetically modified MSCs from hFVIIIBD transgenic mice.
血友病 A (HA) 是一种遗传性 X 连锁隐性出血性疾病，由凝血因子 VIII (FVIII) 缺乏引起。先前的研究表明，通过表达 FVIII 的逆转录病毒修饰间充质干细胞 (MSCs) 可能导致 HA 动物的表型校正。本研究旨在研究一种替代基因治疗策略，该策略可能导致 HA 小鼠中 FVIII 转基因的持续表达。从野生型小鼠的单细胞胚胎中微注射 B 结构域缺失的人 FVIII (hFVIIIBD) 载体，以生成转基因小鼠系，从中分离 hFVIIIBD-MSCs，并将其移植到 HA 小鼠中。RT-PCR 和实时 PCR 分析表明，受者 HA 小鼠的多个器官中表达 hFVIIIBD。免疫组织化学显示受者 HA 小鼠的多个器官存在 hFVIIIBD 阳性染色。ELISA 表明，受者小鼠的血浆 hFVIIIBD 水平在植入后第 3 周达到峰值（77ng/mL），并在 5 周观察期内持续表达。hFVIIIBD-MSCs 移植后，受者 HA 小鼠的血浆 FVIII 活性从 0%增加到 32%。与未治疗的 HA 小鼠相比，hFVIIIBD-MSCs 移植的 HA 小鼠的 APTT（活化部分凝血活酶时间）值降低（45.5s 与 91.3s）。我们的研究表明，使用来自 hFVIIIBD 转基因小鼠的基因修饰 MSCs 可有效纠正 HA 小鼠的表型。
