Pang Jialun, Wu Yong, Li Zhuo, Hu Zhiqing, Wang Xiaolin, Hu Xuyun, Wang Xiaoyan, Liu Xionghao, Zhou Miaojin, Liu Bo, Wang Yanchi, Feng Mai, Liang Desheng
State Key Laboratory of Medical Genetics, School of Life Sciences, Central South University, Changsha, Hunan, China.
State Key Laboratory of Medical Genetics, School of Life Sciences, Central South University, Changsha, Hunan, China.
Biochem Biophys Res Commun. 2016 Mar 25;472(1):144-9. doi: 10.1016/j.bbrc.2016.02.083. Epub 2016 Feb 24.
Hemophilia A (HA) is a monogenic disease due to lack of the clotting factor VIII (FVIII). This deficiency may lead to spontaneous joint hemorrhages or life-threatening bleeding but there is no cure for HA until very recently. In this study, we derived induced pluripotent stem cells (iPSCs) from patients with severe HA and used transcription activator-like effector nickases (TALENickases) to target the factor VIII gene (F8) at the multicopy ribosomal DNA (rDNA) locus in HA-iPSCs, aiming to rescue the shortage of FVIII protein. The results revealed that more than one copy of the exogenous F8 could be integrated into the rDNA locus. Importantly, we detected exogenous F8 mRNA and FVIII protein in targeted HA-iPSCs. After they were differentiated into endothelial cells (ECs), the exogenous FVIII protein was still detectable. Thus, it is showed that the multicopy rDNA locus could be utilized as an effective target site in patient-derived iPSCs for gene therapy. This strategy provides a novel iPSCs-based therapeutic option for HA and other monogenic diseases.
甲型血友病（HA）是一种由于缺乏凝血因子VIII（FVIII）导致的单基因疾病。这种缺乏可能导致自发性关节出血或危及生命的出血，但直到最近，HA仍无法治愈。在本研究中，我们从重度HA患者中获得诱导多能干细胞（iPSC），并使用转录激活样效应核酸酶（TALENickases）靶向HA-iPSC中多拷贝核糖体DNA（rDNA）位点的凝血因子VIII基因（F8），旨在挽救FVIII蛋白的短缺。结果显示，多个拷贝的外源F8可以整合到rDNA位点。重要的是，我们在靶向的HA-iPSC中检测到了外源F8 mRNA和FVIII蛋白。在它们分化为内皮细胞（EC）后，仍可检测到外源FVIII蛋白。因此，表明多拷贝rDNA位点可作为患者来源iPSC中基因治疗的有效靶点。该策略为HA和其他单基因疾病提供了一种基于iPSC的新型治疗选择。
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