Targeted addition of mini-dystrophin into rDNA locus of Duchenne muscular dystrophy patient-derived iPSCs
Targeted addition of mini-dystrophin into rDNA locus of Duchenne muscular dystrophy patient-derived iPSCs
复制标题
将微型肌营养不良蛋白定向添加到杜氏肌营养不良症患者来源的 iPSC 的 rDNA 基因座中
DOI：
10.1016/j.bbrc.2021.01.056
复制
发表时间：
2021
影响因子：
3.1
通讯作者：
Liang Desheng
中科院分区：
文献类型：
--
作者：
Zeng Baitao;Zhou Miaojin;Liu Bo;Shen Fei;Xiao Rou;Su Jiasun;Hu Zhiqing;Zhang Yiti;Gu Ao;Wu Lingqian;Liu Xionghao;Liang Desheng
杜氏肌营养不良症（DMD）是最常见的致死性肌肉疾病，每5000名男婴中就有1人患病。它是由X连锁肌营养不良基因（DMD）突变引起的，目前没有有效的治疗方法。基因添加是一种很有前途的策略，因为它对所有基因突变类型的患者具有普遍性。在这项研究中，我们描述了一种位点特异性基因添加策略，诱导多能干细胞（iPSCs）来自DMD患者与外显子50缺失。通过使用转录激活因子样效应子切口酶（TALENickases），通过同源重组以高靶向效率将微型肌养蛋白盒精确靶向核糖体RNA基因（rDNA）位点。靶向克隆保留了主要的多能性，并分化为心肌细胞。值得注意的是，在遗传校正的iPSC及其衍生的心肌细胞中，肌养蛋白表达和膜定位得以恢复。更重要的是，在修饰的心肌细胞中观察到增强的自发收缩。这些结果为DMD基因治疗的有效靶向基因添加提供了原理证明，并代表了DMD精确治疗的重要一步。
Duchenne muscular dystrophy (DMD), the most common lethal muscular disorder, affects 1 in 5000 male births. It is caused by mutations in the X-linkeddystrophingene (DMD), and there is no effective treatment currently. Gene addition is a promising strategy owing to its universality for patients with all gene mutations types. In this study, we describe a site-specific gene addition strategy in induced pluripotent stem cells (iPSCs) derived from a DMD patient with exon 50 deletion. By using transcription activator-like effector nickases (TALENickases), the mini-dystrophin cassette was precisely targeted at the ribosomal RNA gene (rDNA) locus via homologous recombination with high targeting efficiency. The targeted clone retained the main pluripotent properties and was differentiated into cardiomyocytes. Significantly, the dystrophin expression and membrane localization were restored in the genetic corrected iPSCs and their derived cardiomyocytes. More importantly, the enhanced spontaneous contraction was observed in modified cardiomyocytes. These results provide a proof of principle for an efficient targeted gene addition for DMD gene therapy and represents a significant step toward precisely therapeutic for DMD.