Department of Molecular Biology.
Senator Paul D. Wellstone Muscular Dystrophy Cooperative Research Center, and.
JCI Insight. 2017 Sep 21;2(18). doi: 10.1172/jci.insight.95918.
Dystrophin maintains the integrity of striated muscles by linking the actin cytoskeleton with the cell membrane. Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene (DMD) that result in progressive, debilitating muscle weakness, cardiomyopathy, and a shortened lifespan. Mutations of dystrophin that disrupt the amino-terminal actin-binding domain 1 (ABD-1), encoded by exons 2-8, represent the second-most common cause of DMD. In the present study, we compared three different strategies for CRISPR/Cas9 genome editing to correct mutations in the ABD-1 region of the DMD gene by deleting exons 3-9, 6-9, or 7-11 in human induced pluripotent stem cells (iPSCs) and by assessing the function of iPSC-derived cardiomyocytes. All three exon deletion strategies enabled the expression of truncated dystrophin protein and restoration of cardiomyocyte contractility and calcium transients to varying degrees. We show that deletion of exons 3-9 by genomic editing provides an especially effective means of correcting disease-causing ABD-1 mutations. These findings represent an important step toward eventual correction of common DMD mutations and provide a means of rapidly assessing the expression and function of internally truncated forms of dystrophin-lacking portions of ABD-1.
肌营养不良蛋白通过将肌动蛋白细胞骨架与细胞膜连接来维持横纹肌的完整性。杜氏肌营养不良症（DMD）是由肌营养不良蛋白基因（DMD）的突变引起的，导致进行性、衰弱性肌肉无力、心肌病和寿命缩短。肌营养不良蛋白的突变破坏了由外显子 2-8 编码的氨基末端肌动蛋白结合域 1（ABD-1），这是 DMD 的第二大常见原因。在本研究中，我们比较了三种不同的 CRISPR/Cas9 基因组编辑策略，以通过删除人诱导多能干细胞（iPSC）中的 DMD 基因 ABD-1 区的外显子 3-9、6-9 或 7-11，从而纠正 ABD-1 区的突变，并评估 iPSC 衍生的心肌细胞的功能。所有三种外显子缺失策略都能够表达截断的肌营养不良蛋白，并在不同程度上恢复心肌细胞的收缩性和钙瞬变。我们表明，通过基因组编辑删除外显子 3-9 提供了纠正致病 ABD-1 突变的特别有效手段。这些发现代表了朝着最终纠正常见 DMD 突变迈出的重要一步，并提供了一种快速评估 ABD-1 缺失内部截断形式的表达和功能的方法。
