Department of Molecular Biology, Hamon Center for Regenerative Science and Medicine, Senator Paul D. Wellstone Muscular Dystrophy Cooperative Research Center, University of Texas Southern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA.
Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
Sci Transl Med. 2017 Nov 29;9(418). doi: 10.1126/scitranslmed.aan8081.
Duchenne muscular dystrophy (DMD) is a severe, progressive muscle disease caused by mutations in the dystrophin gene. The majority of DMD mutations are deletions that prematurely terminate the dystrophin protein. Deletions of exon 50 of the dystrophin gene are among the most common single exon deletions causing DMD. Such mutations can be corrected by skipping exon 51, thereby restoring the dystrophin reading frame. Using clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9), we generated a DMD mouse model by deleting exon 50. These ΔEx50 mice displayed severe muscle dysfunction, which was corrected by systemic delivery of adeno-associated virus encoding CRISPR/Cas9 genome editing components. We optimized the method for dystrophin reading frame correction using a single guide RNA that created reframing mutations and allowed skipping of exon 51. In conjunction with muscle-specific expression of Cas9, this approach restored up to 90% of dystrophin protein expression throughout skeletal muscles and the heart of ΔEx50 mice. This method of permanently bypassing DMD mutations using a single cut in genomic DNA represents a step toward clinical correction of DMD mutations and potentially those of other neuromuscular disorders.
杜氏肌营养不良症（DMD）是一种严重的进行性肌肉疾病，由肌营养不良蛋白基因突变引起。大多数 DMD 突变是导致肌营养不良蛋白过早终止的缺失。肌营养不良蛋白基因外显子 50 的缺失是导致 DMD 的最常见的单个外显子缺失之一。通过跳过外显子 51，可以纠正这种突变，从而恢复肌营养不良蛋白的阅读框。我们使用成簇的规则间隔的短回文重复序列/CRISPR 相关 9（CRISPR/Cas9），通过删除外显子 50 产生了 DMD 小鼠模型。这些 ΔEx50 小鼠表现出严重的肌肉功能障碍，通过系统递送编码 CRISPR/Cas9 基因组编辑组件的腺相关病毒得到纠正。我们使用单链向导 RNA 优化了肌营养不良蛋白阅读框校正的方法，该方法产生了重新框架突变并允许跳过外显子 51。与肌特异性表达 Cas9 结合，这种方法可在 ΔEx50 小鼠的骨骼肌和心脏中恢复高达 90%的肌营养不良蛋白表达。这种使用单个 DNA 碱基对切割永久性绕过 DMD 突变的方法，代表了朝着临床纠正 DMD 突变以及潜在的其他神经肌肉疾病突变迈出的一步。
