Research Center for Industries of the Future, Westlake University, Hangzhou 310024, Zhejiang, China; Center for Genome Editing, Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou 310024, Zhejiang, China; Key Laboratory of Growth Regulation and Translational Research of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou 310024, Zhejiang, China; Institute of Basic Medical Sciences, Westlake Institute for Advanced Study, Hangzhou 310024, Zhejiang, China; Shanghai Institute of Nutrition and Health, Shanghai Institutes for Biological Sciences, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
Research Center for Industries of the Future, Westlake University, Hangzhou 310024, Zhejiang, China; Center for Genome Editing, Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou 310024, Zhejiang, China; Key Laboratory of Growth Regulation and Translational Research of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou 310024, Zhejiang, China; Institute of Basic Medical Sciences, Westlake Institute for Advanced Study, Hangzhou 310024, Zhejiang, China.
Cell Rep. 2023 Nov 28;42(11):113340. doi: 10.1016/j.celrep.2023.113340. Epub 2023 Oct 30.
Duchenne muscular dystrophy (DMD) is a severe genetic disease caused by the loss of the dystrophin protein. Exon skipping is a promising strategy to treat DMD by restoring truncated dystrophin. Here, we demonstrate that base editors (e.g., targeted AID-mediated mutagenesis [TAM]) are able to efficiently induce exon skipping by disrupting functional redundant exonic splicing enhancers (ESEs). By developing an unbiased and high-throughput screening to interrogate exonic sequences, we successfully identify novel ESEs in DMD exons 51 and 53. TAM-CBE (cytidine base editor) induces near-complete skipping of the respective exons by targeting these ESEs in patients' induced pluripotent stem cell (iPSC)-derived cardiomyocytes. Combined with strategies to disrupt splice sites, we identify suitable single guide RNAs (sgRNAs) with TAM-CBE to efficiently skip most DMD hotspot exons without substantial double-stranded breaks. Our study thus expands the repertoire of potential targets for CBE-mediated exon skipping in treating DMD and other RNA mis-splicing diseases.
杜氏肌营养不良症（DMD）是一种由肌营养不良蛋白缺失引起的严重遗传疾病。外显子跳跃是一种有前途的治疗 DMD 的策略，通过恢复截短的肌营养不良蛋白来实现。在这里，我们证明碱基编辑器（例如，靶向 AID 介导的诱变 [TAM]）能够通过破坏功能冗余的外显子剪接增强子（ESEs）来有效地诱导外显子跳跃。通过开发一种无偏和高通量的筛选方法来研究外显子序列，我们成功地在 DMD 外显子 51 和 53 中鉴定出了新的 ESE。TAM-CBE（胞嘧啶碱基编辑器）通过靶向这些 ESE 在患者诱导多能干细胞（iPSC）衍生的心肌细胞中诱导相应外显子的近乎完全跳跃。结合破坏剪接位点的策略，我们使用 TAM-CBE 鉴定出合适的单指导 RNA（sgRNA），可有效地跳过大多数 DMD 热点外显子，而不会产生大量双链断裂。因此，我们的研究扩展了 CBE 介导的外显子跳跃治疗 DMD 和其他 RNA 错剪接疾病的潜在靶标范围。
