Müthel Stefanie, Marg Andreas, Ignak Busem, Kieshauer Janine, Escobar Helena, Stadelmann Christian, Spuler Simone
Max-Delbrück-Center for Molecular Medicine in the Helmholtz Association (MDC), 13125 Berlin, Germany.
Muscle Research Unit at the Experimental and Clinical Research Center, a Cooperation Between the Max-Delbrück-Center for Molecular Medicine in the Helmholtz Association (MDC) and the Charité-Universitätsmedizin Berlin, 13125 Berlin, Germany.
Mol Ther Nucleic Acids. 2023 Feb 5;31:494-511. doi: 10.1016/j.omtn.2023.02.005. eCollection 2023 Mar 14.
With thousands of patients worldwide, c.550delA is the most frequent mutation causing severe, progressive, and untreatable limb girdle muscular dystrophy. We aimed to genetically correct this founder mutation in primary human muscle stem cells. We designed editing strategies providing CRISPR-Cas9 as plasmid and mRNA first in patient-derived induced pluripotent stem cells and applied this strategy then in primary human muscle stem cells from patients. Mutation-specific targeting yielded highly efficient and precise correction of c.550delA to wild type for both cell types. Most likely a single cut generated by SpCas9 resulted in a 5' staggered overhang of one base pair, which triggered an overhang-dependent base replication of an A:T at the mutation site. This recovered the open reading frame and the DNA sequence was repaired template-free to wild type, which led to mRNA and protein expression. Off-target analysis using amplicon sequencing of 43 predicted sites demonstrates the safety of this approach. Our study extends previous usage of single cut DNA modification since our gene product has been repaired into the wild-type sequence with the perspective of a real cure.
在全球数千名患者中，c.550delA是导致严重、进行性且无法治疗的肢带型肌营养不良症的最常见突变。我们旨在对原代人类肌肉干细胞中的这种奠基者突变进行基因校正。我们首先在患者来源的诱导多能干细胞中设计了以质粒和信使核糖核酸形式提供CRISPR-Cas9的编辑策略，然后将该策略应用于患者的原代人类肌肉干细胞。针对突变的靶向操作对两种细胞类型都产生了高效且精确的将c.550delA校正为野生型的效果。很可能是SpCas9产生的单次切割导致了一个碱基对的5' 交错末端，这触发了突变位点处A:T的末端依赖性碱基复制。这恢复了开放阅读框，并且DNA序列在无模板的情况下被修复为野生型，从而导致信使核糖核酸和蛋白质表达。使用43个预测位点的扩增子测序进行的脱靶分析证明了该方法的安全性。我们的研究扩展了之前单次切割DNA修饰的应用，因为我们的基因产物已从治疗的角度被修复为野生型序列。
