Zhang Yu, Li Hui, Nishiyama Takahiko, McAnally John R, Sanchez-Ortiz Efrain, Huang Jian, Mammen Pradeep P A, Bassel-Duby Rhonda, Olson Eric N
Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
Senator Paul D. Wellstone Muscular Dystrophy Specialized Research Center, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
Mol Ther Nucleic Acids. 2022 Aug 1;29:525-537. doi: 10.1016/j.omtn.2022.07.024. eCollection 2022 Sep 13.
Duchenne muscular dystrophy (DMD) is a lethal neuromuscular disease caused by mutations in the X-linked dystrophin () gene. Exon deletions flanking exon 51, which disrupt the dystrophin open reading frame (ORF), represent one of the most common types of human DMD mutations. Previously, we used clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) gene editing to restore the reading frame of exon 51 in mice and dogs with exon 50 deletions. Due to genomic sequence variations between species, the single guide RNAs (sgRNAs) used for DMD gene editing are often not conserved, impeding direct clinical translation of CRISPR-Cas therapeutic gene-editing strategies. To circumvent this potential obstacle, we generated a humanized DMD mouse model by replacing mouse exon 51 with human exon 51, followed by deletion of mouse exon 50, which disrupted the dystrophin ORF. Systemic CRISPR-Cas9 gene editing using an sgRNA that targets human exon 51 efficiently restored dystrophin expression and ameliorated pathologic hallmarks of DMD, including histopathology and grip strength in this mouse model. This unique DMD mouse model with the human genomic sequence allows assessment of clinically relevant gene editing strategies as well as other therapeutic approaches and represents a significant step toward therapeutic translation of CRISPR-Cas9 gene editing for correction of DMD.
杜兴氏肌营养不良症（DMD）是一种由X连锁肌营养不良蛋白（）基因突变引起的致死性神经肌肉疾病。位于外显子51侧翼的外显子缺失会破坏肌营养不良蛋白的开放阅读框（ORF），这是人类DMD突变最常见的类型之一。此前，我们利用成簇规律间隔短回文重复序列（CRISPR）和CRISPR相关蛋白（Cas）基因编辑技术，在患有外显子50缺失的小鼠和犬中恢复了外显子51的阅读框。由于物种间基因组序列的差异，用于DMD基因编辑的单向导RNA（sgRNA）通常不保守，这阻碍了CRISPR-Cas治疗性基因编辑策略的直接临床转化。为了规避这一潜在障碍，我们通过将小鼠外显子51替换为人外显子51，然后删除破坏肌营养不良蛋白ORF的小鼠外显子50，构建了一种人源化DMD小鼠模型。在该小鼠模型中，使用靶向人外显子51的sgRNA进行全身CRISPR-Cas9基因编辑，有效地恢复了肌营养不良蛋白的表达，并改善了DMD的病理特征，包括组织病理学和握力。这种具有人类基因组序列的独特DMD小鼠模型能够评估临床相关的基因编辑策略以及其他治疗方法，代表了CRISPR-Cas9基因编辑用于纠正DMD的治疗性转化的重要一步。
