Xiang Xi, Zhao Xiaoying, Pan Xiaoguang, Dong Zhanying, Yu Jiaying, Li Siyuan, Liang Xue, Han Peng, Qu Kunli, Jensen Jonas Brorson, Farup Jean, Wang Fei, Petersen Trine Skov, Bolund Lars, Teng Huajing, Lin Lin, Luo Yonglun
Lars Bolund Institute of Regenerative Medicine, Qingdao-Europe Advanced Institute for Life Sciences, BGI-Qingdao, BGI-Shenzhen, Qingdao 266555, China.
Department of Biomedicine, Aarhus University, Aarhus 8000, Denmark.
Mol Ther Nucleic Acids. 2021 Mar 13;24:403-415. doi: 10.1016/j.omtn.2021.03.005. eCollection 2021 Jun 4.
CRISPR gene therapy is one promising approach for treatment of Duchenne muscular dystrophy (DMD), which is caused by a large spectrum of mutations in the dystrophin gene. To broaden CRISPR gene editing strategies for DMD treatment, we report the efficient restoration of dystrophin expression in induced myotubes by SpCas9 and dual guide RNAs (gRNAs). We first sequenced 32 deletion junctions generated by this editing method and revealed that non-homologous blunt-end joining represents the major indel type. Based on this predictive repair outcome, efficient in-frame deletion of a part of exon 51 was achieved in HEK293T cells with plasmids expressing SpCas9 and dual gRNAs. More importantly, we further corrected a frameshift mutation in human (exon45del) fibroblasts with SpCas9-dual gRNA ribonucleoproteins. The edited DMD fibroblasts were transdifferentiated into myotubes by lentiviral-mediated overexpression of a human transcription factor. Restoration of expression at both the mRNA and protein levels was confirmed in the induced myotubes. With further development, the combination of SpCas9-dual gRNA-corrected DMD patient fibroblasts and transdifferentiation may provide a valuable therapeutic strategy for DMD.
CRISPR基因疗法是治疗杜氏肌营养不良症（DMD）的一种有前景的方法，该疾病由肌营养不良蛋白基因中的大量突变引起。为了拓宽用于DMD治疗的CRISPR基因编辑策略，我们报告了通过SpCas9和双导向RNA（gRNAs）在诱导的肌管中有效恢复肌营养不良蛋白表达的情况。我们首先对通过这种编辑方法产生的32个缺失连接进行了测序，并揭示非同源平端连接是主要的插入缺失类型。基于这种预测的修复结果，在表达SpCas9和双gRNAs的质粒的HEK293T细胞中实现了外显子51一部分的有效框内缺失。更重要的是，我们用SpCas9-双gRNA核糖核蛋白进一步校正了人（外显子45缺失）成纤维细胞中的移码突变。通过慢病毒介导的人类转录因子过表达，将编辑后的DMD成纤维细胞转分化为肌管。在诱导的肌管中证实了mRNA和蛋白质水平上的表达恢复。随着进一步发展，SpCas9-双gRNA校正的DMD患者成纤维细胞与转分化的结合可能为DMD提供一种有价值的治疗策略。
