DYS△45-55基因修复的人脂肪干细胞源性肌肉祖细胞移植DMD模型鼠促进肌肉再生的研究

Duchenne muscular dystrophy (DMD) is a common fatal genetic disease caused by dystrophin gene mutation and there is no effective treatment for this disease. Our previous study indicated that adipose stem cells can be efficiently transduced into myogenic progenitors, which have the same characteristics as satellite cells. Transplantation of the cells to mdx mouse confirmed its therapeutic effect in vivo. It has been reported that a class of common deletions in the exon 45–55 mutation hotspot region within the dystrophin gene leads to the expression of a truncated, but functional, dystrophin protein. We successfully employed the CRISPR/Cas9 system to restore the expression of dystrophin gene in DMD patient derived muscle stem cells by deleting the mutational hotspot at exons 45-55. Therefore, we propose the following hypothesis: CRISPR / Cas9 technology was employed to correct dystrophin gene of DMD patient derived adipose stem cells, which can be transduced into myogenic progenitors after induction. These myogenic progenitors will be transplanted into mdx mice. We intended to construct a cell model of CRISPR / Cas9- DYS△45-55-ADSCs-MPs in vitro and then explore the effect of transplantation and the mechanism of muscle regeneration. In this study, we combined gene editing and stem cell transplantation, which provided a new strategy for the individualized treatment of DMD.

Duchenne型肌营养不良症（DMD）是因dystrophin基因（DYS）突变引起的致死性遗传病，目前无有效疗法。我们前期成功将同种鼠源脂肪干细胞高效诱导成具有肌卫星细胞特性的肌肉祖细胞，且移植DMD模型鼠疗效显著。文献报道，DYS基因45-55号外显子是热点突变区，敲除该区可恢复DYS表达及功能。我们预实验采用CRISPR/Cas9成功敲除DMD患者来源肌肉干细胞的DYS热点突变区，并观察到DYS基因的再表达。由此，我们提出科学假说：CRISPR/Cas9修复的DMD患者来源脂肪干细胞，经高效诱导成肌肉祖细胞后移植治疗DMD模型鼠。我们首先构建人CRISPR/Cas9-DYS△45-55-ASCs-MPs细胞模型，然后动物验证移植后其在体内存活、肌肉再生和对运动功能影响并探讨成肌机制。本研究将基因修复和干细胞移植相结合，为自体干细胞治疗DMD患儿的临床转化提供新的方案和策略。

Duchenne型肌营养不良症（DMD）是因dystrophin基因突变引起的致死性遗传病，目前无有效疗法。我们首先构建人CRISPR/Cas9-DYS△45-55-SCs细胞模型，然后动物验证移植后其在体内存活、肌肉再生和对运动功能影响并探讨成肌机制。本研究运用Crispr/Cas9技术修正DMD患者来源的干细胞，恢复dystrophin蛋白的表达，并进行脱靶和表达谱的分析。然后将修正的干细胞移植入DMD模型鼠体内，结果表明人源干细胞能在鼠体内分化为成熟的肌纤维。同时证实了基因编辑的DMD来源的肌纤维恢复了dystrophin的表达和功能。本研究采用基因编辑联合干细胞移植的方法为肌营养不良症提供新的治疗策略。

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