假肥大型肌营养不良症Dystrophin基因重复及点突变的修复研究

假肥大型肌营养不良症由dystrophin基因突变所致，目前在临床上尚无有效治疗手段。基因治疗是其研究的焦点，主要策略有外显子跳跃、终止密码子通读及Crispr/Cas9基因编辑等，这些技术主要针对外显子缺失和无义突变，极少涉及重复及其它点突变。我们前期应用Crispr/Cas9技术在18-25号外显子重复突变患者来源的成肌细胞上成功实现了重复序列的完整删除，并表达出全长dystrophin蛋白。此外，还建立了各种dystrophin基因突变型成肌细胞及ips细胞系，引进了mdx小鼠模型。本项目将基于良好的前期基础，采用Crispr/Cas9技术从成肌细胞及iPS细胞两个方面对dystrophin基因各种重复突变进行修复；同时，应用最新报导的单碱基修复技术BE3和ABE系统，从成肌细胞和mdx小鼠水平对dystrophin基因各种类型点突变进行原位精确修复，为本病的治疗提供新的方向和依据。

Pseudohypertrophic muscular dystrophy is caused by mutations in dystrophin gene. There is currently no effective clinical treatment. Most of researches are focus on gene therapy, and the main strategies include exon skipping, stop codon read-through and Crispr/Cas9 gene editing. However, these technologies are mainly used for deletion and nonsense mutations, rarely involving duplication and point mutations. Our previous study successfully removed large duplicated DNA sequence and expressed full-length dystrophin protein with the application of Crispr/Cas9 technology on myoblast cells carried exon 18-25 duplications. In addition, myoblasts and iPS cell lines with various mutations in dystrophin gene were also established, and the mdx mouse model was also introduced. Based on the well-prepared foundation, this project will use Crispr/Cas9 technology on both myoblast and iPS cells to repair a variety of duplicated mutations of dystrophin gene; At the same time, with the application of the latest reported single-base editing technology BE3 and ABE system, accurate repairing for point mutations in dystrophin gene will be performed on myoblasts and mdx mice, which may provide a new direction and basis for the treatment of this disease in the future.