CRISPR-Cas系统介导的dystrophin基因转录激活研究

Pseudohypertrophy muscular dystrophy is an X-linked recessive disorder caused by dystrophin gene mutations, including Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). The disease is highly disabling with poor prognosis. Since drug treatment cannot reverse the progression of the disease, gene therapy has become a research hot spot. However, BMD is still lack of effective gene therapy. Moreover, even though a variety of gene editing technologies for DMD can restore functional dystrophin protein after gene repairing, the expression of protein is low, which is difficult to significantly improve the motor function of patients. In recent years, CRISPR/Cas9 system for transcriptional activation has been proved to be able to activate the expression of targeted endogenous gene. Therefore, our project intends to activate dystrophin gene expression by CRISPR/ Cas9 system. For BMD, dystrophin gene is activated to upgrade the level of the truncated and functional dystrophin on patient-derived myoblasts and iPS cells (induced pluripotent stem cells); for DMD, dystrophin gene is further activated to improve the restored dystrophin protein after gene repairing on patient-derived myoblasts. At the same time, transcriptional activation on dystrophin gene is performed in Mdx mice after gene editing. Finally, we provide new methods and evidence through the in vitro and in vivo tests for the future treatment of DMD/BMD.

假肥大型肌营养不良症包括杜氏型（DMD）和贝氏型（BMD），由dystrophin基因突变致病。药物无法治愈该病，基因治疗已成为研究热点。然而，BMD目前缺乏有效的基因治疗方式；针对DMD的多种基因编辑技术虽然在基因修复后能重新获得dystrophin蛋白，但蛋白量低，难以明显改善患者症状。近期CRISPR/Cas9系统介导的基因激活技术被证实能高效促进目的基因表达。本项目拟运用CRISPR/Cas9介导的基因转录激活技术对dystrophin基因进行表达调控：在BMD成肌细胞和iPSC水平调控dystrophin基因转录表达，上调dystrophin蛋白；在DMD成肌细胞水平先进行有效的基因修复后，进一步激活dystrophin基因转录表达，以提高重新获得的dystrophin蛋白表达量；同时在基因修复后Mdx鼠上验证体内dystrophin基因转录激活作用，为该病的治疗提供新的方法。

假肥大型肌营养不良症包括杜氏型（DMD）和贝氏型（BMD），由dystrophin基因突变致病。药物无法治愈该病，基因治疗已成为研究热点。然而，BMD目前缺乏有效的基因治疗方式；针对DMD的多种基因编辑技术虽然在基因修复后能重新获得dystrophin蛋白，但蛋白量低，难以明显改善患者症状。本研究紧密围绕运用CRISPR/Cas介导的基因转录激活技术对dystrophin基因进行表达调控这一核心问题开展。在主要工作中，首先构建了DMD/BMD各基因突变类型和非DMD/BMD对照组成肌细胞模型平台。接着设计并筛选出了有效的sgRNA。进一步运用CRISPR/Cas介导的靶基因激活SAM系统，在BMD患者来源的成肌细胞和正常对照成肌细胞中成功实现对dystrophin基因的转录激活作用，证实该靶基因激活系统能够有效激活全长dystrophin基因和整码突变的dystrophin基因，明显提高dystrophin基因的转录和蛋白表达水平，为BMD患者和症状性女性DMD携带者的基因治疗奠定基础。对于DMD移码突变的dystrophin基因转录激活探索，本研究运用CRISPR/Cas9技术成功修复DMD成肌细胞大片段重复型移码突变，使其重新表达dystrophin蛋白。在DMD成肌细胞移码突变修复后的基础上，运用CRISPR/Cas介导的靶基因激活SAM系统增加dystrophin蛋白表达。但由于经历移码突变修复和转录激活两个过程中的多次慢病毒感染导致体外成肌细胞无法耐受，转录激活技术运用于移码突变的dystrophin基因调控方式有待进一步改善和研究。同时，本研究总结分析了BMD患者临床表现和基因型特征，拓展了BMD疾病的基因谱，也为进一步推进BMD疾病综合管理提供依据。

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