以诱导多能干细胞为模型研究MYH7基因突变引起肥厚型心肌病的机制

Hypertrophic cardiomyopathy (HCM) is the most common hereditary heart disorder, which is known to be caused by monogenic mutation of sarcomeric protein coding genes. However, the pathogenesis of HCM is not well understood due to lack of effective disease models. The patient specific induced pluripotent stem cells (iPSCs) derived cardiomyocyte have been utilized to model cardiovascular diseases as a promising tool. Since the integration of the mutant protein in to the sarcomere is the direct cause of HCM, we hypothesize that abolishing the mutant gene function by gene knockout can restore the cardiomyocyte function and prevent the onset of HCM. In this proposal: (1) We will first generate iPSCs from epithelial cells isolated from the urine samples of patients carrying MYH7 R403Q heterozygous mutation (MYH7403/+); (2) We will apply CRISPR/Cas9 to generate isogenic iPSCs containing single mutant or wild type allele (MYH7+/-, MYH7403/-); (3) We will test if the single wild type allele can restore the cardiomyocyte function of MYH7+/- cell; (4) We will utilize the amplified effects of mutant protein in MYH7403/- cardiomyocyte and further investigate the pathogenesis of HCM. In summary, the novel methods applied in this study will not only bring new insight into the pathogenesis of monogenic disease, but also promote the drug discovery and gene therapy for HCM.

以往研究发现原发型肥厚型心肌病（HCM）主要由编码收缩蛋白的单基因突变引起，但由于缺乏有效的疾病模型，其病理发展过程还不清楚。我们的假说是突变基因编码的蛋白参与组成肌小节等结构后，造成了心肌收缩相关功能的紊乱，从而引发疾病。关键科学问题是能否通过特异性的敲除突变等位基因，恢复心肌功能并消除HCM。诱导多能干细胞（iPSCs）心肌模型的出现，为验证这一假说提供了可能。以经典的MYH7 R403Q突变杂合子（MYH7 403/+）iPSCs为基础，采用Cas9基因组编辑技术制备同基因iPSCs，获得只含单个突变或野生型等位基因的iPSCs（MYH7 +/-, MYH7 403/-），并检测HCM表型是否得到相应的纠正或加重。本课题研究的意义在于通过基因组编程技术，达到消除遗传性背景差异，同时放大致病基因对疾病的影响，从而快速准确的确定发病机理，并为探索潜在的药物和基因治疗方法提供了可能。

以往研究发现原发型肥厚型心肌病（HCM）主要由编码收缩蛋白的单基因突变引起，但由于缺乏有效的疾病模型，其病理发展过程还不清楚。我们的假说是突变基因编码的蛋白参与组成肌小节等结构后，造成了心肌收缩相关功能的紊乱，从而引发疾病。关键科学问题是能否通过特异性的敲除突变等位基因，恢复心肌功能并消除HCM。诱导多能干细胞（iPSCs）心肌模型的出现，为验证这一假说提供了可能。以经典的MYH7 R403Q突变杂合子（MYH7 403/+）iPSCs为基础，采用Cas9基因组编辑技术制备同基因iPSCs，获得只含单个突变或野生型等位基因的iPSCs（MYH7 +/-, MYH7 403/-），并检测HCM表型是否得到相应的纠正或加重。本课题研究的意义在于通过基因组编程技术，达到消除遗传性背景差异，同时放大致病基因对疾病的影响，从而快速准确的确定发病机理，并为探索潜在的药物和基因治疗方法提供了可能。