采用高特异性CRISPRa体系靶向治疗肺动脉高压

肺动脉高压（PAH）是一种可由多种基因突变和基因表达失调导致的致命性心肺血管疾病，目前尚缺乏根治性药物。采用CRISPR-Cas9体系介导的靶向转录激活技术（CRISPRa）激活PAH患者及其大鼠模型中下调的SERCA2a基因，可为这种疾病提供新颖的治疗方法。然而CRISPRa体系潜在的脱靶效应和靶向递送仍然是其在临床治疗中应用的瓶颈。本项目首先在肺动脉平滑肌细胞中，采用CRISPRa体系靶向结合SERCA2a基因的组织特异性启动子或增强子，转录激活其原位表达；然后通过RNA-seq和ChIP-seq检测其脱靶效应，构建高特异性的SERCA2a-CRISPRa体系。在大鼠PAH模型中，通过气管或雾化给药SERCA2a-CRISPRa-AAV，结合血流动力学检测和右心肥厚指数及肺血管重构等指标验证其治疗效果，进一步探究其治疗机制，为PAH的基因治疗提供实验数据和理论基础。

Pulmonary arterial hypertension (PAH) is a fatal cardiopulmonary vascular disease caused by a variety of gene mutations and gene dysregulation and there is still a lack of radical drugs. Using CRISPR-Cas9 system-mediated targeted gene activation system (CRISPRa), to up-regulate SERCA2a gene in PAH patients and its rat models, can provide a novel treatment for this disease. However, the potential off-target effects and low targeted delivery efficiency of the CRISPRa system are still the bottleneck of its application in clinical. To construct a SERCA2a-CRISPRa system with high specificity, we apply CRISPRa system to activate the tissue-specific promoter and enhancer of SERCA2a gene in pulmonary artery smooth muscle cells, transcriptionally activate the in-situ expression of the SERCA2a gene, and detect its off-target effects through RNA-seq and ChIP-seq. In rat PAH model, SERCA2a-CRISPRa-AAV was administered through trachea or aerosol, to verify its therapeutic effects and mechanism by hemodynamic testing, right heart hypertrophy index, as well as pulmonary vascular remodeling. This will provide experimental data and theoretical basis for the gene therapy of PAH.