Division of Pediatric Cardiothoracic Surgery, Department of Surgery, University of California at San Francisco, San Francisco, Calif.
Department of Hygiene, Kawasaki Medical University, Okayama, Japan.
J Thorac Cardiovasc Surg. 2022 Apr;163(4):1479-1490.e5. doi: 10.1016/j.jtcvs.2020.05.040. Epub 2020 May 29.
Permanent loss of cardiomyocytes after myocardial infarction results in irreversible damage to cardiac function. The present study aims to enhance the cardiomyogenic efficiency of cardiosphere-derived cells (CDCs) to develop into large populations of cardiomyocytes by intrinsic activation of cardio-specific differentiation factors (Gata4, Mef2c, Nkx2-5, Hand2, and Tnnt2) by a CRISPR/dCas9 assisted transcriptional enhancement system.
Exhaustive screening was performed to identify the specific sequences in endogenous regulatory regions (enhancers and promoters) responsible for transcriptional activation of the target genes, via a CRISPR/dCas9 system fused with transcriptional activator VP64 (CRISPR-dCas9-VP64). In a rat model of acute myocardial infarction, we compared the regenerative potential and functional benefits of CDCs with or without transcriptional activation.
We identified a panel of specific CRISPR RNA targeting the enhancers and promoters, which demonstrated significantly higher expression of differentiation factors of Gata4, Hand2, and Tnnt2. The group of CDCs with transcriptional activator VP64 (CDC with VP64) showed significant improvement in the left ventricular ejection fraction (61.9% vs 52.5% and 44.1% in the CDC without transcriptional activation group and control) and decreased scar area in the heart.
We have identified endogenous regulatory regions responsible for an intrinsic activation of cardio-specific differentiation factors assisted via a CRISPR/dCas9 gene transcriptional system. The CRISPR/dCas9 system may provide an efficient and effective means of regulating Tnnt2 gene activation within stem cells. Subsequently, this system can be used to enhance transplanted CDCs differentiation potential within ischemic myocardia to better therapeutic outcomes of patients with ischemic heart disease.
心肌梗死后心肌细胞的永久性丧失导致心脏功能的不可逆损伤。本研究旨在通过 CRISPR/dCas9 辅助转录增强系统，使内源性心脏特异性分化因子（Gata4、Mef2c、Nkx2-5、Hand2 和 Tnnt2）的内在激活，将心脏球源性细胞（CDCs）的心肌生成效率提高到可产生大量心肌细胞的水平。
通过 CRISPR/dCas9 系统融合转录激活因子 VP64（CRISPR-dCas9-VP64），对靶基因的内源性调节区（增强子和启动子）中的特定序列进行了详尽的筛选，以确定其对转录激活的作用。在急性心肌梗死大鼠模型中，我们比较了经或未经转录激活的 CDCs 的再生潜力和功能益处。
我们确定了一组特定的 CRISPR RNA 靶向增强子和启动子，这些 RNA 显示出 Gata4、Hand2 和 Tnnt2 分化因子的表达显著增加。具有转录激活因子 VP64（CDC with VP64）的 CDCs 组在左心室射血分数方面（与未进行转录激活的 CDCs 组和对照组相比，分别为 61.9%、52.5%和 44.1%）和心脏瘢痕面积方面均有显著改善。
我们已经确定了内源性调节区，这些区域可通过 CRISPR/dCas9 基因转录系统辅助心脏特异性分化因子的内在激活。CRISPR/dCas9 系统可能为调节干细胞内 Tnnt2 基因的激活提供一种高效、有效的方法。随后，该系统可用于增强移植 CDCs 在缺血心肌中的分化潜能，从而改善缺血性心脏病患者的治疗效果。
