• Toshikazu Sano, Tatsuo Ito, Shuta Ishigami, Srinivas Bandaru, and Shunji Sano.
• Division of Pediatric Cardiothoracic Surgery, Department of Surgery, University of California at San Francisco, San Francisco, Calif.
• J. Thorac. Cardiovasc. Surg. 2022 Apr 1; 163 (4): 14791490.e51479-1490.e5.

ObjectivePermanent 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.MethodsExhaustive 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.ResultsWe 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.ConclusionsWe 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.Published by Elsevier Inc.

24 keywords...

hide…