Abstract 16620: Enhancing transplanted Cardiac Progenitor Cells homing and survival in Ischemic Heart By CRISPR-dcas9/sgRNA-based Endogenous CXCR4 Promoter Activation

Abstract

Cardiac progenitor cell (CPC) therapy is promising for repairing the damaged heart, limited stem cell survivability post-transplantation has precluded widespread use of this therapy. Recent reports indicate that interactions between the CXC chemokine stromal cell-derived factor 1 and its receptor CXC chemokine receptor 4 (CXCR4) critically mediate the ischemia-induced recruitment of bone marrow-derived circulating stem/progenitor cells, but the expression of CXCR4 in cardiac progenitor cells is very low. Strategies to overexpress CXCR4 genes in CPC have been shown to improve the survival of donor cells within the ischemic myocardium. The CRISPR/dCas9-VP64/sgRNA activation system (CAS), was recently developed as a simple and efficient method in activation of endogenous loci by targeting their promoter region in mammalian cells. With single guide chimeric RNA's (sgRNA) targeting proximal promoter region of CXCR4, we found that the CAS activation system can upregulate protein level of endogenous CXCR4 gene expression about 140 fold by qRT-PCR. The CAS -CXCR4 improves CPC response to SDF-1 induced migration and survival against H2O2 induced oxidative stress. Half million CAS-Control or CAS-CXCR4 CPC were intramyocardially injected into the peri-infarction zone after myocardial infarction (MI) induced by left anterior descending coronary artery (LAD) ligation in mice. The CAS- CXCR4 gene modification improved CPC survival by bioluminescence imaging (BLI), and CAS-CXCR4 CPC also exhibited improved left ventricular (LV) ejection fraction (EF) at 3 wk after MI (n=6, P<0.05). Our results demonstrate that activation of endogenous CXCR4 expression by CRISPR/dCas9-VP64 activation system using sgRNA is very effective in improving homing and survival of donor CPC, and attenuate cardiomyopathy after MI.