Genome Editing by CRISPR/Cas-9 to Reprogram Cardiac Mesenchymal Stromal Cells

Background and Aim: The environment of the failing and infarcted myocardium drives resident and transplanted mesenchymal stromal cells (MSCs) toward a pro-inflammatory phenotype and restricts their survival and reparative effects in a mechanism mediated by the toll-like receptor 4 (TLR4). CRISPR/Cas9 is a promising tool for genome-editing DNA in cells with single-base-pair precision, which raises hopes for therapeutic genome editing in the clinic. We aimed to test the hypothesis that ex-vivo disruption of the human TLR4 gene by CRISPR/Cas9-mediated genome editing would switch the MSCs to an anti-inflammatory, reparative phenotype that could prevent remodeling of the left ventricle post-myocardial infarction.

Methods and Results: For gene editing, we used a recombinant Cas9 nucleoprotein attached to a guide-RNA that creates a double-strand DNA break at desired locations in the genome to induce a knock-out of the downstream gene. CRISPR was electroporated into the cells to target the TLR4 gene. To evaluate the inflammatory response we tested secretome secretions from the cells.

Our preliminary results show a 21% success rate in editing the genome of the MSCs. In response, these cells were found to have significantly reduced inflammatory cytokine secretion, specifically of IL-1α (p<0.00001). Other cytokines with reduced secretion were IL-6, IL-15, IL-17, and IL-23 (p=0.2000 for all). Additionally, after finding a significant change in the exosome secretion profile from the cells (p<0.0001), we used exosomes to treat human-cardiac MSCs in a scratch assay. Exosomes from edited cells increased the fibroblast migration rate (83% scratch closure compared to 50%, after 24-hour incubation, p<0.0001).

Conclusion: Our preliminary results suggest, for the first time, that human TLR4 gene-editing by CRISPR/Cas9 blocks inflammatory secretions and facilitates a reparative response by human-cardiac MSCs. The precise and efficient genome editing of TLR4 could provide a new strategy to improve MSC-based cell therapy to improve cardiac remodeling and function.