CRISPR/Cas9-based knockout of the TLR4 gene enhances secretion of extracellular vesicles with anti-Inflammatory properties from human 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 is a promising tool for genome editing of DNA in cells, which raises hope for therapeutic genome editing in the clinic. We hypothesize that ex-vivo knockout (KO) of the human TLR4 gene by CRISPR would switch human cardiac MSCs (hMSCs) to an anti-inflammatory, reparative phenotype that could prevent remodeling of the left ventricle after myocardial infarction.

Methods and Results: For gene editing, we electroporated a Cas9 nucleoprotein aimed to KO the TLR4 gene. To assess the inflammatory response, we analyzed cell secretome.

We achieved up to a 68% (out of 400,000 cells) success rate in editing the genome of hMSCs (R²=0.93). The TLR4 KO hMSCs secreted extracellular vesicles (EVs) compared with unedited hMScs (p<0.001) and decreased the secretion of most pro-inflammatory (e.g. IL-1α) and pro-fibrotic (e.g. IL-10) cytokines from edited compared with unedited hMSCs. Additionally, EVs from TLR4 KO cells stimulated hMSC migration by “wound healing” scratch assay (p<0.001).

Conclusion: Our preliminary results show, for the first time, that CRISPR-based KO of the human TLR4 gene in hMSCs inhibits inflammatory cytokine secretion and facilitates a reparative response by human-cardiac MSCs in vitro. The precise and efficient ex vivo gene editing could provide a newly engineered cell line to improve the outcome of hMSC-based cell therapy.