Gene editing of human mesenchymal stromal cells using CRISPR-Cas9 to improve the outcome of cell therapy

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 the 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 (Fig. 1A).

Methods and Results: We achieved up to 68% (out of 400,000 cells) success rate in editing the genome of hMSCs (R²=0.93). The TLR4 KO hMSCs secreted more extracellular vesicles (EVs) compared with unedited hMScs (Fig. 1B, 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 (Fig. 1C). Additionally, we found that the CD47 ‘Don’t eat me signal’ was expressed significantly higher in the edited cell group (Fig. 1D, p=0.0048). Finally, EVs from the edited cells stimulated higher hMSC migration by scratch assay (p<0.001).

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