CRISPR-Cas9-based gene editing of human mesenchymal stromal cells
to improve the outcome of cell therapy

Background: myocardial-infarction (MI) drives resident and transplanted mesenchymal stromal cells (MSC) toward a pro-inflammatory phenotype, thereby restricting their survival and reparative effects in a mechanism mediated by toll-like receptor 4 (TLR4). By utilizing CRISPR for genome editing we offer a new approache for improved efficacy of cell-therapy for heart failure.

Purpose: We aimed to enhance MSC-based cell-therapy in the heart. We hypothesized that ex-vivo knockout of the human TLR4 gene by CRISPR would switch primary cardiac-human MSC (hMSC) to a reparative phenotype that could prevent heart-failure after MI.

Methods and results: We achieved 68% success rate of gene-editing in hMSC from patients with ischemic heart disease. Deleting TLR4 in hMSC as measured by protein mass spectrometry and multiplex ELISA, significantly reduced pro-inflammatory and extracellular-matrix protein secretion. Furthermore, TLR4-KO hMSC secreted more extracellular-vesicles compared with control, indicating more cell-to-cell interactions after editing. Notably, editing of the cells did not alter hMSC characteristics such as proliferation-rate or surface markers. These findings suggest that editing the cells would improve their ability to treat the myocardium after MI. Additionaly, we evaluated the therapeutic potential of the edited hMSC in a mouse model of MI. Our preliminary results indicate that edited hMSC can prevent cardiac remodeling and improve function.

Conclusions: We show, that CRISPR-based deletion of the TLR4-gene in hMSC inhibits pro-inflammatory and extracellular-matrix protein secretion, and facilitates a reparative response by hMSC in-vitro and in-vivo when treating the heart. This approach could provide a novel treatment to improve the outcome of hMSC-based cell-therapy.