CRISPR-Cas9 Editing of TLR4 Improves the Outcome of Cardiac Cell Therapy

We developed and optimized a new electroporation protocol for CRISPR-Cas9 gene editing. This protocol achieved up to 68% success rate, when applied to isolated hMSCs from the heart and epicardial fat of patients with ischemic heart disease. While cell editing resulted lowered TLR4 expression in hMSCs, it did not affect classical markers of hMSCs and proliferation rate. Advanced protein mass spectrometry analysis revealed that edited cells secreted fewer proteins involved in inflammation and in extracellular matrix organization. The immunomodulatory effect of TLR4 editing was apparent in both free and EV-encapsulated proteins. Furthermore, edited cells expressed less NF-ƙB and secreted lower amounts of extracellular vesicles, pro-inflammatory and pro-fibrotic cytokines than unedited hMSCs. Cell therapy with edited and unedited hMSCs improved survival, left ventricular (LV) remodeling, and cardiac function after myocardial infarction (MI) in mice. Postmortem histologic analysis revealed clusters of edited cells that survived in the scar tissue 28 days after MI. Morphometric analysis showed that implantation of edited cells increased the area of myocardial islands in the scar tissue, reduced the occurrence of transmural scar, increased scar thickness, and markedly decreased expansion index, compared with unedited cells or saline.