CRISPR-Cas9 editing of TLR4 to improve the outcome of cardiac cell therapy

Abstract

Inflammation and fibrosis limit the reparative properties of human mesenchymal stromal cells (hMSCs). We hypothesized that disrupting the toll-like receptor 4 (TLR4) gene would switch hMSCs toward a reparative phenotype and improve the outcome of cell therapy for infarct repair. We developed and optimized an improved electroporation protocol for CRISPR-Cas9 gene editing. This protocol achieved a 68% success rate when applied to isolated hMSCs from the heart and epicardial fat of patients with ischemic heart disease. While cell editing lowered TLR4 expression in hMSCs, it did not affect classical markers of hMSCs, proliferation, and migration rate. Protein mass spectrometry analysis revealed that edited cells secreted fewer proteins involved in inflammation. Analysis of biological processes revealed that TLR4 editing reduced processes linked to inflammation and extracellular organization. Furthermore, edited cells expressed less NF-ƙB and secreted lower amounts of extracellular vesicles and pro-inflammatory and pro-fibrotic cytokines than unedited hMSCs. Cell therapy with both edited and unedited hMSCs improved survival, left ventricular 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 decreased expansion index. We show, for the first time, that CRISPR-Cas9-based disruption of the TLR4-gene reduces pro-inflammatory polarization of hMSCs and improves infarct healing and remodeling in mice. Our results provide a new approach to improving the outcomes of cell therapy for cardiovascular diseases.

Bridging the Gap: Investigating the Link between Inflammasomes and Postoperative Cognitive Dysfunction

“… CRISPR/Cas9 gene editing: This technique allows for altering specific genes and gene products, making it possible to examine the role of inflammasomes in POCD with more focus. With this method, inflammasome-related genes in certain cell types may be selectively targeted or animal models created without key inflammasome components [ 180 , 181 ]. Imaging techniques: Inflammasome activity in the brain and other organs may be monitored in real-time using imaging techniques such as positron emission tomography (PET) and magnetic resonance imaging (MRI) [ 122 , 182 ].…”

“…CRISPR/Cas9 gene editing: This technique allows for altering specific genes and gene products, making it possible to examine the role of inflammasomes in POCD with more focus. With this method, inflammasome-related genes in certain cell types may be selectively targeted or animal models created without key inflammasome components [ 180 , 181 ].…”

Applications of Genome Editing Technologies in CAD Research and Therapy with a Focus on Atherosclerosis

“…Schary et al utilised CRISPR/Cas9 to edit hMSCs to lower TLR4 expression. Mice administered with the edited hMSCs post MI exhibited improved survival, infarct healing, and remodelling [ 146 ]. While results of the study are promising, concerns regarding immune responses to hMSC implantation at the site of injury remain.…”