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.
BackgroundProgress has been achieved with the introduction of biologics for the management of inflammatory/autoimmune diseases such as rheumatoid arthritis (RA), however such medications induce immune suppression, which is nonselective to the pathogenesis of the disease, resulting in higher rates of infections. Therefore, there are unmet medical needs in the treatment of such diseases, which should be addressed by novel approaches. Accumulating evidence suggests that extracellular vesicles (EVs) play a role in the establishment, maintenance and modulation of autoimmune processes.ObjectivesIn the current study, we hypothesized that isolation of circulating autologous tissue-specific homing EVs from RA patients - may improve the delivery of current FDA-approved anti-inflammatory drugs, which will be encapsulated into these EVs. The drug-loaded EVs will be injected back to the diseased subjects and will naturally find their way to the inflamed tissue.ResultsIndeed, we found that autologous labeled EVs, expressing joint/synovia-specific homing receptors (e.g. αVβ3 integrin), derived from blood of diseased arthritic mice (Collagen antibody-induced arthritis model), can migrate toward the inflamed synovia, usingin vivoimaging system (IVIS). Moreover, we show that these EVs strongly expresses glucose transporter 1 (mGLUT1) which in turn, improve their therapeutic potential to be loaded with anti-inflammatory drugs using glucose-coated gold nanoparticles (GNPs). Finally, we show that EVs derived from plasma of RA patients overexpresses αVβ3 integrin and taken up by LPS/TNFα-induced activated human synovial cell linein vitro.ConclusionOverall, we show the potential of autologous circulating EVs of RA patients to serves as natural nano-carrier for current FDA-approved drugs. We believe that this strategy will increase the specificity and efficiency of current treatment, therefore it will reduce side effects and will improve the quality of life of RA patients and potentially other autoimmune disease patients.REFERENCES:NIL.Acknowledgements:NIL.Disclosure of InterestsNone Declared.
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