Friday, April 19, 2024
4/19/2024
Project Summary/Abstract Cardiovascular disease associated with myocardial infarction (MI) remains a major cause of death worldwide. Cell reprogramming into cardiovascular progenitor cells (CPCs), bypassing the process of pluripotency induction, has provided a promising approach for cardiac repair through simultaneous neovascularization and cardiomyogenesis. One significant gap in current research is an unclear understanding of the mechanisms of direct reprogramming. The activation of endogenous genetic loci (that are occluded by repressive chromatin marks) in starting cells has been considered an important criterion of high-quality fully reprogrammed cells. Therefore, we attempt to obtain a new source of CPCs through direct manipulation of endogenous genes. To this end, we propose a combined use of reprogramming technologies and CRISPR-based tools. CRISPR-induced gene activation is superior to conventional techniques employing cDNA overexpression due to its effectiveness, simplicity of design and avoidance of the need for additional transduction of transcription factor complexes. Our preliminary data demonstrates that CRISPR-induced cells possess the properties of CPCs including clonality, self- renewal, and cardiac tri-potentiality. Importantly, the CRISPR-induced CPCs (iCPCs) can blunt the worsening of heart function and reduce infarct size after transplantation in MI mice. These findings are the basis for a further systematic investigation of CRISPR-iCPCs in a preclinical setting. Accordingly, three Specific Aims are proposed to develop a technical platform to generate new CRISPR-iCPCs, study cell-reprogramming mechanisms, and evaluate the application of iCPCs for MI therapy. In Aim-1, a new non-viral CRISPR system will be developed for generation of iCPCs using a clinically acceptable gene vector system and the biological and functional features of new iCPCs will be characterized. In Aim-2, epigenetic factors mediating iCPC generation will be the focus of experiments. Long non-coding RNAs and targeted epigenetic mediators will be investigated to dissect the mechanisms of cellular reprogramming. In Aim-3, iCPCs will be implanted using cell patch techniques, and their cell fate decisions will be tracked in the infarcted heart. The extent and role of differentiated iCPCs will be determined by in vivo imaging and distinguished from other consequences using a cell ablation approach. In conclusion, this proposal will provide a new regeneration strategy for MI therapy through convergence of CRISPR technology and tissue engineering in CPC development.
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