Project Grant K99HL175039

POST-TRANSCRIPTIONAL MECHANISMS OF DIRECT CARDIAC REPROGRAMMING - PROJECT SUMMARY HEART ATTACKS, OR MYOCARDIAL INFARCTIONS (MI), REMAIN A LEADING CAUSE OF DEATH WORLDWIDE. AS ADULT HUMAN HEARTS HAVE LIMITED REGENERATIVE CAPACITY, CARDIOMYOCYTES (CMS) LOST AFTER MI ARE REPLACED BY FIBROTIC SCAR TISSUE, LEADING TO CARDIAC DYSFUNCTION AND OFTEN HEART FAILURE. A PROMISING APPROACH TO REPLACE LOST CMS IS DIRECT REPROGRAMMING OF CARDIAC FIBROBLASTS (CFS) INTO INDUCED CARDIOMYOCYTE-LIKE CELLS (ICMS) USING THE SPECIFIC CARDIAC LINEAGE TRANSCRIPTION FACTORS MEF2C, GATA4 AND TBX5 (MGT). OVER THE LAST DECADE, CONSIDERABLE PROGRESS HAS BEEN MADE IN UNDERSTANDING THE TRANSCRIPTIONAL AND EPIGENETIC REGULATION OF ICM GENERATION. HOWEVER, POST- TRANSCRIPTIONAL PROCESSES, INCLUDING TRANSLATION AND MRNA MODIFICATION, ARE ALSO IMPORTANT TO REGULATE GENE EXPRESSION. POST-TRANSCRIPTIONAL REGULATORS HAVE RECENTLY BEEN SHOWN TO PLAY CRITICAL ROLES IN CELL FATE DETERMINATION, BUT THEIR ROLES IN DIRECT CARDIAC REPROGRAMMING ARE LARGELY UNKNOWN. RECENTLY, DR. XIE IDENTIFIED THE RNA BINDING PROTEIN YBX1 AND THE M6A READER IGF2BP1 AS CRITICAL POST-TRANSCRIPTIONAL BARRIERS TO ICM INDUCTION. EXCITINGLY, UPON REMOVING YBX1, A SINGLE FACTOR, TBX5, COULD SUCCESSFULLY REPROGRAM FIBROBLASTS INTO ICMS. THUS, IN AIM 1, DR. XIE WILL USE CHIP-SEQ AND ATAC-SEQ TO CHARACTERIZE THE MECHANISMS BY WHICH THE INNOVATIVE COCKTAIL TBX5+SIYBX1 GENERATES ICMS. THEN, TAKING ADVANTAGE OF THE REDUCED CARGO SIZE OF THIS COCKTAIL, DR. XIE WILL DEVELOP A CLINICALLY RELEVANT LIPID NANOPARTICLE-MRNA DELIVERY SYSTEM TO ACHIEVE IN VIVO ICM CONVERSION IN A MOUSE MI MODEL. POST-TRANSCRIPTIONAL MRNA M6A METHYLATION IS ANOTHER REGULATOR OF GENE EXPRESSION IMPORTANT IN CELL FATE MAINTENANCE. IN ADDITION TO SERVING AS A BARRIER TO CARDIAC REPROGRAMMING, DR. XIE FOUND THAT THE M6A BINDING PROTEIN, IGF2BP1, FORMED CELLULAR GRANULES IN CFS. SHE, THEREFORE, HYPOTHESIZES THAT IGF2BP1 SEQUESTERS OR OTHERWISE REGULATES THE STABILITY OF M6A MRNAS IN CFS BY REGULATING CELLULAR RNP GRANULE DYNAMICS AND THAT LOSS OF IGF2BP1 LEADS TO A SHIFT IN MRNA METABOLISM PROMOTING CARDIAC FATE CONVERSION. TO TEST THESE HYPOTHESES, AIM 2 IS TO IDENTIFY M6A ALTERNATIONS ASSOCIATED WITH ICM GENERATION, MAP PRECISE RNA BINDING SITES OF IGF2BP1 AND ELUCIDATE THE UNDERLYING MECHANISM THROUGH WHICH IGF2BP1-MEDIATED CELLULAR GRANULE FORMATION REGULATES THE INDUCTION OF ICMS. THE PROPOSED WORK WILL NOT ONLY FILL CRITICAL GAPS IN OUR MECHANISTIC UNDERSTANDING OF HOW POST-TRANSCRIPTIONAL REGULATION IMPACTS CARDIAC FATE CONVERSION, BUT ALSO ADVANCE ICM REPROGRAMMING CLOSER TO THERAPEUTIC APPLICATION THROUGH THE DEVELOPMENT OF AN LNP-MRNA DELIVERY SYSTEM. WITH AN EXCELLENT MENTORING TEAM AND A STIMULATING RESEARCH ENVIRONMENT AT UNC CHAPEL HILL, DR. XIE WILL LEARN CUTTING-EDGE TECHNIQUES AND HONE HER PROFESSIONAL SKILLS, WHICH WILL PROVIDE A SOLID FOUNDATION FOR HER FUTURE RESEARCH IN STUDYING THE MECHANISMS OF CARDIOVASCULAR DISEASE AND TO USE INSIGHTS FROM THIS WORK TO DEVELOP NOVEL THERAPIES TO IMPROVE PATIENTS' LIVES. THIS K99/R00 AWARD WILL SUPPORT THE DEVELOPMENT OF HER UNIQUE AND COMPREHENSIVE RESEARCH PROGRAM AND CATALYZE THE TRANSITION TO INDEPENDENCE.