MAPPING MICRORNA-TARGET INTERACTIONS IN THE ZONATED LIVER - ABSTRACT THE LENS OF METABOLIC ZONATION IS EXTREMELY PERTINENT TO LIVER PATHOPHYSIOLOGY, AND CAN BE APPLIED TO BOTH ACUTE AND CHRONIC/PROGRESSIVE CONDITIONS LIKE ACUTE LIVER DAMAGE AND MASLD/MASH. THE LONG TERM GOAL IS TO BETTER UNDERSTAND THE ROLE OF MICRORNAS IN LIVER PHYSIOLOGY, MICRORNA DYSREGULATION IN PATHOPHYSIOLOGY, AND TO IMPROVE CLINICAL OUTCOMES FOR PATIENTS WITH ACUTE AND CHRONIC LIVER DISEASES. THE OVERALL OBJECTIVE OF THIS APPLICATION IS TO UNRAVEL THE SPECIFIC MECHANISM BY WHICH MICRORNAS MAINTAIN METABOLIC ZONATION BY MAPPING AND VALIDATING MICRORNA-TARGET INTERACTIONS ACROSS THE LOBULE WITH HIGH FIDELITY TO IDENTIFY NEW BIOMARKERS AND INFORM THERAPEUTICS IN THE CONTEXT OF THE HEALTHY AND DISEASED LIVER. THE CENTRAL HYPOTHESIS IS THAT THE PHENOMENON OF METABOLIC ZONATION IS AT LEAST PARTIALLY CONTROLLED BY MIRNA-TARGET NETWORKS THAT CAN BE TAKEN ADVANTAGE OF BOTH THERAPEUTICALLY AND DIAGNOSTICALLY. THE CENTRAL HYPOTHESIS WILL BE TESTED BY PURSUING TWO SPECIFIC AIMS: 1) MAPPING AND VALIDATION OF MIRNA-TARGET INTERACTIONS IN LIVER CELLS ALONG THE LOBULAR AXIS WITH HIGH FIDELITY AND 2) ASSESSING DYSREGULATION OF MIRNA-TARGET NETWORKS IN A MODEL OF ACUTE AND CHRONIC LIVER DISEASE. UNDER THE FIRST AIM, WE WILL USE SPY3-AGO2-PULLDOWN AND SEQUENCING (SAPSEQ) IN MICE WITH A CRE-INDUCIBLE SPY3-TAG ON MICRORNA EFFECTOR PROTEIN AGO2 (CRE +ISPY MICE) TO CREATE A SINGLE HIGH-FIDELITY MAP OF DIFFERENTIAL MIRNA-TARGET REGULATION IN SPATIALLY SORTED HEPATOCYTES, VALIDATE MIRNA ZONATION USING MIRNA-SENSING REPORTER CONSTRUCTS AND INDEPENDENTLY VALIDATE NODE-LEVEL AND GLOBAL MIRNA-TARGET INTERACTIONS USING N-ACETYLGALACTOSAMINE CONJUGATED MIRNA SPONGING ASOS AND VECTORS THAT DELIVER MIRNA-BLOCKING PEPTIDE DDT6B. FOR THE SECOND AIM, WE WILL USE SPATIAL SORTING AND SAPSEQ TO EVALUATE MIRNA-TARGET CHANGES IN RESPONSE TO PERICENTRAL TOXIN APAP AND PERIPORTAL TOXIN ALLYL ALCOHOL OVER TIME IN CRE+ ISPY MICE, MIRNA-TARGET CHANGES IN CRE+ ISPY MICE FED A CHOLINE-DEFICIENT, L-AMINO ACID DEFINED, HIGH FAT DIET OVER TIME (INDUCES MASLD/MASH), AND USE SERUM SAPSEQ TO EVALUATE CIRCULATING MIRNA CHANGES IN BOTH CONDITIONS. OUR PROPOSAL IS INNOVATIVE AS IT UTILIZES TECHNICAL ADVANCES IN MICRORNA BIOLOGY AND AGO2 STRUCTURE TO EXPAND OUR UNDERSTANDING OF LIVER PHYSIOLOGY WITH APPLICATIONS IN ADDRESSING PATHOLOGIES. ULTIMATELY, THIS WORK WILL HAVE A SIGNIFICANT, DIRECT, AND POSITIVE IMPACT IN IMPROVING TREATMENT OPTIONS FOR PATIENTS WITH CHRONIC LIVER DISEASES AND DIAGNOSTICS FOR ACUTE LIVER DISEASES. BEYOND THE LIVER, IT WILL HAVE WIDE-REACHING EFFECTS IN THE FIELDS OF CELL, GENE, AND IMMUNOTHERAPY. THE PROPOSED WORK WILL BE CONDUCTED AT THE SCRIPPS RESEARCH INSTITUTE, UNDER GUIDANCE OF MENTORS IN RNA BIOLOGY, LIVER BIOCHEMISTRY, AND CLINICAL CARE. THIS PROPOSAL WILL BE VITAL TO SUPPLEMENT MY DUAL-DEGREE TRAINING TO BECOME AN INDEPENDENT PHYSICIAN-SCIENTIST AND DOMAIN EXPERT IN CLINICAL RNA BIOLOGY.