Project Grant F32HL188945

THERAPEUTIC TARGETING OF THE 5'-UNTRANSLATED REGION IN TITIN CARDIOMYOPATHY - PROJECT SUMMARY HETEROZYGOUS TRUNCATING FRAMESHIFT AND NONSENSE MUTATIONS IN THE MYOFILAMENT PROTEIN TITIN (TTN) ARE THE MOST COMMON CAUSE OF NONISCHEMIC DILATED CARDIOMYOPATHY (DCM), WHICH IS A LEADING CAUSE OF CONGESTIVE HEART FAILURE. TITIN IS INDISPENSABLE FOR SARCOMERE FUNCTION SINCE IT IS A MOLECULAR SCAFFOLD FOR SARCOMERE ASSEMBLY, SIGNALING, AND HOMEOSTASIS. CURRENTLY, THERE ARE NO CURRENT TTN TARGETED THERAPIES AVAILABLE. PRIOR STUDIES DEMONSTRATED INCREASING TTN PROTEIN EXPRESSION VIA CRISPR ACTIVATION OR PROTEOSOME INHIBITION RESCUED THE IMPAIRED TTN TRUNCATING VARIANT (TTNTV) CONTRACTILE PHENOTYPE. UPSTREAM OPEN READINGS FRAMES (UORFS) IN THE 5'-UNTRANSLATED REGION (5'-UTR) HAVE BEEN SHOWN TO NEGATIVELY REGULATE TITIN TRANSLATION IN A MOUSE TITIN 5'-UTR LUCIFERASE REPORTER. FURTHERMORE, MUTATION OF THE UORFS IMPROVED TITIN TRANSLATIONAL EFFICIENCY. FOR THIS PROJECT, I HYPOTHESIZE THAT DISRUPTION OF THE HUMAN UORFS INCREASES NATIVE TITIN TRANSLATION AND RESCUES THE TITIN TRUNCATING DILATED CARDIOMYOPATHY PHENOTYPE. AIM 1 WILL TEST THE HYPOTHESIS THAT THE UORFS NEGATIVELY REGULATE TTN TRANSLATION IN HUMAN INDUCED PLURIPOTENT STEM CELL-DERIVED CARDIOMYOCYTES (HIPSC-CMS). CRISPR/CAS9 GENOME EDITING WILL BE USED TO MUTATE THE UORFS ELEMENTS TO CREATE THE TITIN OVEREXPRESSION MODEL, TTN-UORF-/- HIPSC-CM. IN CONTROL AND TTN-UORF-/- HIPSC-CMS, TTN PROTEIN LEVELS WILL BE ASSESSED BY VERTICAL GEL ELECTROPHORESIS (VAGE), PULSE LABEL ASSAYS. PARALLEL RIBO-SEQ AND RNA-SEQ WILL ASSESS TRANSLATIONAL EFFICIENCY. AIM 2 WILL TEST IF MUTAGENESIS OF THE UORFS IN A TTNTV DISEASE MODEL RESCUES FUNCTION. AIM 2 WILL USE CRISPR/CAS9 GENOME EDITING TO GENERATE THE DISEASE TTNTV EXON 329 TRUNCATION MODEL (TTN-329+/- HIPSC LINE) AND DUAL TTN- UORF-/--329+/- HIPSC-CM RESCUE MODEL. TOTAL TTN PROTEIN LEVELS WILL BE ASSESSED BY WESTERN BLOTTING AND VAGE. PARALLEL RIBO-SEQ AND RNA-SEQ WILL BE USED TO ASSESS TTN TRANSLATIONAL EFFICIENCY. CARDIOMYOCYTE FUNCTION WILL BE QUANTIFIED BY SINGLE CELL CONTRACTILITY ASSAYS AND 3D ENGINEERED HEART TISSUE FORCE ASSAYS. THE FINDINGS WILL INCREASE THE BIOLOGICAL UNDERSTANDING OF THE TTN 5'-UTR AND ITS POTENTIAL ROLE AS A NOVEL THERAPEUTIC TARGET IN TTNTV DCM. DR. CADAR'S GOAL IS TO BECOME AN INDEPENDENT PHYSICIAN-SCIENTIST FOCUSED ON INHERITED CARDIOMYOPATHIES. DR. CADAR'S TRAINING PLAN WILL INCLUDE HANDS-ON TRAINING IN RIBO-SEQ AND RNA-SEQ (INSTRUMENTATION AND ANALYSIS) IN COLLABORATION WITH DR. SEZEN MEYDAN, SCIENTIFIC WRITING, PROFESSIONAL DEVELOPMENT, BIOINFORMATIC COURSEWORK, AND CLINICAL GENETIC TRAINING. VANDERBILT UNIVERSITY IS AN EXCEPTIONAL TRAINING ENVIRONMENT WITH RESOURCES TO SUPPORT DR. CADAR'S DEVELOPMENT TOWARDS INDEPENDENCE.