BS-452758-1 SUPPRESSION-REPLACEMENT GENE THERAPY REVERSE REMODELS THE INTRACELLULAR CALCIUM HANDLING DISEASE PHENOTYPE OF -MEDIATED HYPERTROPHIC CARDIOMYOPATHY

Hypertrophic cardiomyopathy (HCM), characterized by otherwise unexplained left ventricular hypertrophy, has a prevalence of 1:500. Pathogenic variants in TNNT2-encoded cardiac troponin T causes thin filament subtype sarcomeric HCM. A key cellular feature the pathological remodeling TNNT2-mediated HCM involves dysregulation intracellular calcium handling cardiomyocytes (CMs). To develop TNNT2 “suppression-replacement (SupRep)” gene therapy to reverse perturbations associated with filament, using induced pluripotent stem cell derived (iPSC-CMs) from patient TNNT2-R288P-mediated Custom-designed targeting shRNAs were tested for knockdown (KD) efficiency TSA201 cells and RT-qPCR. dual-component SupRep was created cloning into single construct custom-designed shRNA that produced greatest KD (suppression) “shRNA-immune” (shIMM) cDNA (replacement). Patient-specific TNNT2-R288P CRISPR/Cas9 variant-corrected isogenic control (IC) iPSC-CMs generated The indicator, Fluo-4, used detect free calcium. Our lead TNNT2-shRNA had an 80% TNNT2. treated TNNT2-SupRep showed similar efficiency. Compared IC iPSC-CMs, increased irregular events (30.4% vs. 10.9%, p<0.0001), prolonged transient duration 90 (CTD90, 1.01 ± 0.03 s 0.89 0.02 s, p<0.0001) peak 90% decay time (0.57 0.49 0.01 p=0.0012). untreated cells, transfection therapy, decrease 14%, shortened CTD90 (1.01 0.91 p=0.003), 0.50 p=0.02). There no significant difference events, CTD90, or between iPSC-CMs. Here, we provide first proof-of-principle almost fully restored impaired properties patient-derived back normal state achieved gene-edited cure.