BS-452757-2 A SINGLE SUPPRESSION AND REPLACEMENT GENE THERAPY FOR ALL THREE CARDIAC CALMODULINOPATHIES

Calmodulin (CaM)-mediated long QT syndrome (CaM-LQTS) and CaM-mediated catecholaminergic polymorphic ventricular tachycardia (CaM-CPVT) are genetic arrhythmia disorders (calmodulinopathies) characterized by a high prevalence of life-threatening arrhythmias occurring very early in life. Three distinct genes (CALM1, CALM2, CALM3) encode for the identical CaM protein. Conventional pharmacotherapies fail to adequately protect against potentially lethal cardiac events patients with CaM-LQTS or CaM-CPVT. To develop single construct CALM suppression-replacement (SupRep) gene therapy rescue prolonged action potential duration (APD) patient-derived induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from three CALM1-, CALM2-, CALM3-mediated CaM-LQTS. Five custom-designed CALM3-targeting shRNAs were tested knockdown (KD) efficiency using TSA201 cells RT-qPCR. A dual-component tandem SupRep was created cloning into CALM3-shRNAs that produce KD (suppression) each respective “shRNA-immune” (shIMM) CALM1 cDNA (replacement). Patient-specific CALM1-F142L, CALM2-D130G, CALM3-D130G variant iPSC-CMs generated severe CRISPR/Cas9 CALM2-D130G variant-corrected isogenic control (CALM2-WT) created. FluoVolt voltage-sensing dye used measure APD at 90% repolarization (APD90). Following shRNA testing, candidate identified (89% KD), CALM2 (70% CALM3 (87% KD). The APD90 significantly (647±9 ms) compared CALM2-WT (359±12 ms, p<0.0001). Transfection CALM-SupRep shortened average 457±19 ms (66% attenuation, Additionally, transfection CALM1-F142L (665±9 410±15 p<0.0001) (656±12 294±4 We provide first proof-of-principle pathologically patient-specific CALM3-variant iPSC-CM lines. This may be able treat all calmodulinopathies (CaM-LQTS CaM-CPVT) regardless which CaM-encoding affected.