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