Background: Type 1 long QT syndrome (LQT1) is caused by loss-of-function variants in the KCNQ1 -encoded K v 7.1 potassium channel α-subunit which is essential for cardiac repolarization, providing the slow delayed rectifier current (IKs). No current therapies target the molecular cause of LQT1. Methods: A dual-component "suppression-and-replacement" (SupRep) KCNQ1 gene therapy was created by cloning a KCNQ1 shRNA and a "shRNA-immune" (shIMM) KCNQ1 cDNA modified with silent variants in the shRNA target site, into a single construct. The ability of KCNQ1-SupRep gene therapy to suppress and replace LQT1-causative variants in KCNQ1 was evaluated via heterologous expression in TSA201 cells. For a human in vitro cardiac model, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were generated from four patients with LQT1 (KCNQ1-Y171X, -V254M, -I567S, and -A344A/spl) and an unrelated healthy control. CRISPR-Cas9 corrected isogenic control iPSC-CMs were made for two LQT1 lines (correction of KCNQ1-V254M and KCNQ1-A344A/spl). FluoVolt voltage dye was used to measure the cardiac action potential duration (APD) in iPSC-CMs treated with KCNQ1-SupRep. Results: In TSA201 cells, KCNQ1-SupRep achieved mutation-independent suppression of wild-type KCNQ1 and three LQT1-causative variants (KCNQ1-Y171X, -V254M, and -I567S) with simultaneous replacement of KCNQ1-shIMM as measured by allele-specific qRT-PCR and western blot. Using FluoVolt voltage dye to measure the cardiac APD in the four LQT1 patient-derived iPSC-CMs, treatment with KCNQ1-SupRep resulted in shortening of the pathologically prolonged APD at both 90% (APD 90 ) and 50% (APD 50 ) repolarization resulting in APD values similar to those of the two isogenic controls. Conclusions: This study provides the first proof-of-principle gene therapy for complete correction of LQTS. As a dual-component gene therapy vector, KCNQ1-SupRep successfully suppressed and replaced KCNQ1 to normal wild-type levels. In TSA201 cells, co-transfection of LQT1-causative variants and KCNQ1-SupRep caused mutation-independent suppression-and-replacement of KCNQ1 . In LQT1 iPSC-CMs, KCNQ1-SupRep gene therapy shortened the APD, thereby eliminating the pathognomonic feature of LQT1.

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Background: The CACNA1C -encoded cardiac L-type calcium channel (Cav1.2) is essential for cardiocyte action potential duration (APD). We previously reported the CACNA1C-p.R518C variant associated with prolonged QT intervals, cardiomyopathy, and sudden cardiac death in several pedigrees. Methods: To characterize a patient-derived human induced pluripotent stem cell–derived cardiomyocyte (hiPSC-CM) CACNA1C-p.R518C model, CACNA1C-p.R518C hiPSC-CMs were generated from a 13-year-old man (QTc, >480 ms) with a family history of sudden cardiac death. An isogenic hiPSC-CM gene-corrected control was created using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat–associated 9). APD and calcium handling were assessed by live cell imaging with Arclight voltage and Fluo-4 calcium indicators, respectively. The APD and L-type calcium channel biophysical properties were further assessed by whole-cell patch clamp technique. Results: The Bazett formula-corrected, Arclight-measured APD 90 of CACNA1C-p.R518C hiPSC-CMs was significantly longer (622±11 ms; n=92) than the isogenic control hiPSC-CMs (453±5 ms; n=62; P <0.0001). Patch clamp assessment of CACNA1C-p.R518C hiPSC-CMs paced at 1 Hz confirmed a prolonged APD 90 (689±29 ms; n=10) compared with the patient’s isogenic control hiPSC-CMs (434±30 ms; n=8; P <0.05). Fluo-4–measured calcium transient decay time suggested calcium mishandling in CACNA1C-p.R518C. Patch clamp analysis revealed increased L-type calcium channel window current, slow decay time at various voltages, and increased late calcium current for CACNA1C-p.R518C hiPSC-CMs when compared with isogenic control hiPSC-CMs. Conclusions: Using patient-specific hiPSC-CM mutant and isogenic control lines, we demonstrate that the CACNA1C-p.R518C variant is the self-sufficient, monogenetic substrate for the patient’s long-QT syndrome phenotype. These data further bolster the conclusion that CACNA1C is a bona fide, definite evidence long-QT syndrome susceptibility gene.

Background: MRAS was identified recently as a novel Noonan syndrome (NS)-susceptibility gene. Phenotypically, both patients with NS, harboring pathogenic MRAS variants, displayed severe cardiac hypertrophy. This study aimed to demonstrate both the necessity and sufficiency of a patient-specific variant (p.Gly23Val-MRAS) to cause NS through the generation and characterization of patient-specific, isogenic control, and disease modeled induced pluripotent stem cell (iPSC) lines. Methods: iPSCs were derived from a patient with a p.Gly23Val-MRAS variant to assess the effect of MRAS variants on pathogenesis of NS-associated cardiac hypertrophy. CRISPR/Cas9 gene editing was used to correct the pathogenic p.Gly23Val-MRAS variant in patient cells (isogenic control) and to introduce the pathogenic variant into unrelated control cells (disease modeled) to determine the necessity and sufficiency of the p.Gly23Val-MRAS variant to elicit the disease phenotype in iPSC-derived cardiomyocytes (iPSC-CMs). iPSC-CMs were analyzed by microscopy and immunofluroesence, single-cell RNAseq, Western blot, room temperature-quantitative polymerase chain reaction, and live-cell calcium imaging to define an in vitro phenotype of MRAS -mediated cardiac hypertrophy. Results: Compared with controls, both patient and disease modeled iPSC-CMs were significantly larger and demonstrated changes in gene expression and intracellular pathway signaling characteristic of cardiac hypertrophy. Additionally, patient and disease modeled iPSC-CMs displayed impaired Ca 2+ handling, including increased frequency of irregular Ca 2+ transients and changes in Ca 2+ handling kinetics. Conclusions: p.Gly23Val-MRAS is both necessary and sufficient to elicit a cardiac hypertrophy phenotype in iPSC-CMs that includes increased cell size, changes in cardiac gene expression, and abnormal calcium handling-providing further evidence to establish the monogenetic pathogenicity of p.Gly23Val-MRAS in NS with cardiac hypertrophy.