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.

Background - The KCNH2 -encoded Kv11.1 (hERG) potassium channel is a critical regulator of cardiomyocyte (CM) action potential duration (APD). The majority of type 2 long QT syndrome (LQT2) stems from trafficking defective KCNH2 mutations. Recently, FDA-approved cystic fibrosis protein trafficking chaperone, lumacaftor (LUM), has been proposed as novel therapy for LQT2. Here, we test the efficacy of LUM treatment in patient-specific induced pluripotent stem cell-cardiomyocytes (iPSC-CMs) derived from two patients with known LQT2 trafficking defective mutations and a patient with novel KCNH2 variant, p.R685P. Methods - Patient-specific iPSC-CM models of KCNH2-G604S, KCNH2-N633S, and KCNH2-R685P were generated from three unrelated patients diagnosed with severe LQT2 (QTc > 500 ms). LUM efficacy was also tested by ANEPPS, FluoVolt, and ArcLight voltage dye-based APD90 measurements. Results - All three mutations were hERG trafficking defective in iPSC-CMs. While LUM treatment failed to rescue the hERG trafficking defect in TSA201 cells, LUM rescued channel trafficking for all mutations in the iPSC-CM model. All three mutations conferred a prolonged APD90 compared to control. While LUM treatment rescued the phenotype of KCNH2-N633S and KCNH2-R685P, LUM paradoxically prolonged the APD90 in KCNH2-G604S iPSC-CMs. LUM-mediated APD90 rescue were affected by IKr blocker consistent with the increase of IKr by LUM is the underlying mechanism of the LQT2 rescue. Conclusions - While LUM is an effective hERG channel trafficking chaperone and may be therapeutic for LQT2, we urge caution. Without understanding the functionality of the mutant channel to be rescued, LUM therapy could be harmful.

No abstract

Background - Prior epidemiological studies demonstrated that the p.D85N-KCNE1 common variant reduces repolarization reserve and predisposes to drug-induced QT prolongation/torsades de pointes. We sought to develop a cellular model for drug-induced long QT syndrome (DI-LQTS) using a patient-specific induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM). Methods - p.D85N-KCNE1 iPSCs were generated from a 23-year-old female with an exaggerated QTc response to metoclopramide (ΔQTc of 160 ms). CRISPR/Cas9 technology was used to generate "gene-corrected" isogenic iPSCs. Field potential duration (FPD) and action potential duration (APD) were measured from iPSC-CMs. Results - At baseline, p.D85N-KCNE1 iPSC-CMs displayed significantly longer FPD (281 ± 15 ms, n=13 vs. 223 ± 8.6 ms, n=14, p<0.01) and APD 90 (579 ± 22 ms, n=24 vs. 465 ± 33 ms, n=26, p<0.01) than isogenic-control iPSC-CMs. Dofetilide at a concentration of 2nM increased significantly FPD (379 ± 20 ms, n=13, p<0.01) and APD 90 (666 ± 11 ms, n=46, p<0.01) in p.D85N-KCNE1 iPSC-CMs, but not in isogenic-control. The effect of dofetilide on APD 90 (616 ± 54 ms, n=7 vs. 526 ± 54 ms, n=10, p<0.05) was confirmed by Patch-clamp. Interestingly, treatment of p.D85N-KCNE1 iPSC-CMs with estrogen at a concentration of 1nM exaggerated further dofetilide-induced APD 90 prolongation (696 ± 9 ms, n=81, p<0.01) and caused more early afterdepolarizations (EADs) (11.7%) compared to isogenic control (APD 90 : 618 ± 8 ms, n=115 and EADs: 2.6%, p<0.05) Conclusions - This iPSC-CM study provides further evidence that the p.D85N-KCNE1 common variant in combination with environmental factors such as QT prolonging drugs and female sex is pro-arrhythmic.