Suppression-Replacement KCNQ1 Gene Therapy for Type 1 Long QT Syndrome.

BACKGROUND: Type 1 long QT syndrome (LQT1) is caused by loss-of-function variants in the KCNQ1-encoded Kv7.1 potassium channel α-subunit that is essential for cardiac repolarization, providing the slow delayed rectifier current. 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 short hairpin RNA and a short hairpin RNA-immune KCNQ1 cDNA modified with synonymous variants in the short hairpin RNA target site, into a single construct. The ability of KCNQ1-SupRep gene therapy to suppress and replace LQT1-causative variants in KCNQ1 was evaluated by means of heterologous expression in TSA201 cells. For a human in vitro cardiac model, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were generated from 4 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 2 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 3 LQT1-causative variants (KCNQ1-Y171X, -V254M, and -I567S) with simultaneous replacement of short hairpin RNA-immune KCNQ1 as measured by allele-specific quantitative reverse transcription polymerase chain reaction and Western blot. Using FluoVolt voltage dye to measure the cardiac APD in the 4 LQT1 patient-derived iPSC-CMs, treatment with KCNQ1-SupRep resulted in shortening of the pathologically prolonged APD at both 90% and 50% repolarization, resulting in APD values similar to those of the 2 isogenic controls. CONCLUSIONS: This study provides the first proof-of-principle gene therapy for complete correction of long QT syndrome. As a dual-component gene therapy vector, KCNQ1-SupRep successfully suppressed and replaced KCNQ1 to normal wild-type levels. In TSA201 cells, cotransfection 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.

Follow-up of 316 molecularly defined pediatric long-QT syndrome patients: clinical course, treatments, and side effects.

BACKGROUND: Inherited long-QT syndrome (LQTS) is associated with risk of sudden death. We assessed the clinical course and the fulfillment of current treatment strategies in molecularly defined pediatric LQTS type 1 and (LQT1) and type 2 (LQT2) patients. METHODS AND RESULTS: Follow-up data covering a mean of 12 years were collected for 316 genotyped LQT1 and LQT2 patients aged 0 to 18 years. No arrhythmic deaths occurred during the follow-up. Finnish KCNQ1 and KCNH2 founder mutations were associated with fewer cardiac events than other KCNQ1 and KCNH2 mutations (hazard ratio [HR], 0.33; P=0.03 and HR, 0.16; P=0.01, respectively). QTc interval ≥500 ms increased the risk of cardiac events compared with QTc <470 ms (HR, 3.32; P=0.001). Treatment with ß-blocker medication was associated with reduced risk of first cardiac event (HR, 0.23; P=0.001). Noncompliant LQT2 patients were more often symptomatic than compliant LQT2 patients (18% and 0%, respectively; P=0.03). Treatment with implantable cardioverter defibrillator was rare (3%) and resulted in reinterventions in 44% of cases. CONCLUSIONS: Severe cardiac events are uncommon in molecularly defined and appropriately treated pediatric LQTS mutation carriers. ß-Blocker medication reduces the risk of cardiac events and is generally well tolerated in this age group of LQTS patients.

The incorporation of an ion channel gene mutation associated with the long QT syndrome (Q9E-hMiRP1) in a plasmid vector for site-specific arrhythmia gene therapy: in vitro and in vivo feasibility studies.

The present studies investigated the cardiac potassium channel missense mutation, Q9E-hMiRP1, for potential use as a gene therapy construct for cardiac arrhythmias. This gene abnormality is one of a number of mutations that can cause the long QT syndrome (LQTS), a hereditary arrhythmia disorder that is associated with sudden death. However, individuals who carry the Q9E-hMiRP1 variant are predisposed to developing the LQTS only after clarithromycin administration. Because the electrophysiologic mechanism of action of Q9E-hMiRP1 (i.e., diminished potassium currents resulting in delayed myocardial repolarization) is comparable to that of class III antiarrhythmic agents, we examined Q9E-hMiRP1 as a candidate gene therapy construct for site-specific treatment of reentrant atrial cardiac arrhythmias. Our rationale was also based on the hypothetical safety of the atrial use of Q9E-hMiRP1 because LQTS characteristically causes ventricular but not atrial arrhythmias. Furthermore, the possible use of clarithromycin to control the conduction effects of overexpressed Q9E-hMiRP1 pharmacologically was another attractive feature. In our studies we investigated the use of two bicistronic plasmid DNA gene vectors with either hMiRP1 or Q9E-MiRP1 and green fluorescent protein (GFP), plus a C-terminus of the hMiRP1 or of the Q9E-hMiRP1 coding region for the FLAG (MDYKDDDDK) peptide. We generated two stable cell lines using HEK293 and SH-SY5Y (human cell lines), overexpressing the genes of interest, confirmed by real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blots. The expected plasma membrane localization of each overexpressed transgene was confirmed by immunofluorescent confocal fluorescent microscopy using anti-FLAG antibody. Patchclamp studies demonstrated that cells transfected with Q9E-hMiRP1 plasmid DNA exhibited significantly reduced potassium currents but only with clarithromycin administration. A novel plasmid DNA delivery system was formulated for use in our animal studies of the hMiRP1 vectors, which was composed of DNA-anti-DNA antibody-cationic lipid (DAC) heteroplexes. In vitro and in vivo studies using DAC heteroplexes containing anti-DNA antibodies with nuclear targeting capability demonstrated significantly increased transfection compared to naked DNA, and to DNA-cationic lipid complexes. Pig atrial myocardial injections of DAC heteroplexes demonstrated 16% of regional cardiac myocytes transfected using the Q9E-hMiRP1 plasmid, and 15% of cells with the hMiRP1 vector. It is concluded that the present studies support the view that site-specific gene therapy for atrial arrhythmias is feasible using plasmid vectors for overexpressing ion channel mutations that have electrophysiologic effects comparable to class III antiarrhythmic agents.