Haeberlin, Andreas

Abstract Background and Aims Type 1 short QT syndrome (SQT1) is a genetic channelopathy caused by gain-of-function variants in KCNH2, resulting in shortened cardiac repolarization and QT intervals, which predispose patients to ventricular arrhythmias and sudden cardiac death. This study aimed to investigate the therapeutic efficacy of KCNH2-specific suppression-and-replacement (KCNH2-SupRep) gene therapy in a transgenic rabbit model of SQT1. Methods KCNH2-SupRep was developed by combining a KCNH2-shRNA with its corresponding shRNA-immune KCNH2-cDNA into an AAV9 vector, delivered directly into the aortic root (1 × 1010 vg/kg). Therapeutic efficacy was evaluated in vivo by electrocardiogram, ex vivo by optical mapping, and at cellular levels by patch-clamp, calcium imaging, and qPCR in ventricular cardiomyocytes (VCMs). Results In vivo, KCNH2-SupRep normalized the heart rate-corrected QT interval (QTc) in SQT1 rabbits, without affecting repolarization heterogeneity. Ex vivo, KCNH2-SupRep corrected the action potential duration (APD90) and resolved the increased apicobasal APD90 heterogeneity observed in untreated (UT)-SQT1 hearts, supporting an antiarrhythmic effect, which was further validated by reduced re-entry formation in silico. At cellular levels, KCNH2-SupRep prolonged APD90 in VCMs from SupRep-SQT1 rabbits closer to wildtype levels compared with UT- and sham-SQT1. Additionally, KCNH2-SupRep restored the cellular surrogate of the electro-mechanical window and normalized IKr in nearly 50% of VCMs, in line with a 50%–60% suppression of the mutant KCNH2 transcript. Conclusions This proof-of-concept study is the first to demonstrate the efficacy of gene therapy for SQT1 in a medium-sized animal model. KCNH2-SupRep gene therapy successfully corrected the pathologic phenotype in vivo, ex vivo, and at cellular levels in transgenic SQT1 rabbits.

Abstract Background and Aims Type 1 long QT syndrome (LQT1) is caused by pathogenic variants in the KCNQ1-encoded Kv7.1 potassium channels, which pathologically prolong ventricular action potential duration (APD). Herein, the pathologic phenotype in transgenic LQT1 rabbits is rescued using a novel KCNQ1 suppression-replacement (SupRep) gene therapy. Methods KCNQ1-SupRep gene therapy was developed by combining into a single construct a KCNQ1 shRNA (suppression) and an shRNA-immune KCNQ1 cDNA (replacement), packaged into adeno-associated virus serotype 9, and delivered in vivo via an intra-aortic root injection (1E10 vg/kg). To ascertain the efficacy of SupRep, 12-lead electrocardiograms were assessed in adult LQT1 and wild-type (WT) rabbits and patch-clamp experiments were performed on isolated ventricular cardiomyocytes. Results KCNQ1-SupRep treatment of LQT1 rabbits resulted in significant shortening of the pathologically prolonged QT index (QTi) towards WT levels. Ventricular cardiomyocytes isolated from treated LQT1 rabbits demonstrated pronounced shortening of APD compared to LQT1 controls, leading to levels similar to WT (LQT1-UT vs. LQT1-SupRep, P < .0001, LQT1-SupRep vs. WT, P = ns). Under β-adrenergic stimulation with isoproterenol, SupRep-treated rabbits demonstrated a WT-like physiological QTi and APD90 behaviour. Conclusions This study provides the first animal-model, proof-of-concept gene therapy for correction of LQT1. In LQT1 rabbits, treatment with KCNQ1-SupRep gene therapy normalized the clinical QTi and cellular APD90 to near WT levels both at baseline and after isoproterenol. If similar QT/APD correction can be achieved with intravenous administration of KCNQ1-SupRep gene therapy in LQT1 rabbits, these encouraging data should compel continued development of this gene therapy for patients with LQT1.

Abstract Background Short QT syndrome type 1 (SQT1) is a genetic channelopathy characterized by gain-of-function variants in the KCNH2-encoded potassium channel underlying the IKr current, leading to a pathologically shortened cardiac action potential duration (APD). Patients with SQT1 manifest shortening of the QT duration on 12-lead ECG and are prone to cardiac arrhythmias that can culminate in sudden cardiac death. Purpose To investigate the effect of a KCNH2-specific suppression-replacement (KCNH2-SupRep) gene therapy on the pathogenic phenotype in transgenic SQT1 rabbits. Methods A novel dual-component KCNH2-SupRep gene therapy was developed by combining a custom-designed KCNH2 shRNA (suppression) and an shRNA-immune (shIMM) KCNH2 cDNA (replacement) into AAV9. In vivo AAV9-mediated KCNH2-SupRep / sham treatment was performed by intra-aortic root injection of the KCNH2-SupRep AAV9 (1E10 vg/kg) / NaCl 0.9% during balloon occlusion using a Swan Ganz catheter. Three weeks after administration, 12-lead ECGs were assessed in adult transgenic SQT1 (KCNH2-N588K, gain of IKr due to impaired inactivation) and wild-type (WT) rabbits to determine the effect of KCNH2-SupRep / sham treatment on the rabbit’s QTc duration and QT index. Patch-clamp recordings were performed in isolated ventricular cardiomyocytes (CMs) to evaluate the effect of KCNH2-SupRep / sham treatment on the CM’s action potential duration at 90% repolarization (APD90). Results After in vivo AAV9-mediated KCNH2-SupRep gene therapy injection (1E10 vg/kg) in 7 SQT1 rabbits, QTc duration and QT index significantly increased in all SQT1 rabbits (QTc: untreated(UT)-SQT1: 153.2±3.8ms vs treated-SQT1: 166.5±4.6ms, p=0.0002, N=7 each; QTi: UT-SQT1: 92.5±1.9% vs treated-SQT1: 99.7±2.7%, p=0.0007, N=7 each), reaching a QT index of ~100% typically observed in WT rabbits. No such changes were observed in the sham-SQT1 rabbits. Furthermore, cardiomyocytes isolated from 7 SQT1 rabbits that received in vivo SupRep gene therapy demonstrated APD90 prolongation closer to WT rabbits compared to UT-SQT1 and sham-SQT1 rabbits (APD90: UT-SQT1: 314.2±11.9ms, WT: 404.5±13.8ms, treated-SQT1: 362.9±10.6ms; UT-SQT1 vs WT: p<0.0001; UT-SQT1 vs treated-SQT1: p=0.019; WT vs Treated-SQT1: p=0.031). In line with these observations, first IKr measurements demonstrate smaller IKr currents in SupRep-SQT1 than in sham-SQT1 CMs. Conclusion In vivo KCNH2-SupRep gene therapy normalizes the pathologically shortened QTc duration and QT index in transgenic SQT1 rabbits. Moreover, KCNH2-SupRep treatment prolongs APD90 in SQT1 compared to UT-SQT1 and sham-SQT1 reaching levels closer to WT.

Abstract Background Long-QT syndrome type 1 (LQT1) is a genetic channelopathy characterized by loss-of-function variants in the repolarizing potassium channel KCNQ1/IKs which lead to QT-prolongation and predisposes patients to adverse events such as cardiac arrhythmias. Clincally, during catecholamine infusion or exercise stress test, patients with LQT1 present a paradoxical QT prolongation. Purpose To investigate whether a gene-therapy approach targeting the defective K+-channel, through a KCNQ1 suppression-and-replacement (SupRep) strategy, can restore the healthy phenotype at baseline and under catecholamine infusion in LQT1 transgenic rabbits. Methods Our proprietary dual component suppression-and-replacement (SupRep) gene therapy was created by combining a custom-designed KCNQ1 shRNA and a shRNA-immune (shIMM) KCNQ1 cDNA into AAV9. In vivo KCNQ1-SupRep gene therapy was performed by targeted intra-aortic root injection during balloon occlusion. Two or three weeks post gene therapy, 12-lead ECGs were assessed in adult transgenic LQT1 (KCNQ1-Y315S, loss-of IKs) and wild-type (WT) rabbits to determine the effect of SupRep gene therapy on QT-duration at baseline and under catecholamine infusion (10nM isoproterenol, 5mL/h). Ventricular cardiomyocytes (VCMs) were isolated to investigate the effect of SupRep gene therapy on action potential duration (APD) and calcium transients (Ca2+T) at baseline and under isoproterenol infusion. Results At the cellular level, VCMs isolated from SupRep treated LQT1 rabbits showed a significant shortening of the APD90 as compared to UT-LQT1 (p<0.0001). At the whole animal level, at baseline, the QTi (%) of the UT-LQT1 transgenic rabbits (122±3) was significantly longer compared to the WT rabbits (105±2, p<0.0001). Treatment with AAV9-KCNQ1-SupRep shortened significantly the QTi of the transgenic LQT1 rabbits as compared to the UT-LQT1 rabbits (111±2, p=0.0115). To further investigate the effect of SupRep gene therapy, 12-lead ECGs were obtained in UT-LQT1, sham-treated LQT1, SupRep-treated LQT1, and WT rabbits under ISO infusion. While reaching a 20% increase in heart rate during ISO stimulation, the UT-LQT1 and the sham treated LQT1 rabbits showed a marked QT prolongation, with ΔQTi of 31.4% and 24.5%, respectively, as compared to baseline. In contrast, the SupRep-treated LQT1 rabbit behaved nearly identical to the WT rabbit during ISO infusion, showing a ΔQTi of 16.9% as compared to 16.5% in the WT rabbit. Similarly, in contrast to the UT-LQT1, VCMs isolated from SupRep-treated LQT1 rabbits demonstrated a normalization of the APD and Ca2+T under ISO stimulation, showing a significantly shortened APD90 (p<0.0001) and Ca2+T90 (p=0.0025) which was similar to what was observed in WT VCMs. Conclusion In vivo KCNQ1 suppression-and-replacement gene therapy normalizes the pathologically prolonged APD/QT interval and restores a physiological response to catecholamine infusion in treated LQT1 transgenic rabbits.