Zehender, Manfred

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 Funding Acknowledgements Type of funding sources: Private company. Main funding source(s): Pfizer Background Type 1 long QT syndrome (LQT1) is a genetic channelopathy characterized by both haploinsufficient and dominant-negative loss-of-function pathogenic variants in the KCNQ1-encoded Kv7.1 K+ channels conducting IKs. Patients with LQT1 may manifest QT prolongation and ventricular arrhythmias that can culminate in sudden cardiac death. Purpose With this project, we aim to investigate whether our mutation independent, KCNQ1-specific suppression-replacement (SupRep) gene therapy can restore the deficient K+-channel and thereby restore the healthy phenotype. Methods Our proprietary dual component SupRep gene therapy was created by combining a custom-designed KCNQ1 shRNA and a shRNA-immune (shIMM) KCNQ1 cDNA into AAV9. In vivo AAV9-mediated KCNQ1SupRep gene therapy was performed by targeted intra-aortic root construct injection (1E12 vg/kg body weight) during balloon occlusion using a Swan Ganz catheter. 2 weeks after in vivo 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 therapy on the rabbit’s QTc. Patch-clamp and calcium transient measurements were performed in isolated ventricular cardiomyocytes to evaluate the effect of SupRep on the cardiomyocyte’s action potential duration (APD90) and Ca2+ transient duration (Ca2+90) both, in vitro – after plasmid transfection, and in vivo – after AAV9-mediated gene therapy. AAV9-KCNQ1-construct expression was verified with immunohistochemistry targeting the GFP-tag. Results After injection of 3 LQT1 rabbits so far, no significant changes were observed in the LQT1 rabbits’ QTc before and after in vivo AAV9-mediated gene therapy. However, at the cellular level, LQT1 cardiomyocytes transfected in vitro with the SupRep-plasmid demonstrated a significant reduction of APD90 (ms, 1Hz stimulation at 37°) compared to LQT1 control cells (SupRep-in-vitro, 358±21 vs. control 447±22, p<0.05). No differences were observed between sham-plasmid transfected cardiomyocytes and LQT1 control cells after one day of culturing. Similarly, cardiomyocytes isolated from the 3 LQT1 rabbits that underwent in vivo SupRep gene therapy demonstrated pronounced shortening of both APD90 and Ca2+90 compared to LQT1 controls, leading to levels similar to WT controls (APD90, 1Hz, 37°: LQT1: 530±18, WT: 445±18, LQT1-SupRep: 375±25, p<0.0001 LQT1 vs. LQT1-SupRep, p=ns LQT1-SupRep vs. WT) (Ca2+90: LQT1: 487±23, WT: 346±16, LQT1-SupRep: 393±19, p<0.0001 LQT1 vs. LQT1-SupRep, p=ns LQT1-SupRep vs. WT). Conclusion In vivo KCNQ1-suppression-replacement gene therapy normalizes cellular action potential and calcium transient duration in transgenic LQT1 rabbits. Further in vivo experiments will be conducted to evaluate whether this therapeutic correction at the cardiomyocyte level will translate into normalization of the LQT1 rabbits’ QTc both at rest and during stress testing.