PO-02-210 AAV9-MEDIATED KCNQ1-SUPPRESSION-REPLACEMENT GENE THERAPY IN TRANSGENIC RABBITS WITH TYPE 1 LONG QT SYNDROME

Abstract

Type 1 long QT syndrome (LQT1) is characterized by both haploinsufficient and dominant-negative loss-of-function pathogenic variants in the KCNQ1-encoded Kv7.1 K+ channels conducting IKs current, which contributes to the cardiac action potential. No current therapies target the molecular cause of LQT1. To rescue the pathologic phenotype in the transgenic KCNQ1-Y315S LQT1 rabbit model using our proprietary KCNQ1-specific suppression-replacement (SupRep) gene therapy. Our dual component SupRep gene therapy was created by combining into a single construct a custom-designed KCNQ1 shRNA (suppression) and a cardiac-specific troponin C (cTnC) promoter driven shRNA-immune KCNQ1 cDNA (replacement). The KCNQ1-SupRep construct was packaged into AAV9 (AAV9-KCNQ1-SupRep), a nominally immunogenic and cardiotropic viral vector with limited off-target delivery. AAV9-KCNQ1-SupRep was injected in vivo via a targeted intra-aortic root injection (1E12 vg/kg bodyweight) during balloon occlusion using a Swan Ganz catheter. Two weeks post in vivo gene therapy, 12-lead ECGs were assessed in adult transgenic LQT1 and wildtype (WT) rabbits to ascertain the effect of SupRep on the rabbit’s QTc. Patch-clamp and calcium transient measurements were performed in isolated ventricular cardiomyocytes (CMs) to evaluate the effect of SupRep on the CM’s action potential duration (APD90) and Ca2+ transient duration (Ca2+90) post in vivo gene therapy. AAV9-KCNQ1-SupRep expression was verified with immunohistochemistry. After injection of 3 LQT1 rabbits so far, no statistically significant changes were observed in the LQT1 rabbits’ QTc pre/post in vivo AAV9-KCNQ1-SupRep. However, CMs isolated from the 3 LQT1 rabbits that underwent in vivo SupRep gene therapy demonstrated pronounced shortening (ms) 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). In vivo AAV9-KCNQ1-SupRep gene therapy normalizes cellular AP and calcium transient duration in transgenic LQT1 rabbits. Further in vivo experiments will be conducted to evaluate whether this therapeutic correction at the CM level will translate into normalization of the LQT1 rabbits’ QTc both at rest and during stress testing.