EN-510-02 SUPPRESSION AND REPLACEMENT GENE THERAPY FOR KCNH2-MEDIATED ARRHYTHMIAS

Abstract

KCNH2-mediated arrhythmias are caused by either loss-of-function (type 2 long QT syndrome, LQT2) or gain-of-function (type 1 short QT syndrome, SQT1) pathogenic variants in the KCNH2-encoded Kv11.1 potassium channel which is essential for the rapid delayed rectifier current (IKr) that contributes to the cardiac action potential (AP). No current therapies target the molecular cause of either LQT2 or SQT1. To rescue the pathologic phenotype in cell models of LQT2 and SQT1 using our novel gene therapy. A dual-component “suppression-and-replacement” (SupRep) KCNH2 gene therapy was created by cloning into a single construct a custom-designed KCNH2 shRNA that produces ∼80% knockdown (suppression) and a “shRNA-immune” (shIMM) KCNH2 cDNA (replacement). Patient-derived induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) and their CRISPR-Cas9 variant-corrected isogenic control (IC) iPSC-CMs were made for 3 LQT2- (G604S, G628S, N633S) and 1 SQT1- (N588K) causative variants. All 4 variant lines were treated with KCNH2-SupRep or non-targeting control shRNA (sham). FluoVolt voltage dye was used to measure the APD at 90% repolarization (APD90). KCNH2-SupRep achieved mutation-independent rescue of the pathologic phenotype in both LQT2 and SQT1. For LQT2-causative variants, treatment with KCNH2-SupRep resulted in shortening of the pathologically prolonged APD90 to near curative (IC-like) APD90 levels (G604S IC, 471±25ms; G628S IC, 429±16ms; N633S IC, 405±55ms) compared to treatment with sham (G604S: SupRep-treated, 452±76ms vs. sham-treated, 550±41ms, p<0.0001; G628S: SupRep-treated, 491±38ms vs. sham-treated, 674±20ms, p<0.0001; N633S: SupRep-treated, 399±105ms vs. sham-treated, 577±39ms, p<0.0001). Conversely, for the SQT1-causative N588K, treatment with KCNH2-SupRep resulted in therapeutic prolongation of the pathologically shortened APD90 (IC: 429±16ms; SupRep-treated: 396±61ms; sham-treated: 274±12ms). We provide the first proof-of-principle gene therapy for correction of both LQT2 and SQT1. Akin to our sentinel discovery of SupRep gene therapy for LQT1, KCNH2-SupRep gene therapy successfully corrected/normalized the pathologic APD90, thereby eliminating the pathognomonic feature of both LQT2 and SQT1.