Suppression and Replacement Gene Therapy for KCNH2-Mediated Arrhythmias

Background: KCNH2-mediated arrhythmia syndromes are caused by 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 K(v)11.1 potassium channel, which is essential for the cardiac action potential. Methods: A dual-component "suppression-and-replacement" (SupRep) KCNH2 gene therapy was created by cloning into a single construct a custom-designed KCNH2 short hairpin RNA with similar to 80% knockdown (suppression) and a "short hairpin RNA-immune" KCNH2 cDNA (replacement). Induced pluripotent stem cell-derived cardiomyocytes and their CRISPR-Cas9 variant-corrected isogenic control (IC) induced pluripotent stem cell-derived cardiomyocytes were made for 2 LQT2- (G604S, N633S) and 1 SQT1- (N588K) causative variants. All variant lines were treated with KCNH2-SupRep or non-targeting control short hairpin RNA (shCT). The action potential duration (APD) at 90% repolarization (APD(90)) was measured using FluoVolt voltage dye. Results: KCNH2-SupRep achieved variant-independent rescue of both pathologic phenotypes. For LQT2-causative variants, treatment with KCNH2-SupRep resulted in shortening of the pathologically prolonged APD(90) to near curative (IC-like) APD(90) levels (G604S IC, 471 +/- 25 ms; N633S IC, 405 +/- 55 ms) compared with treatment with shCT (G604S: SupRep-treated, 452 +/- 76 ms versus shCT-treated, 550 +/- 41 ms; P<0.0001; N633S: SupRep-treated, 399 +/- 105 ms versus shCT-treated, 577 +/- 39 ms, P<0.0001). Conversely, for the SQT1-causative variant, N588K, treatment with KCNH2-SupRep resulted in therapeutic prolongation of the pathologically shortened APD(90) (IC: 429 +/- 16 ms; SupRep-treated: 396 +/- 61 ms; shCT-treated: 274 +/- 12 ms). Conclusions: We provide the first proof-of-principle gene therapy for correction of both LQT2 and SQT1. KCNH2-SupRep gene therapy successfully normalized the pathologic APD(90), thereby eliminating the pathognomonic feature of both LQT2 and SQT1.