Suppression-Replacement Gene Therapy for Type 1 Long QT Syndrome.

Background: Type 1 long QT syndrome (LQT1) is caused by loss-of-function variants in the -encoded K7.1 potassium channel α-subunit that is essential for cardiac repolarization, providing the slow delayed rectifier current. No current therapies target the molecular cause of LQT1.

Methods: A dual-component suppression-and-replacement (SupRep) gene therapy was created by cloning a short hairpin RNA and a short hairpin RNA-immune cDNA modified with synonymous variants in the short hairpin RNA target site, into a single construct. The ability of KCNQ1-SupRep gene therapy to suppress and replace LQT1-causative variants in was evaluated by means of heterologous expression in TSA201 cells. For a human in vitro cardiac model, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were generated from 4 patients with LQT1 (KCNQ1-Y171X, -V254M, -I567S, and -A344A/spl) and an unrelated healthy control. CRISPR-Cas9 corrected isogenic control iPSC-CMs were made for 2 LQT1 lines (correction of KCNQ1-V254M and KCNQ1-A344A/spl). FluoVolt voltage dye was used to measure the cardiac action potential duration (APD) in iPSC-CMs treated with KCNQ1-SupRep.

Results: In TSA201 cells, KCNQ1-SupRep achieved mutation-independent suppression of wild-type and 3 LQT1-causative variants (KCNQ1-Y171X, -V254M, and -I567S) with simultaneous replacement of short hairpin RNA-immune as measured by allele-specific quantitative reverse transcription polymerase chain reaction and Western blot. Using FluoVolt voltage dye to measure the cardiac APD in the 4 LQT1 patient-derived iPSC-CMs, treatment with KCNQ1-SupRep resulted in shortening of the pathologically prolonged APD at both 90% and 50% repolarization, resulting in APD values similar to those of the 2 isogenic controls.

Conclusions: This study provides the first proof-of-principle gene therapy for complete correction of long QT syndrome. As a dual-component gene therapy vector, KCNQ1-SupRep successfully suppressed and replaced to normal wild-type levels. In TSA201 cells, cotransfection of LQT1-causative variants and KCNQ1-SupRep caused mutation-independent suppression and replacement of . In LQT1 iPSC-CMs, KCNQ1-SupRep gene therapy shortened the APD, thereby eliminating the pathognomonic feature of LQT1.