Background:
Type 1 long QT syndrome (LQT1) is caused by loss-of-function variants in the
KCNQ1
-encoded K
v
7.1 potassium channel α-subunit which is essential for cardiac repolarization, providing the slow delayed rectifier current (IKs). No current therapies target the molecular cause of LQT1.
Methods:
A dual-component "suppression-and-replacement" (SupRep)
KCNQ1
gene therapy was created by cloning a
KCNQ1
shRNA and a "shRNA-immune" (shIMM)
KCNQ1
cDNA modified with silent variants in the shRNA target site, into a single construct. The ability of KCNQ1-SupRep gene therapy to suppress and replace LQT1-causative variants in
KCNQ1
was evaluated via heterologous expression in TSA201 cells. For a human in vitro cardiac model, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were generated from four 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 two 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
KCNQ1
and three LQT1-causative variants (KCNQ1-Y171X, -V254M, and -I567S) with simultaneous replacement of KCNQ1-shIMM as measured by allele-specific qRT-PCR and western blot. Using FluoVolt voltage dye to measure the cardiac APD in the four LQT1 patient-derived iPSC-CMs, treatment with KCNQ1-SupRep resulted in shortening of the pathologically prolonged APD at both 90% (APD
90
) and 50% (APD
50
) repolarization resulting in APD values similar to those of the two isogenic controls.
Conclusions:
This study provides the first proof-of-principle gene therapy for complete correction of LQTS. As a dual-component gene therapy vector, KCNQ1-SupRep successfully suppressed and replaced
KCNQ1
to normal wild-type levels. In TSA201 cells, co-transfection of LQT1-causative variants and KCNQ1-SupRep caused mutation-independent suppression-and-replacement of
KCNQ1
. In LQT1 iPSC-CMs, KCNQ1-SupRep gene therapy shortened the APD, thereby eliminating the pathognomonic feature of LQT1.
Even pore-localizing missense variants at highly conserved sites in KCNQ1 -encoded K v 7.1 channels may have wild-type function and not cause type 1 long QT syndrome: Do not rely solely on the genetic test company's interpretation
