Single Construct Suppression and Replacement Gene Therapy for the Treatment of All -, -, and -Mediated Arrhythmia Disorders.

BACKGROUND

CaM (calmodulin)-mediated long-QT syndrome is a genetic arrhythmia disorder (calmodulinopathies) characterized by a high prevalence of life-threatening ventricular arrhythmias occurring early in life. Three distinct genes (, , and ) encode for the identical CaM protein. Conventional pharmacotherapies fail to adequately protect against potentially lethal cardiac events in patients with calmodulinopathy.

METHODS

Five custom-designed -, -, and -targeting short hairpin RNAs (shRNAs) were tested for knockdown (KD) efficiency using TSA201 cells and reverse transcription-quantitative polymerase chain reaction. A dual-component suppression and replacement (SupRep) gene therapy (CALM-SupRep) was created by cloning into a single construct -, -, and KD (suppression) of each respective gene and a shRNA-immune cDNA (replacement). CALM1-F142L, CALM2-D130G, and CALM3-D130G induced pluripotent stem cell-derived CMs were generated from patients with CaM-mediated long-QT syndrome. A voltage-sensing dye was used to measure action potential duration at 90% repolarization (APD90).

RESULTS

Following shRNA KD efficiency testing, a candidate shRNA was identified for (86% KD), (71% KD), and (94% KD). The APD90 was significantly prolonged in CALM2-D130G (647±9 ms) compared with CALM2-WT (359±12 ms; <0.0001). Transfection with -SupRep shortened the average APD90 of CALM2-D130G to 457±19 ms (66% attenuation; <0.0001). Additionally, transfection with -SupRep shortened the APD90 of CALM1-F142L (665±9 to 410±15 ms; <0.0001) and CALM3-D130G (978±81 to 446±6 ms; <0.001).

CONCLUSIONS

We provide the first proof-of-principle suppression-replacement gene therapy for CaM-mediated long-QT syndrome. The CALM-SupRep gene therapy shortened the pathologically prolonged APD90 in -, -, and CaM-mediated long-QT syndrome induced pluripotent stem cell-derived CM lines. The single CALM-SupRep construct may be able to treat all calmodulinopathies, regardless of which of the 3 CaM-encoding genes are affected.