Department of Molecular Pharmacology and Experimental Therapeutics (Windland Smith Rice Sudden Death Genomics Laboratory) (S.K.H., C.S.J.K., D.J.T., M. Gencarelli, K.E.T., M. Gluscevic, M.J.A.), Mayo Clinic, Rochester, MN.
Department of Cardiovascular Medicine (Division of Heart Rhythm Services, Windland Smith Rice Genetic Heart Rhythm Clinic) (M.J.A.), Mayo Clinic, Rochester, MN.
Circ Arrhythm Electrophysiol. 2024 Aug;17(8):e012036. doi: 10.1161/CIRCEP.123.012036. Epub 2024 Jul 29.
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.
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).
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).
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.
钙调蛋白（CaM）介导的长 QT 综合征是一种遗传性心律失常疾病（钙调蛋白病），其特征是在生命早期发生危及生命的室性心律失常的高发率。三个不同的基因（、和）编码相同的 CaM 蛋白。传统的药物治疗未能充分保护钙调蛋白病患者免受潜在致命的心脏事件的影响。
使用 TSA201 细胞和逆转录定量聚合酶链反应测试了 5 种定制的靶向、和的短发夹 RNA（shRNA）的敲低（KD）效率。通过将、和 KD（抑制）克隆到单个构建体中，创建了双组分抑制和替代（SupRep）基因治疗（CALM-SupRep），每个基因的一个 shRNA-免疫 cDNA（替代）。从钙调蛋白介导的长 QT 综合征患者中生成了诱导多能干细胞衍生的 CM 的 CaM1-F142L、CaM2-D130G 和 CaM3-D130G。使用电压敏感染料测量动作电位复极至 90%（APD90）的持续时间。
在进行 shRNA KD 效率测试后，确定了候选 shRNA，用于（86% KD）、（71% KD）和（94% KD）。与 CALM2-WT（359±12 ms；<0.0001）相比，CALM2-D130G 的 APD90 显著延长（647±9 ms）。转染-SupRep 将 CALM2-D130G 的平均 APD90 缩短至 457±19 ms（66%衰减；<0.0001）。此外，转染-SupRep 将 CALM1-F142L（665±9 至 410±15 ms；<0.0001）和 CALM3-D130G（978±81 至 446±6 ms；<0.001）的 APD90 缩短。
我们提供了钙调蛋白介导的长 QT 综合征的第一个抑制-替代基因治疗的原理证明。CALM-SupRep 基因治疗缩短了、和钙调蛋白介导的长 QT 综合征诱导多能干细胞衍生的 CM 系中病理性延长的 APD90。单一的 CALM-SupRep 构建体可能能够治疗所有钙调蛋白病，而与受影响的 3 个 CaM 编码基因中的哪一个无关。
