Single Construct Suppression and Replacement Gene Therapy for the Treatment of All CALM1-, CALM2-, and CALM3-Mediated Arrhythmia Disorders.

IF 9.1 1区医学 Q1 CARDIAC & CARDIOVASCULAR SYSTEMS

Circulation. Arrhythmia and electrophysiology Pub Date : 2024-08-01 Epub Date: 2024-07-29 DOI:10.1161/CIRCEP.123.012036

Samantha K Hamrick, C S John Kim, David J Tester, Manuela Gencarelli, Kathryn E Tobert, Martina Gluscevic, Michael J Ackerman

{"title":"Single Construct Suppression and Replacement Gene Therapy for the Treatment of All CALM1-, CALM2-, and CALM3-Mediated Arrhythmia Disorders.","authors":"Samantha K Hamrick, C S John Kim, David J Tester, Manuela Gencarelli, Kathryn E Tobert, Martina Gluscevic, Michael J Ackerman","doi":"10.1161/CIRCEP.123.012036","DOIUrl":null,"url":null,"abstract":"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 (CALM1, CALM2, and CALM3) 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 CALM1-, CALM2-, and CALM3-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) CALM gene therapy (CALM-SupRep) was created by cloning into a single construct CALM1-, CALM2-, and CALM3-specific shRNAs that produce KD (suppression) of each respective gene and a shRNA-immune CALM1 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 CALM1 (86% KD), CALM2 (71% KD), and CALM3 (94% KD). The APD90 was significantly prolonged in CALM2-D130G (647±9 ms) compared with CALM2-WT (359±12 ms; P<0.0001). Transfection with CALM-SupRep shortened the average APD90 of CALM2-D130G to 457±19 ms (66% attenuation; P<0.0001). Additionally, transfection with CALM-SupRep shortened the APD90 of CALM1-F142L (665±9 to 410±15 ms; P<0.0001) and CALM3-D130G (978±81 to 446±6 ms; P<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 CALM1-, CALM2-, and CALM3-variant 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.","PeriodicalId":10319,"journal":{"name":"Circulation. Arrhythmia and electrophysiology","volume":" ","pages":"e012036"},"PeriodicalIF":9.1000,"publicationDate":"2024-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Circulation. Arrhythmia and electrophysiology","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1161/CIRCEP.123.012036","RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/7/29 0:00:00","PubModel":"Epub","JCR":"Q1","JCRName":"CARDIAC & CARDIOVASCULAR SYSTEMS","Score":null,"Total":0}

引用次数: 0

Abstract

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 (CALM1, CALM2, and CALM3) 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 CALM1-, CALM2-, and CALM3-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) CALM gene therapy (CALM-SupRep) was created by cloning into a single construct CALM1-, CALM2-, and CALM3-specific shRNAs that produce KD (suppression) of each respective gene and a shRNA-immune CALM1 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 CALM1 (86% KD), CALM2 (71% KD), and CALM3 (94% KD). The APD90 was significantly prolonged in CALM2-D130G (647±9 ms) compared with CALM2-WT (359±12 ms; P<0.0001). Transfection with CALM-SupRep shortened the average APD90 of CALM2-D130G to 457±19 ms (66% attenuation; P<0.0001). Additionally, transfection with CALM-SupRep shortened the APD90 of CALM1-F142L (665±9 to 410±15 ms; P<0.0001) and CALM3-D130G (978±81 to 446±6 ms; P<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 CALM1-, CALM2-, and CALM3-variant 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.

查看原文本刊更多论文

用于治疗所有 CALM1、CALM2 和 CALM3 导致的心律失常疾病的单一构建抑制和替代基因疗法。

背景：CaM（钙调素）介导的长 QT 综合征是一种遗传性心律失常疾病（钙调素病），其特点是在生命早期高发危及生命的室性心律失常。三个不同的基因（CALM1、CALM2 和 CALM3）编码相同的 CaM 蛋白。传统的药物疗法无法充分保护钙调蛋白病患者免受可能致命的心脏事件的影响：方法：使用 TSA201 细胞和反转录定量聚合酶链反应测试了五种定制设计的 CALM1、CALM2 和 CALM3 靶向短发夹 RNA（shRNA）的基因敲除（KD）效率。通过克隆CALM1、CALM2和CALM3特异性shRNA（可对各基因产生KD（抑制））和shRNA免疫CALM1 cDNA（替代）），创建了双组分抑制和替代（SupRep）CALM基因疗法（CALM-SupRep）。CALM1-F142L、CALM2-D130G和CALM3-D130G诱导多能干细胞衍生CM由CaM介导的长QT综合征患者产生。使用电压传感染料测量90%复极化时的动作电位持续时间（APD90）：结果：经过 shRNA KD 效率测试，确定了 CALM1（86% KD）、CALM2（71% KD）和 CALM3（94% KD）的候选 shRNA。与 CALM2-WT 相比，CALM2-D130G 的 APD90 明显延长（647±9 ms）（359±12 ms；PCALM-SupRep 将 CALM2-D130G 的平均 APD90 缩短至 457±19 ms，衰减率为 66%；PCALM-SupRep 将 CALM1-F142L 的 APD90 缩短（665±9 至 410±15 ms；PPConclusions）：我们提供了首个针对 CaM 介导的长 QT 综合征的抑制替代基因疗法的原理验证。CALM-SupRep基因疗法缩短了CALM1-、CALM2-和CALM3-变异型CaM介导的长QT综合征诱导多能干细胞衍生CM株中病理性延长的APD90。单一的CALM-SupRep构建体可能能够治疗所有钙调蛋白病，无论3个CaM编码基因中的哪一个受到影响。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

求助全文

约1分钟内获得全文求助全文

来源期刊

Circulation. Arrhythmia and electrophysiology 医学-心血管系统

CiteScore

13.70

自引率

4.80%

发文量

187

审稿时长

4-8 weeks

期刊介绍： Circulation: Arrhythmia and Electrophysiology is a journal dedicated to the study and application of clinical cardiac electrophysiology. It covers a wide range of topics including the diagnosis and treatment of cardiac arrhythmias, as well as research in this field. The journal accepts various types of studies, including observational research, clinical trials, epidemiological studies, and advancements in translational research.