Samantha K Hamrick, C S John Kim, David J Tester, Manuela Gencarelli, Kathryn E Tobert, Martina Gluscevic, Michael J Ackerman
{"title":"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。","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":"<p><strong>Background: </strong>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 (<i>CALM1</i>, <i>CALM2</i>, and <i>CALM3</i>) encode for the identical CaM protein. Conventional pharmacotherapies fail to adequately protect against potentially lethal cardiac events in patients with calmodulinopathy.</p><p><strong>Methods: </strong>Five custom-designed <i>CALM1</i>-, <i>CALM2</i>-, and <i>CALM3</i>-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) <i>CALM</i> gene therapy (CALM-SupRep) was created by cloning into a single construct <i>CALM1</i>-, <i>CALM2</i>-, and <i>CALM3-specific shRNAs that produce</i> KD (suppression) of each respective gene and a shRNA-immune <i>CALM1</i> 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).</p><p><strong>Results: </strong>Following shRNA KD efficiency testing, a candidate shRNA was identified for <i>CALM1</i> (86% KD), <i>CALM2</i> (71% KD), and <i>CALM3</i> (94% KD). The APD90 was significantly prolonged in CALM2-D130G (647±9 ms) compared with CALM2-WT (359±12 ms; <i>P</i><0.0001). Transfection with <i>CALM</i>-SupRep shortened the average APD90 of CALM2-D130G to 457±19 ms (66% attenuation; <i>P</i><0.0001). Additionally, transfection with <i>CALM</i>-SupRep shortened the APD90 of CALM1-F142L (665±9 to 410±15 ms; <i>P</i><0.0001) and CALM3-D130G (978±81 to 446±6 ms; <i>P</i><0.001).</p><p><strong>Conclusions: </strong>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 <i>CALM1</i>-, <i>CALM2</i>-, and <i>CALM3-variant</i> 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.</p>","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":"{\"title\":\"Single Construct Suppression and Replacement Gene Therapy for the Treatment of All <i>CALM1</i>-, <i>CALM2</i>-, and <i>CALM3</i>-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\":\"<p><strong>Background: </strong>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 (<i>CALM1</i>, <i>CALM2</i>, and <i>CALM3</i>) encode for the identical CaM protein. Conventional pharmacotherapies fail to adequately protect against potentially lethal cardiac events in patients with calmodulinopathy.</p><p><strong>Methods: </strong>Five custom-designed <i>CALM1</i>-, <i>CALM2</i>-, and <i>CALM3</i>-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) <i>CALM</i> gene therapy (CALM-SupRep) was created by cloning into a single construct <i>CALM1</i>-, <i>CALM2</i>-, and <i>CALM3-specific shRNAs that produce</i> KD (suppression) of each respective gene and a shRNA-immune <i>CALM1</i> 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).</p><p><strong>Results: </strong>Following shRNA KD efficiency testing, a candidate shRNA was identified for <i>CALM1</i> (86% KD), <i>CALM2</i> (71% KD), and <i>CALM3</i> (94% KD). The APD90 was significantly prolonged in CALM2-D130G (647±9 ms) compared with CALM2-WT (359±12 ms; <i>P</i><0.0001). Transfection with <i>CALM</i>-SupRep shortened the average APD90 of CALM2-D130G to 457±19 ms (66% attenuation; <i>P</i><0.0001). Additionally, transfection with <i>CALM</i>-SupRep shortened the APD90 of CALM1-F142L (665±9 to 410±15 ms; <i>P</i><0.0001) and CALM3-D130G (978±81 to 446±6 ms; <i>P</i><0.001).</p><p><strong>Conclusions: </strong>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 <i>CALM1</i>-, <i>CALM2</i>-, and <i>CALM3-variant</i> 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.</p>\",\"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. 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Single Construct Suppression and Replacement Gene Therapy for the Treatment of All CALM1-, CALM2-, and CALM3-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 (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.
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.
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