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

Samantha K Hamrick1, C S John Kim1, David J Tester1

  • 1Department 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.

Circulation. Arrhythmia and Electrophysiology
|July 29, 2024
PubMed
Abstract

Insights

This study introduces a novel gene therapy for calmodulin (CaM)-mediated long-QT syndrome. The CALM-SupRep therapy effectively reduced prolonged cardiac action potentials in patient-derived cells, offering a potential unified treatment for this genetic arrhythmia.

Area of Science:

  • Cardiovascular Genetics
  • Molecular Therapy
  • Gene Regulation

Background:

  • Calmodulin (CaM)-mediated long-QT syndrome (calmodulinopathy) is a severe genetic arrhythmia characterized by early-onset, life-threatening ventricular arrhythmias.
  • Three genes (CALM1, CALM2, CALM3) encode the identical CaM protein, and conventional treatments are insufficient for managing lethal cardiac events.
  • The disorder necessitates innovative therapeutic strategies to address the underlying genetic defects.

Purpose of the Study:

  • To develop and evaluate a novel suppression-replacement gene therapy (CALM-SupRep) for CaM-mediated long-QT syndrome.
  • To assess the efficacy of CALM-SupRep in correcting the prolonged action potential duration (APD90) in patient-derived cells.
  • To establish a proof-of-principle for a single gene therapy construct targeting all three CaM-encoding genes.

Main Methods:

  • Five custom short hairpin RNAs (shRNAs) targeting CALM1, CALM2, and CALM3 were tested for knockdown (KD) efficiency.
  • A dual-component CALM-SupRep gene therapy construct was engineered, combining gene-specific shRNAs for suppression and an shRNA-immune CALM1 cDNA for replacement.
  • Induced pluripotent stem cell-derived cardiomyocytes from patients with CALM1, CALM2, and CALM3 variants were used to measure APD90 using voltage-sensing dye.

Main Results:

  • Effective shRNA-mediated KD was achieved for CALM1 (86%), CALM2 (71%), and CALM3 (94%).
  • Significant prolongation of APD90 was observed in patient-derived cells (e.g., CALM2-D130G: 647±9 ms vs. WT: 359±12 ms).
  • CALM-SupRep treatment markedly shortened the prolonged APD90 in all tested cell lines (e.g., CALM2-D130G reduced by 66%, CALM1-F142L by 38%, CALM3-D130G by 54%).

Conclusions:

  • This study presents the first proof-of-principle for a suppression-replacement gene therapy for CaM-mediated long-QT syndrome.
  • The CALM-SupRep gene therapy effectively normalized the prolonged APD90 in patient-derived cells with CALM1, CALM2, and CALM3 variants.
  • A single CALM-SupRep construct holds potential for treating all forms of calmodulinopathy, irrespective of the affected CaM-encoding gene.
Keywords:
RNA, small interferingarrhythmias, cardiaccalmodulingenetic therapylong QT syndrome

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