Gene Therapy for Cardiac Arrhythmias: Mechanisms, Modalities and Therapeutic Applications

Authors, Journal, Affiliations, Type, DOI

• Paschalis Karakasis, Panagiotis Theofilis, Panayotis K. Vlachakis, Nikias Milaras, Kallirhoe Kalinderi, Dimitrios Patoulias, Antonios P. Antoniadis, Nikolaos Fragakis
• Medical Sciences (MDPI), 2025, Vol. 13, Article 102
• Aristotle University of Thessaloniki; National and Kapodistrian University of Athens; International Hellenic University, Thessaloniki
• Review article (open access, CC BY)
• DOI: https://doi.org/10.3390/medsci13030102

Overview

This comprehensive review covers the mechanistic rationale, therapeutic modalities, delivery platforms, and translational frontiers of gene therapy for cardiac arrhythmias. It addresses both inherited channelopathies (LQTS, BrS, CPVT, SQTS) and acquired arrhythmias (AF, post-infarction VT), as well as biological pacemaker development. Five gene therapy modalities are discussed: gene silencing (ASOs, RNAi, CRISPRi), gene replacement (GRT), direct genome editing (CRISPR-Cas9, base/prime editing), pathway modulation, and the novel suppression-and-replacement (SupRep) strategy. AAV9 is the predominant delivery platform, though mRNA-based non-viral approaches are gaining ground. Major limitations include vector packaging size constraints, immune responses, and the translational gap between animal models and human electrophysiology.

Keywords

cardiac arrhythmias, gene therapy, ion channelopathies, genetic modulation, atrial fibrillation, ventricular tachycardia, viral vectors, genome editing

Key Takeaways

Introduction

• Current pharmacologic and device therapies for arrhythmias are largely palliative, addressing downstream electrical manifestations without correcting the molecular substrate. Gene therapy offers disease-modifying potential.
• Pharmacogenetics (variants in SCN5A, KCNQ1, KCNA5, CYP2D6, CYP3A5) inform arrhythmia risk and drug response — a complementary strategy to gene therapy within precision electrophysiology.

Gene Therapy Modalities (Figure 1)

• Gene silencing therapy (GST): Suppresses pathogenic alleles via antisense oligonucleotides (ASOs), RNA interference (RNAi/siRNA/shRNA), or CRISPRi. Targets gain-of-function mutations.
• Gene replacement therapy (GRT): Delivers wild-type cDNA to restore protein function. Targets loss-of-function/haploinsufficiency. Limited by gene size (AAV capacity ~4.7 kb).
• Direct genome editing: CRISPR-Cas9 (double-strand breaks), base editing (nucleotide substitution without DNA breaks), prime editing (insertions/deletions). Enables permanent correction.
• Pathway modulation: Targets downstream signaling nodes (e.g., CaMKII inhibition, calmodulin modification) independent of the specific mutation — mutation-agnostic approach.
• Suppression-and-replacement (SupRep): Simultaneously silences endogenous mutant transcripts (shRNA) and delivers a knockdown-resistant wild-type transgene. Addresses heterozygous dominant-negative pathophysiology.

Atrial Fibrillation

• Electrical remodeling in AF: ↓ICa,L, ↑IK1, ↑IKACh → shortened APD → re-entry. Gene therapy targets these to restore physiological APD.
• AAV-mediated dominant-negative KCNH2 (G628S) transfer in porcine models prolonged atrial APD and suppressed AF inducibility; relative risk of AF 0.44 (Amit et al. 2010; Soucek et al. 2012).
• Dominant-negative Kir2.1AAA suppressed IK1 → prolonged atrial ERP; reduced AF episodes from 2.7 to 0.4/minute (Bikou et al. 2011).
• Cx40/Cx43 overexpression restored atrial conduction velocity and reduced AF inducibility by >80% (Igarashi et al. 2011).
• TGF-β dominant-negative receptor overexpression in canine models reduced atrial fibrosis and improved conduction uniformity (Kunamalla et al. 2016).
• siRNA inhibition of caspase-3 reduced cardiomyocyte apoptosis and delayed AF progression in porcine models.
• Gαi2 C-terminal peptide inhibition attenuated vagally induced AF in canine models.

Post-Infarction Ventricular Arrhythmias

• SkM1 (SCN4A, skeletal sodium channel) overexpression in infarct border zones restored conduction velocity (47.2 vs. 27.3 cm/s) and suppressed re-entrant VT. SkM1 remains active at depolarized potentials where native Nav1.5 is inactivated.
• Cx43 overexpression in infarct border zones reduced sustained VT inducibility by 50% and increased conduction velocity (Trappe 2013; Hu 2014).
• TBX18 mRNA (non-viral) in the EPICCURE trial (NCT03370887): safe myocardial injection during CABG; exploratory signals of improved LVEF (Anttila 2023, phase 2a).

CPVT Gene Therapy

• Direct RYR2 replacement is infeasible — coding sequence ~15,000 nt far exceeds AAV packaging capacity (~4.7 kb).
• AAV9-CASQ2 replacement in CASQ2 knockout mice abolished adrenergically induced VT; effects sustained ≥3 months (Denegri 2012); single AAV9 injection in CASQ2-R33Q knock-in mice showed >85% arrhythmia reduction sustained for ≥1 year (Denegri 2014).
• CASQ2 overexpression in RYR2-mediated CPVT1 mice also suppressed arrhythmias (Santiago Castillo 2023) — pathway-directed approach bypassing direct RYR2 targeting.
• Allele-specific siRNA silencing of mutant CASQ2-R33Q reduced mRNA/protein by ~60%, prevented arrhythmias (Bongianino 2017).
• CRISPR-Cas9 (AAV9-SaCas9) targeting RYR2-R4496C: ~41% editing efficiency, 0/7 treated vs. 7/8 control had arrhythmias — first in-vivo demonstration of safe CRISPR-mediated RYR2 repair (Pan 2023).
• CaMKII inhibitory peptide (AIP) via AAV9 suppressed Ca²⁺ dysregulation and arrhythmias in CASQ2 knockout mice — mutation-agnostic pathway modulation (Bezzerides 2019).
• Modified calmodulin delivery via AAV9 increased RYR2 refractoriness and prevented arrhythmic events (CASQ2-R33Q model).

LQTS Gene Therapy

• Allele-specific RNAi for KCNQ1/KCNH2 is hindered by extreme mutational heterogeneity (hundreds of variants, no single predominant mutation).
• SupRep therapy (AAV9, shRNA + shRNA-immune cDNA) for LQT1: normalized QTi and APD90 in transgenic rabbits, restored β-adrenergic QT response (Bains 2024; Dotzler 2023).
• SupRep for LQT2 (KCNH2): normalized QTc from 470 ms to 414 ms in rabbit model; suppressed EADs and TdP inducibility (Bains 2023).
• SupRep for SQTS1 (gain-of-function KCNH2): shRNA-only (no replacement needed) prolonged QTc from 283 to 333 ms; reduced TdP inducibility from 5/7 to 1/8 animals.
• SupRep balance is critical: excess suppression without replacement worsens LQTS; excess replacement without suppression risks SQTS phenotype.
• LQT3: adenine base editing (ABE8e-SpRY) via dual AAV9 corrected SCN5A-M1875T in mice — 54% editing efficiency, QTc normalized, late INa reduced 66%; even ~20% editing prevented arrhythmias (Qi 2024).

Brugada Syndrome Gene Therapy

• SCN5A direct replacement is infeasible: coding sequence ~6,048 bp approaches/exceeds AAV capacity.
• MOG1 (Ran GEF, Nav1.5 chaperone) overexpression via AAV9 enhanced Nav1.5 cell-surface trafficking and reversed the arrhythmic phenotype in SCN5A-G1746R knock-in mice — pathway-targeted workaround (Yu 2022).

ARVC Gene Therapy

• PKP2 loss-of-function mutations disrupt desmosomes → fibrosis → biventricular dysfunction → arrhythmias.
• AAV9-PKP2 replacement restored plakophilin-2, desmosomal proteins (DSP, DSG2, JUP), Cx43; prevented ARVC phenotype; 100% survival at 6 months even with late-stage administration (Bradford 2023; van Opbergen 2024).
• Three clinical-stage candidates: LX2020 (Lexeo Therapeutics), RP-A601 (Rocket Pharmaceuticals), TN-401 (Tenaya Therapeutics).

Biological Pacemakers

• TBX18 transcription factor overexpression (AAV or synthetic mRNA) reprograms ventricular cardiomyocytes into sinoatrial-node-like pacemaker cells.
• TBX18 mRNA in porcine complete AV block: autonomically responsive pacing (rate-adaptive), persisted ≥2 weeks without electronic pacing, no immune response (Wolfson 2024).
• Dominant-negative Kir2.1AAA suppresses IK1 → spontaneous depolarization; risk of excessive APD prolongation.
• Mutant HCN2 overexpression with enhanced catecholamine sensitivity mimics sinoatrial autonomic modulation.
• hiPSC-derived sinoatrial node-like cells via transgene-independent developmental pathway modulation.
• Optogenetics (channelrhodopsins): non-invasive temporal/spatial cardiac excitability control — preclinical stage.

Delivery Platforms and Routes

• AAV9 is the dominant vector; cardiotropic; broad myocardial distribution.
• Intravenous: least invasive but significant liver off-target accumulation.
• Intracoronary: improves cardiac uptake; less effective with coronary disease.
• Intramyocardial: targeted but invasive; limited spread; risk of localized arrhythmias.
• Lipid nanoparticles (LNPs) encapsulating mRNA: non-viral, transient expression, low immunogenicity (demonstrated in EPICCURE trial with VEGF-A mRNA, Anttila 2023).

Limitations and Future Directions

• AAV packaging limit (~4.7 kb) precludes direct replacement of large genes (RYR2 ~15,000 nt, SCN5A ~6,048 bp); workarounds: dual-vector systems, trans-splicing, mini-gene constructs — inconsistent efficiency.
• Immune responses to AAV vectors limit re-dosing; strategies: transient immunosuppression, capsid re-engineering, extracellular vesicle shielding.
• Genomic integration of AAV (rare) raises insertional mutagenesis risk.
• Murine models have divergent heart rates and ion channel profiles; large-animal models (pigs) are preferred but costly.
• Future: next-generation cardiotropic vectors, mRNA platforms, computational/digital twin modeling for personalized arrhythmia prediction.

Key Concepts Mentioned

• [[concepts/SupRep-Therapy]] — novel dual-function gene therapy platform for dominant-negative channelopathies
• [[concepts/AAV-Gene-Delivery]] — AAV9 as primary cardiac gene therapy vector; packaging constraints and delivery routes
• [[concepts/Gene-Silencing-Therapy]] — ASOs, RNAi, siRNA, shRNA, CRISPRi for pathogenic allele suppression
• [[concepts/Biological-Pacemaker]] — gene-based approach to restore cardiac automaticity
• [[concepts/CRISPR-Cas9-in-Channelopathies]] — first in-vivo RYR2 CRISPR repair; base editing for LQT3
• [[concepts/iPSC-Derived-Cardiomyocytes]] — used for SupRep validation and drug screening
• [[concepts/Ion-Channel-Mutations]] — gain/loss-of-function variants as gene therapy targets
• [[concepts/Left-Cardiac-Sympathetic-Denervation]] — mentioned as conventional comparator to gene therapy

Key Entities Mentioned

• [[entities/CPVT]] — most advanced area; CASQ2 GRT, RYR2 CRISPR, CaMKII pathway modulation
• [[entities/Long-QT-Syndrome]] — SupRep for LQT1/LQT2; base editing for LQT3
• [[entities/Brugada-Syndrome]] — MOG1 overexpression as SCN5A size-constraint workaround
• [[entities/Short-QT-Syndrome]] — SupRep (silencing-only) for SQTS1 gain-of-function KCNH2
• [[entities/Atrial-Fibrillation]] — dominant-negative KCNH2, Cx40/43, TGF-β inhibition strategies
• [[entities/ARVC]] — PKP2 GRT; three clinical-stage candidates
• [[entities/RYR2]] — CRISPR repair, pathway modulation (CASQ2 overexpression, CaMKII inhibition)
• [[entities/SCN5A]] — base editing for LQT3; MOG1 trafficking strategy for BrS; size limits direct AAV delivery
• [[entities/KCNQ1]] — SupRep validated in rabbit LQT1 model
• [[entities/KCNH2]] — SupRep for LQT2; silencing for SQTS1; dominant-negative for AF

Limitations of the Document

• SupRep therapy requires precise dosing balance: over-suppression worsens LQTS, over-replacement induces SQTS — optimal therapeutic window not yet established in humans
• RYR2 and SCN5A both exceed standard AAV packaging capacity — no clinically validated direct replacement solution exists
• AAV immune responses may prevent re-dosing; no established immunosuppression protocol for cardiac gene therapy
• TBX18-based biological pacemakers show preclinical promise but long-term safety, immunogenicity, and integration stability in humans remain unknown
• Murine model findings may not translate to human electrophysiology due to heart rate and ion channel profile differences

Wiki Pages Updated

• wiki/index.md (updated)
• wiki/log.md (updated)
• wiki/sources/gene-therapy-arrhythmia-2025.md (created)
• wiki/concepts/SupRep-Therapy.md (created)
• wiki/concepts/AAV-Gene-Delivery.md (created)
• wiki/concepts/Gene-Silencing-Therapy.md (created)
• wiki/concepts/Biological-Pacemaker.md (created)
• wiki/entities/Atrial-Fibrillation.md (created)
• wiki/entities/ARVC.md (created)
• wiki/concepts/CRISPR-Cas9-in-Channelopathies.md (updated)
• wiki/entities/CPVT.md (updated)
• wiki/entities/Long-QT-Syndrome.md (updated)
• wiki/entities/Brugada-Syndrome.md (updated)
• wiki/entities/RYR2.md (updated)
• wiki/entities/SCN5A.md (updated)
• wiki/entities/KCNQ1.md (updated)
• wiki/entities/KCNH2.md (updated)