Gene Therapy for Cardiac Arrhythmias: Mechanisms, Modalities and Therapeutic Applications
Abstract
1. Introduction
2. Genetic Therapeutic Strategies in the Management of Cardiac Arrhythmias
2.1. Molecular Modulation of Electrophysiological Substrates in Atrial Fibrillation
2.2. Ventricular Arrhythmias
2.2.1. Acquired Ventricular Arrhythmias
2.2.2. Inherited Ventricular Arrhythmias
Author (Year) | Type of Arrhythmia | Type of Study | Vector | Form of Gene Therapy | Main Findings |
---|---|---|---|---|---|
Yu et al. (2022) [112] | Brugada syndrome (BrS), ventricular tachyarrhythmias (VTs), sinus node dysfunction (SND), cardiac conduction disease (CCD) | Preclinical in vivo (murine KI models and hiPSC-derived cardiomyocytes | AAV9 | AAV9-mediated overexpression of MOG1 (20 kDa protein chaperone for NaV1.5) | AAV9-MOG1 significantly increased NaV1.5 surface expression and peak INa density (e.g., from −7.68 ± 0.52 to −16.02 ± 1.27 pA/pF in Scn5aG1746R/+ mice; p < 0.001). It reversed abnormal ECG features (abolished J waves, sinus arrest, and VT), restored shortened APD (APD100 and APD90), and eliminated late phase 3 EADs. It downregulated Kcnd3 and Cacna1c expression. In SCN5A-D1275N KI mice, it rescued contractile dysfunction (EF and FS restored), reduced sinus pauses and heart block incidence, and increased stroke volume and cardiac output (SV: 24.55 μL vs. 31.42 μL; p = 0.0133). Similar rescue of INa density was seen in hiCMs (e.g., −11.33 to −15.81 pA/pF; p = 0.022). |
Santiago Castillo et al. (2023) [94] | Catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1) | In silico model + in vivo (RyR2 R4496C/+ KI mice) | AAV (adeno-associated viral vector) | AAV-mediated overexpression of CASQ2 (cardiac calsequestrin) | In silico: Arrhythmia suppression achieved with 1.4-fold CASQ2 overexpression for Class 1 RyR2 mutations and 1.8-fold for Class 2. In vivo: CASQ2 gene therapy completely prevented arrhythmias upon caffeine/epinephrine challenge (0/12 treated vs. 17/32 untreated mice; p = 0.0012), showing potent antiarrhythmic efficacy and validating CASQ2 overexpression as a novel therapeutic approach in CPVT1. |
Bongianino et al. (2017) [101] | Catecholaminergic polymorphic ventricular tachycardia (CPVT1) | In vivo (CASQ2R33Q/R33Q knock-in mouse model) | Liposome-mediated delivery (Invivofectamine) | Allele-specific silencing of mutant CASQ2 mRNA via siRNA | Systemic delivery of siCASQ2-R33Q (5 mg/kg) significantly reduced mutant CASQ2 mRNA and protein expression by ~60%, without affecting WT CASQ2. After 3 injections (every 2 weeks), treated mice showed normalization of calcium release, reduced spontaneous Ca2+ waves, and prevention of arrhythmias induced by epinephrine/caffeine challenge (0/9 treated vs. 8/12 untreated mice; p = 0.003). Electron microscopy revealed restored junctional SR architecture. No toxicity or off-target effects observed. |
Pan et al. (2023) [102] | Catecholaminergic polymorphic ventricular tachycardia (CPVT1) | In vivo (RyR2R4496C/+ knock-in mouse model) | AAV9 | CRISPR-based gene editing using AAV9-Staphylococcus aureus Cas9 (SaCas9) and guide RNA | AAV9-SaCas9-mediated editing targeted mutant RYR2R4496C in the heart, achieving ~41% editing efficiency in cardiomyocytes. Treated mice exhibited significant reduction in ventricular arrhythmias during epinephrine/caffeine challenge (0/7 treated vs. 7/8 control; p = 0.0014). Restored Ca2+ handling was observed with decreased spontaneous Ca2+ waves and normalized Ca2+ transients. No significant off-target editing detected in vivo, and no adverse effects noted. This study offers first in vivo demonstration of safe, effective CRISPR-mediated RYR2 repair in a CPVT1 model. |
Bezzerides et al. (2019) [103] | Catecholaminergic polymorphic ventricular tachycardia (CPVT1) | In vivo (CASQ2 knockout mice) + isolated cardiomyocytes | AAV9 | AAV9-mediated delivery of CaMKII-inhibitory peptide (AIP) | AAV9-AIP significantly reduced ventricular arrhythmias in CASQ2−/− mice during epinephrine/challenge (event rate 0.67 vs. 2.17 events/min in controls; p < 0.01). Optical mapping showed normalization of Ca2+ transients and suppression of diastolic Ca2+ waves. Treated cardiomyocytes displayed reduced spontaneous Ca2+ oscillations (27% vs. 79% in untreated; p < 0.01). AIP therapy was well tolerated with no off-target contractility or structural effects, demonstrating efficacy of targeted CaMKII inhibition in genetic arrhythmia. |
Denegri et al. (2012) [92] | Catecholaminergic polymorphic ventricular tachycardia (CPVT, CASQ2-related) | In vivo (CASQ2 knockout mice) | AAV9 | AAV9-mediated delivery of wild-type CASQ2 gene | Viral delivery of CASQ2 infected ~50% of myocytes, restored CASQ2, triadin (TrD), and junction (JnC) levels to ~80–90% of wild-type levels. This led to reversal of jSR structural abnormalities, including jSR width normalization (from 37 ± 1.2 nm in KO to 21 ± 0.3 nm in treated; p < 0.001), reduced triggered activity during β-adrenergic stimulation (from 70% to 5% of myocytes; p < 0.001), and suppression of in vivo ventricular tachycardia (from 15/15 to 1/10 mice after epinephrine; p < 0.001). No histological toxicity observed. Demonstrated durable structural and functional rescue. |
Denegri et al. (2014) [93] | Catecholaminergic polymorphic ventricular tachycardia (CPVT1, CASQ2-R33Q model) | In vivo (CASQ2R33Q/R33Q knock-in mice) | AAV9 | AAV9-mediated delivery of wild-type CASQ2 gene | A single intravenous AAV9-CASQ2 injection (3.5 × 1013 vg/kg) restored CASQ2 protein expression to ~60% of WT levels, reversed RyR2 mislocalization, and normalized jSR ultrastructure. Treated mice showed >85% reduction in arrhythmia incidence during exercise + epinephrine challenge (from 92% to 8%; p < 0.001), and >90% reduction in premature ventricular contractions. Intracellular Ca2+ cycling was restored with normalization of Ca2+ transients and reduced spontaneous waves. Effects were sustained for at least 3 months with no observed toxicity, confirming long-term efficacy and safety. |
Bains et al. (2024) [110] | Congenital Long QT Syndrome Type 1 (LQT1) | Preclinical (animal model—transgenic rabbit) | AAV9 | KCNQ1 suppression-and-replacement (SupRep) | The therapy combined shRNA-mediated suppression of endogenous KCNQ1 and replacement with shRNA-immune KCNQ1 cDNA. In vivo administration (1E10 vg/kg, intra-aortic root injection) led to significant QT index (QTi) shortening in LQT1 rabbits (from 122 ± 3% to 110 ± 4%, p = 0.03), normalizing levels close to wild type (WT: 105 ± 2%). APD90 in ventricular cardiomyocytes decreased from 525 ± 15 ms (untreated LQT1) to 394 ± 15 ms (treated), approaching WT values (417 ± 14 ms). Under β-adrenergic stimulation, SupRep-treated animals exhibited normal physiological responses: ΔQTi ~16.5 (vs. 24.5 in sham), and ΔAPD90 ~109 ms. Ca2+ transient duration (Ca2+T90) also normalized (baseline 338 ± 13 ms to 293 ± 17 ms after ISO, p = 0.003). The treatment reduced mutant KCNQ1 mRNA by ~30% and decreased QT dispersion. No significant inflammation or adverse events were observed. |
Dotzler et al. (2023) [108] | Congenital Long QT Syndrome Type 1 (LQT1) | Preclinical (transgenic rabbit model) | AAV9 | Suppression-and-replacement of KCNQ1 using AAV-shRNA + shRNA-immune KCNQ1 cDNA | Combined suppression of endogenous mutant KCNQ1 mRNA and replacement with shRNA-resistant KCNQ1 cDNA via AAV9 resulted in shortened QT interval and normalized electrophysiological parameters. QTc shortened by ~13% post-treatment (from 457 ± 21 ms to 397 ± 17 ms, p < 0.01). Action potential duration at 90% repolarization (APD90) in isolated cardiomyocytes reduced from 499 ± 19 ms (sham) to 399 ± 15 ms (treated), approximating wild-type values. β-adrenergic response (isoproterenol challenge) restored: ΔAPD90 increased by 86 ± 10 ms in treated vs. 39 ± 8 ms in sham (p < 0.01). Ventricular arrhythmia inducibility was significantly reduced. No adverse inflammation or off-target effects observed. |
Bains et al. (2023) [31] | Congenital Long QT Syndrome Type 2 (LQT2) | Preclinical (KCNH2-mutant rabbit model) | AAV9 | Suppression-and-replacement of KCNH2 via shRNA + shRNA-immune KCNH2 cDNA | The AAV9 vector delivered a dual approach: shRNA suppressed endogenous mutant KCNH2, while shRNA-immune KCNH2 cDNA restored normal function. QTc duration was significantly reduced from 470 ± 11 ms in sham to 414 ± 12 ms in treated animals (p < 0.01). Cardiomyocyte APD90 decreased from 519 ± 19 ms (sham) to 424 ± 13 ms (treated), approaching WT values (400 ± 9 ms). β-adrenergic stimulation (isoproterenol) led to preserved physiological APD90 shortening in treated animals (ΔAPD90 = −90 ms) vs. minimal change in sham (−36 ms). Early afterdepolarizations (EADs) were significantly reduced, and inducibility of Torsades de Pointes was suppressed. |
Congenital Short QT Syndrome Type 1 (SQT1) | Preclinical (transgenic rabbit model) | AAV9 | Gain-of-function KCNH2 suppression via shRNA (no replacement) | In SQT1 rabbits (gain-of-function KCNH2 N588K mutation), shRNA targeting mutant KCNH2 significantly prolonged QTc from 283 ± 8 ms to 333 ± 9 ms (p < 0.01), approaching wild-type values (342 ± 7 ms). Cardiomyocyte APD90 increased from 267 ± 11 ms to 316 ± 9 ms. Ventricular effective refractory period (VERP) improved from 142 ± 8 ms to 187 ± 11 ms (p < 0.05). Arrhythmia inducibility was significantly reduced (Torsades observed in 5/7 sham vs. 1/8 treated; p = 0.03). No off-target effects or myocardial inflammation were detected. | |
Qi et al. (2024) [87] | Congenital Long QT Syndrome Type 3 (LQT3) | Preclinical (Scn5a-M1875T knock-in mouse model) | AAV9 | In vivo base editing using ABE8e-SpRY to correct SCN5A-M1875T | AAV9-encoded adenine base editor (ABE8e-SpRY) was delivered systemically at P10. In vivo base editing efficiency reached 54.1% in cardiac tissue. Corrected mice exhibited significantly shorter QTc intervals (from 57.3 ± 1.3 ms to 47.6 ± 1.2 ms; p < 0.0001), normalized action potential duration (APD90 from 58.9 ± 2.1 ms to 45.7 ± 1.4 ms), and restored sodium channel function (reduction in late sodium current by 66%). Arrhythmia burden decreased significantly (6/10 untreated vs. 1/11 treated mice with inducible VT; p < 0.01). No detectable off-target edits or adverse effects were reported. |
Bradford et al. (2023) [113] | Arrhythmogenic right ventricular cardiomyopathy (ARVC) | Preclinical (mouse) | AAV9 | AAV-mediated PKP2 replacement | In PKP2 IVS10-1G>C knock-in mice, early AAV-PKP2 delivery at postnatal day 2 restored PKP2 expression, desmosomal protein levels (DSP, DSG2, JUP), and gap junction protein CX43, fully preventing ARVC phenotype and ensuring 100% survival up to 6 months. Late-stage administration at 4 weeks also significantly improved cardiac function, reduced fibrosis, normalized ECG (QRS duration), eliminated PVCs (0% vs. 60% in controls), and improved survival to 100% at 20 weeks vs. 20% in GFP controls. Rescue occurred even at advanced disease stages. |
van Opbergen et al. (2024) [114] | Arrhythmogenic right ventricular cardiomyopathy (ARVC) | Preclinical (mouse model) | AAV9 | AAV-mediated PKP2a gene transfer | AAV9-mediated delivery of Plakophilin-2a (PKP2a) in PKP2-deficient mice arrested disease progression. Early treatment (2 days after birth) restored intercalated disc protein expression (Cx43, N-cadherin, desmoplakin) and prevented right ventricular dilation and dysfunction. Late-stage therapy (initiated at 4 weeks) reduced arrhythmias, restored conduction velocity, and improved survival (100% vs. 20% in controls at 20 weeks). |
2.3. Gene Therapy Approaches for Biological Pacemaker Development
2.4. Routes of Administration in Cardiac Gene Therapy
3. Limitations and Future Directions
4. Conclusions
Author Contributions
Funding
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Author (Year) | Type of Arrhythmia | Type of Study | Vector | Form of Gene Therapy | Main Findings |
---|---|---|---|---|---|
Amit et al. (2010) [51] | Atrial fibrillation | Preclinical (porcine model) | Adenovirus (AdG628S) | Dominant-negative gene transfer (KCNH2-G628S) | Atrial painting with AdG628S prolonged atrial MAPD90 significantly at day 7 (right atrium: median increase ~80 ms vs. control, p < 0.01), with all treated animals maintaining sinus rhythm (SR) from days 4 to 10. By day 21, loss of transgene expression led to loss of efficacy. Relative risk of AF was 0.44 (95% CI 0.33–0.59; p < 0.01) in treated animals. KCNH2 protein expression was 79% higher in the treated group at day 7. No ventricular proarrhythmia or electrophysiological changes observed. |
Soucek et al. (2012) [50] | Atrial fibrillation | Preclinical (porcine model) | Adenovirus (AdCERG-G627S) | Dominant-negative gene transfer (CERG-G627S) | AdCERG-G627S significantly delayed or prevented persistent AF in pigs (mean time to persistent AF: 12 ± 2.1 days vs. 6.2 ± 1.3 days in controls; p < 0.001). Atrial ERP and MAPD90 were significantly prolonged (ERP: 221.5 ± 4.7 ms vs. 197.0 ± 4.7 ms; p = 0.006). Left ventricular function was preserved (LVEF: 62.1% ± 4.0% vs. 30.3% ± 9.1%; p < 0.001). No ventricular or extracardiac expression observed, supporting atrial-specific safety of the therapy. |
Bikou et al. (2011) [55] | Atrial fibrillation | Preclinical (porcine model) | Adenovirus (AdKir2.1AAA) | Dominant-negative gene transfer (Kir2.1AAA) | AdKir2.1AAA selectively suppressed IK1 current, leading to significant prolongation of atrial ERP (from 131 ± 5 ms to 174 ± 6 ms; p < 0.001). AF inducibility was markedly reduced (AF episodes: 0.4 ± 0.2 vs. 2.7 ± 0.3 in controls; p < 0.001). No significant ventricular ERP prolongation or proarrhythmic effects observed. Protein expression confirmed selective atrial Kir2.1AAA expression without extracardiac transduction. |
Igarashi et al. (2011) [54] | Atrial fibrillation | Preclinical (porcine model) | Adenovirus (AdCx40, AdCx43) | Overexpression (connexin 40 and 43) | AdCx40 and AdCx43 gene transfer preserved atrial conduction velocity (41.2 ± 3.2 cm/s vs. 26.5 ± 3.5 cm/s in controls; p < 0.001) and prevented AF induction at 7 days. AF inducibility was reduced by >80% (induction rate: 12.5% vs. 66.7% in controls; p < 0.01). Connexin expression was successfully upregulated and localized at intercalated disks. No adverse effects observed in ventricular conduction or function. |
Kunamalla et al. (2016) [56] | Atrial fibrillation | Preclinical (canine model) | Lentivirus (LVdnTGFβRII) | Inhibition of TGF-β signaling (dominant-negative receptor) | Posterior LA gene transfer of LVdnTGFβRII significantly reduced atrial fibrosis (collagen volume fraction: 13.9% ± 2.4% vs. 27.1% ± 1.7%; p < 0.05) and preserved conduction velocity. AF duration was significantly shorter (mean 1.3 ± 0.4 s vs. 5.6 ± 1.1 s; p < 0.05), with fewer sustained AF episodes. No off-target effects in ventricular myocardium or extracardiac organs detected. The anti-fibrotic strategy preserved posterior wall structure and conduction. |
Trappe et al. (2013) [57] | Ventricular tachycardia (post-infarction) | Preclinical (porcine model) | Adenovirus (AdCx43) | Overexpression (connexin 43) | AdCx43 gene transfer into the infarct border zone significantly increased Cx43 protein expression (1.8 fold; p < 0.01) and improved electrical coupling. Ventricular conduction velocity was significantly faster (25.8 ± 3.4 vs. 16.2 ± 2.1 cm/s in controls; p < 0.01), and inducibility of sustained VT was reduced by 50%. Reduced arrhythmia burden was associated with homogeneous Cx43 distribution and improved connexin-mediated intercellular coupling. No increase in proarrhythmia or off-target expression observed. |
Aistrup et al. (2009) [58] | Atrial fibrillation | Preclinical in vivo (canine) | Adenoviral vector (AdGαi2ctp) | Gαi2 C-terminal peptide overexpression (dominant-negative G-protein inhibition) | Epicardial atrial gene transfer of AdGαi2ctp significantly reduced AF duration from 13.0 ± 4.2 s (control) to 0.8 ± 0.5 s at 3–5 days post-transduction (p < 0.001). AF inducibility was also markedly decreased. The intervention selectively attenuated I_KAch activation and prolonged action potential duration during vagal stimulation, without affecting sympathetic signaling. Histological assessment confirmed targeted expression with minimal inflammatory response. |
Kapoor et al. (2013) [59] | Bradyarrhythmia (Sinoatrial node dysfunction) | In vitro and in vivo (animal model—guinea pig) | Adenoviral vector | Ectopic overexpression of Tbx18 transcription factor | Tbx18 reprogrammed ventricular myocytes into sinoatrial node-like pacemaker cells (iSAN) with spontaneous beating. In vitro, 73.8% of Tbx18-NRVMs beat spontaneously vs. 28.8% in controls (p < 0.05). Action potential frequency was 95 ± 23 bpm vs. 46 ± 10 bpm in controls. In vivo, 5/7 Tbx18-injected guinea pigs showed ectopic ventricular rhythm; beating rate ~154 bpm vs. ~120 bpm in controls. Converted cells had reduced IK1 (78% decrease), depolarized MDP (−47 ± 10 mV vs. −73 ± 6 mV), increased HCN4 (3.8× more HCN4+ cells), and durable pacemaker phenotype up to 8 weeks despite loss of Tbx18 transcript. |
Hu et al. (2014) [60] | Ventricular tachyarrhythmia post-myocardial infarction | In vivo (canine model of MI) | Adenoviral vector (AdCx43) | Overexpression of connexin 43 (Cx43) in the infarct border zone | Cx43 gene transfer restored gap junction expression and improved conduction velocity post-MI. Inducibility of sustained VT was reduced to 10% in treated animals vs. 70% in controls (p < 0.01). Conduction velocity increased from 13.1 ± 1.6 cm/s (control) to 21.3 ± 1.7 cm/s (treated, p < 0.001). Cx43 protein expression doubled, and immunostaining confirmed localization to intercalated disks. Gene expression peaked at 72 h and declined thereafter, but antiarrhythmic effects were sustained for ≥1 week. |
Lau et al. (2009) [61] | Post-infarction ventricular arrhythmia | In vivo (canine model of myocardial infarction) | Adenoviral vector (AdSkM1-GFP) | Overexpression of skeletal muscle sodium channel SkM1 in the epicardial border zone | SkM1 gene transfer restored fast sodium current in the infarct border zone, improving conduction and reducing arrhythmogenicity. Treated hearts had faster epicardial activation (median CV 47.2 cm/s vs. 27.3 cm/s in controls, p < 0.001) and preserved conduction delay. Transduced cells showed normal excitability at depolarized resting membrane potentials, unlike native cardiac Na+ channels. Histology confirmed successful transgene expression localized to the border zone myocytes. No increase in arrhythmia susceptibility observed post-intervention. |
Wolfson et al. (2024) [62] | Complete AV block (CAVB) | Preclinical in vivo (rat and pig) | Synthetic mRNA (naked, unformulated) | TBX18 mRNA (non-viral gene therapy) | Myocardial injection of TBX18 mRNA in rats and pigs induced de novo biological pacing. In rats, heart rates were significantly higher vs. controls over 14 days (mean HR ~343 bpm vs. ~130 bpm, p < 0.01). In pigs, pacing was rate-adaptive and correlated with physical activity (Pearson’s R = 0.67 vs. 0.21 in controls). TBX18 mRNA achieved focal expression, low inflammation, and significantly reduced backup pacemaker dependency over 28 days. |
Anttila et al. (2023) [63] | Ischemic cardiomyopathy | Randomized controlled trial (phase 2a) | Naked mRNA (AZD8601, VEGF-A mRNA) | mRNA-mediated protein expression (VEGF-A) | In the EPICCURE trial (NCT03370887), 30 epicardial injections of AZD8601 (3 mg total) during CABG were safe, with no arrhythmias, infections, or immune reactions observed. Exploratory outcomes suggested improved LVEF, NT-proBNP reduction, and better KCCQ scores over 6 months in treated vs. placebo groups. This study provides first-in-human evidence of the safety and feasibility of direct myocardial injection of naked VEGF-A mRNA without lipid encapsulation. |
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Karakasis, P.; Theofilis, P.; Vlachakis, P.K.; Milaras, N.; Kalinderi, K.; Patoulias, D.; Antoniadis, A.P.; Fragakis, N. Gene Therapy for Cardiac Arrhythmias: Mechanisms, Modalities and Therapeutic Applications. Med. Sci. 2025, 13, 102. https://doi.org/10.3390/medsci13030102
Karakasis P, Theofilis P, Vlachakis PK, Milaras N, Kalinderi K, Patoulias D, Antoniadis AP, Fragakis N. Gene Therapy for Cardiac Arrhythmias: Mechanisms, Modalities and Therapeutic Applications. Medical Sciences. 2025; 13(3):102. https://doi.org/10.3390/medsci13030102
Chicago/Turabian StyleKarakasis, Paschalis, Panagiotis Theofilis, Panayotis K. Vlachakis, Nikias Milaras, Kallirhoe Kalinderi, Dimitrios Patoulias, Antonios P. Antoniadis, and Nikolaos Fragakis. 2025. "Gene Therapy for Cardiac Arrhythmias: Mechanisms, Modalities and Therapeutic Applications" Medical Sciences 13, no. 3: 102. https://doi.org/10.3390/medsci13030102
APA StyleKarakasis, P., Theofilis, P., Vlachakis, P. K., Milaras, N., Kalinderi, K., Patoulias, D., Antoniadis, A. P., & Fragakis, N. (2025). Gene Therapy for Cardiac Arrhythmias: Mechanisms, Modalities and Therapeutic Applications. Medical Sciences, 13(3), 102. https://doi.org/10.3390/medsci13030102