WO2025238184A1 - Scn10a-short gene therapy to restore cardiac impulse conduction and to protect against cardiac arrhythmia - Google Patents

Abstract

The invention relates to an expression construct, comprising a coding sequence for a C-terminal part of a voltage-gated sodium channel alpha subunit, under control of a promoter that directs expression in cardiomyocytes. The invention further relates to a pharmaceutical composition, comprising said expression construct and a pharmacologically acceptable excipient, and to use of the pharmaceutical composition in a method of treatment of an individual suffering from an arrhythmia and/or heart failure.

Description

P137306PC00 Title: SCN10A-short gene therapy to restore cardiac impulse conduction and to protect against cardiac arrhythmia FIELD The invention relates to methods and means for treatment of conduction disorders, arrhythmias, and cardiomyopathies. INTRODUCTION Ion channels play an important role in all aspects of heart function including rhythmicity and contractility (Priest and McDermott, 2015. Channels 9: 352-359). The SCN5A-encoded α-subunit of the cardiac sodium channel (Nav1.5) largely determines the cardiac sodium current (INa), which is responsible for the initiation and propagation of the cardiac electrical excitation wavefront. A reduction of INa impairs the action potential (AP) upstroke and cardiac conduction, and is implicated in both acquired and inherited arrhythmia syndromes such as Brugada syndrome (BrS), progressive cardiac conduction disease (PCCD), sick sinus syndrome (SSS), atrial fibrillation (AF), ventricular tachycardia or fibrillation (VT/VF), and dilated cardiomyopathy (DCM) (Remme et al., 2008. Trends Cardiovasc Med 18: 78-87; Remme and Bezzina 2010. Cardiovasc Ther 28: 287-294; Boink et al., 2012. Cardiovasc Res 94: 450-459; Pu and Boyden, 1997. Circ Res 81: 110-119). In recent years, gene therapy has emerged as a powerful tool to restore the function of damaged or dysfunctional cells and tissues (Dimmeler et al., 2014. Nat Med 20: 814-821; Mendell et al., 2021. Mol Ther 29: 464-488; Ylä-Herttuala and Baker, 2017. Mol Ther 25: 1095-1106). Restoration of cardiac INa by gene transfer of SCN5A represents a logical approach with the potential to provide a curative treatment. However, the large size of the SCN5A coding sequence is beyond the capacity of adeno-associated viral (AAV) vectors, complicating its clinical applicability in gene therapy. In 2021, Doisne et al. reported efficient expression of human SCN5A in neonatal mice utilizing a dual AAV vector system (Doisne et al., 2021. Front Physiol 12: 661413). Yet, the required use of a high vector dose of over 1 x 10¹⁵ viral genomes (vg)/kg in neonatal mice underscores the difficulty of translating such a strategy to human patients. Indeed, drawbacks of the dual AAV vector strategy, such as the relatively low efficiency, the expression of unwanted products and the need of two vector preparations, hamper clinical translation of this approach (Li and Samulski, 2020. Nat Rev Genet 21: 255-272). The challenges with dual AAV vector systems inspired the search for alternative strategies involving the expression of smaller transgenes, yet these interventions still have their own limitations. For example, overexpression of a bacterial sodium channel effectively improved cardiac conduction in mice (Nguyen et al., 2022. Nat Commun 13: 620). Yet the distinctly different gating properties and anticipated higher immunogenicity pose important translational barriers. A different angle was taken in a recent study that demonstrated the potential value of a chaperone protein RAN Guanine Nucleotide Release Factor, also termed MOG1, as a means to suppress a Brugada syndrome (BrS)-like phenotype in Nav1.5-trafficking defect mouse models (Yu et al., 2022. Sci Transl Med 14: eabf3136). While expression of MOG1 may represent a more translatable approach, the potential pleiotropic effects of MOG1 remain an important concern that could be difficult to address on an individual basis when considering eventual patient treatment. Moreover, MOG1 gene therapy was specifically designed to counter SCN5A trafficking mutations, which limits its application to a relatively small subset of genetic defects. Recently, a naturally occurring cardiac-specific short transcript of SCN10A (SCN10A-short, here designated S10s) was discovered, which modulates the density of the Nav1.5-mediated sodium current (Man et al., 2021. Circulation 144: 229-242). S10s is expressed in the sinus node, atria, and ventricular conduction system of the heart, and comprises the last 7 exons of the neuronal sodium channel gene SCN10A. The predicted coding product of S10s contains the C-terminal portion of the full channel, including part of domain III, the entire domain IV, and the cytosolic C-terminus. Loss of Scn10a-short expression was found to slow cardiac conduction in mice and reduce INa in isolated cardiomyocytes, while overexpression of S10s in HEK293 cells stably expressing SCN5A, increased INa (Man et al., 2021. Circulation 144: 229-242). Cardiac sodium current (INa) reduction is implicated in various arrhythmias including Brugada syndrome and ventricular tachycardia or fibrillation. The present disclosure aims to develop novel strategies to overcome arrhythmia, especially chronical arrhythmia. BRIEF DESCRIPTION OF THE INVENTION In this disclosure, overexpression of S10s, a short carboxy-terminal domain of human neuronal sodium channel SCN10A, is shown to increase INa and action potential upstroke velocity in cardiomyocytes. Gene therapy containing S10s improves cardiac conduction in both healthy and BrS model mice, and prevents conduction block and associated arrhythmias in simulated human heart models. These results suggest that S10s gene therapy has the potential to be broadly applicable for the treatment for cardiac arrhythmias and contractile dysfunction, which may be based on abnormalities in cardiac sodium channel function and/or conduction. S10s gene therapy therefore holds potential in the treatment of abnormalities in cardiac sodium channel function and/or cardiac conduction abnormalities and associated arrhythmias, and/or contractile dysfunction, including DCM and heart failure. The invention therefore provides an expression construct, comprising a coding sequence for a C-terminal part of a voltage-gated sodium channel alpha subunit, under control of a promoter that directs expression of the C-terminal part of a voltage-gated sodium channel alpha subunit in cardiomyocytes. In embodiments, said voltage-gated sodium channel alpha subunit is SCN10A. In embodiments, said coding sequence for a C-terminal part of a voltage-gated sodium channel alpha subunit has at least 60% identity to SEQ ID NO:1. In embodiments, the transcript size of the C-terminal part of a voltage-gated sodium channel alpha subunit is less than 2070 base pairs (bp). In embodiments, said coding sequence for a C-terminal part of a voltage-gated sodium channel alpha subunit is selected from the amino acid sequences depicted in Table 2. In embodiments, said expression construct is a viral construct, preferably a recombinant adeno-associated virus (rAAV) construct, preferably a recombinant AAV6 or an AAV6 variant construct. In embodiment, the promoter of an expression product according to the invention is selected from cardiac troponin C, cardiac troponin I, and cardiac troponin T (cTnT). The invention further provides a pharmaceutical composition, comprising an expression construct as claimed, and a pharmacologically acceptable excipient. In embodiments, said pharmaceutical composition is for use in a method of treatment of an individual suffering from a cardiac conduction abnormality and/or an arrhythmia, such as a chronic/recurrental arrhythmia. In embodiments, said pharmaceutical composition is for use in a method of treatment of an individual suffering from an arrhythmia, such as a chronical arrhythmia. In embodiments, the arrhythmia is a bradyarrhythmia, such as a sinoatrial node exit block, a conduction defect such as an atrioventricular block, a left bundle branch block (LBB), a non-specific ventricular conduction abnormality, a progressive cardiac conduction disease (PCCD), and/or a tachyarrhythmia, such as an ischemia-related arrhythmia, a non-ischemia-related arrhythmia (such as associated with Brugada syndrome), a non-ischemic cardiomyopathy and/or a fibrosis-related or scar-related arrhythmia, such as post-myocardial infarction-related arrhythmia. In embodiments, the arrhythmia is a malignant ventricular arrhythmia. In embodiments, the individual to be treated has previously been treated with an antiarrhythmic drug, by ablation, by stereotactic radiotherapy, or a combination thereof. In embodiments, a pharmaceutical composition as claimed is injected or infused into the myocardium, preferably by intramyocardial injection. In embodiments, the individual is further treated with an antiarrhythmic drug. FIGURE LEGENDS Figure 1. Schematic diagrams of SCN10A-short (S10s), its predicted coding product SCN10A-short (S10s), and the viral vectors used in this study. (A) A UCSC genome browser view of the human SCN10A locus and a zoom-in view of the S10s region. (B) A schematic representation of S10s, the predicted coding product of S10s. It contains the C-terminal portion of the full SCN10A channel, including part of domain III, the entire domain IV, and the cytosolic C-terminus. Adapted from Man et al., 2021. Circulation 144: 229-242. (C) Viral vectors used in this study. S10s vectors contain a bicistronic expression cassette including a self-cleaving P2A- GFP. GFP vectors contain GFP only. MCS vector contains no coding sequence. Figure 2. S10s gene therapy increases sodium current density in cardiomyocytes isolated from wild type mice. (A) Immunofluorescence staining images of P2A-tagged S10s in mouse left ventricles. Scale bars represent 200 µm. (B) Typical sodium currents in cardiomyocytes isolated from mice injected with AAV6-GFP (N = 3 mice, n = 12 cardiomyocytes) or AAV6-S10s (N = 3 mice, n = 11 cardiomyocytes). (C) Average I-V relationships of sodium current amplitude. (D) Peak sodium current at -40 mV. (E) Activation and inactivation curves (triangles and circles, respectively). Data are presented as mean ± SEM. Data were compared using Two-way RM ANOVA with post-hoc Fisher’s LSD test (C) or Mann-Whitney test (D). *p < 0.05; **p < 0.01. Figure 3. S10s gene therapy increases action potential upstroke velocity in cardiomyocytes isolated from wild type mice. (A) Typical examples of action potentials (AP) elicited at 6 Hz stimulation (left panel) and their time derivative near the upstroke (right panel) of cardiomyocytes isolated from mice injected with AAV6-GFP or AAV6-S10s. (B–F) AP parameters of cardiomyocytes isolated from mice injected with AAV6-GFP (N = 3 mice, n = 7 cardiomyocytes) or AAV6-S10s (N = 3 mice, n = 9 cardiomyocytes). (B) Maximal AP upstroke velocity (dV/dtmax). (C) Resting membrane potential (RMP). (D) AP amplitude (APA). (E) AP duration at 50% of repolarization (APD50). (F) AP duration at 90% of repolarization (APD90). Data are presented as mean ± SEM. Data were compared using Mann-Whitney test. **p < 0.01; ns, not significant. Figure 4. S10s gene therapy increases cardiac conduction velocity in wild type mice. (A) Schematic diagram of the experimental design. (B) mRNA expression level of S10s in left ventricles of wild type mice injected with AAV9-GFP or AAV9- S10s. (C) Representative activation maps of ventricles. (D) Epicardial longitudinal conduction velocity of ventricles stimulated at 8 Hz. (E) Typical ECG traces. (F) Average QRS intervals. Data are presented as mean ± SEM. Data were compared using two-way ANOVA with post-hoc Fisher’s LSD test (D) or unpaired t-test (F). **p < 0.01; ns, not significant. Figure 5. S10s gene therapy improves cardiac conduction in Scn5a+/Δ7bp mice. (A) Schematic diagram of the experimental design. (B) mRNA expression level of S10s in left ventricles of Scn5a+/Δ7bp mice injected with AAV vectors. (C) Immunofluorescence staining images of P2A-tagged S10s. Scale bars represent 200 µm. (D) Representative activation maps of ventricles, (E) Epicardial longitudinal conduction velocity (CVL) of ventricles, and (F) Average QRS intervals from Scn5a+/Δ7bp mice injected with AAV9-GFP or AAV9-S10s. (G) Representative activation maps of ventricles, (H) Epicardial CVL of ventricles, and (I) Average QRS intervals from Scn5a+/Δ7bp mice injected with 8 x 10¹² vg/mouse HD_AAV9- MCS or HD_AAV9-S10s. Data are presented as mean ± SEM. Data were compared using two-way ANOVA with post-hoc Fisher’s LSD test (E & H) or unpaired t-test (F & I). *p < 0.05; **p < 0.01; ns, not significant. Figure 6. S10s overexpression increases action potential upstroke velocity in human iPSC-derived cardiomyocytes. (A) Immunofluorescence staining images of GFP and P2A-tagged S10s in hiPSC-CMs. S10s expression was detected in hiPSC- CMs transduced with Lenti-S10s but not in hiPSC-CMs transduced with Lenti- GFP. Scale bars represent 50 µm. (B) Typical examples of APs elicited at 1 Hz (left panel) and their time derivative near the upstroke (right panel) from hiPSC-CMs transduced with Lenti-GFP or Lenti-S10s. (C-G) dV/dtmax, RMP, APA, APD50, and APD90 measured from hiPSC-CMs transduced with Lenti-GFP or Lenti-S10s. Data are presented as mean ± SEM. Data were compared using Mann-Whitney test. **p < 0.01; ns, not significant. Figure 7. S10s gene therapy improves conduction and increases excitability in a simulated strand of human left ventricular cardiomyocytes. (A) Longitudinal conduction velocity in a simulated linear strand of human left ventricular cardiomyocytes as a function of gap junctional conductance (gj). Complete loss-of- function mutation in SCN5A simulated by a 50% decrease in Nav1.5 conductance (‘SCN5A+/−’) and the effect of S10s as a 44% increase in remaining Nav1.5 conductance to 72% of the wild type Nav1.5 conductance (‘SCN5A+/− + S10s’). Individual cells of the 80-cell strand (inset) described according to the human left ventricular cell model by Ten Tusscher (Ten Tusscher et al., 2004. Am J Physiol 286: H1573-1589) as updated by Ten Tusscher and Panfilov (Ten Tusscher and Panfilov, 2006. Physics Med Biol 51: 6141-6156). Myoplasmic resistivity set to 150 Ω∙cm (Meijer van Putten et al., 2015. Front Physiol 6: 7). (B) Stimulus current threshold of the 80-cell strand as a function of gj. (C) Structure of the 80-cell strand with branches at cells #30 and #31 of the strand used to simulate an increased electrical load. gj set to 10 µS in the simulations. (D) AP conduction in the branched strand of panel C at stimulus rates of 50 (top), 100 (middle), and 150 beats/min (bottom). APs of cells #15 (left) and #56 (right) of the strand, as indicated by ‘AP’ in panel C, under control conditions (wild type, light gray solid line) and in case of SCN5A+/− (dark gray line) and SCN5A+/− + S10s (dark grey dashed line). Figure 8. S10s suppresses arrhythmia induction in a 3D human whole-heart model. (A) I-V relationships of INa amplitude. (B) The maximum upstroke velocities of the steady-state action potentials at 1 Hz pacing. (C) Left: reconstructed ventricular model with three different tissue types: non-fibrotic myocardium (medium grey), fibrotic remodelling (light grey) and dense scar (dark grey). Scar region is non-conductive; the non-fibrotic myocardium exhibits altered EP properties due to the SCN5A mutation, while the fibrotic regions include additional fibrosis-induced changes in EP properties. Right: activation patterns of the VT re- entrant circuit induced by validated in-silico rapid pacing protocol at the base of the SCN5A heart model. (D) Top and bottom rows show the comparison of wave front propagation following rapid pacing in the heart model before (top) and after (bottom) S10s gene therapy is applied. Each row of figures shows a series of frames that depict the continuous wave propagation in a portion of the 3D whole-heart model. The images in each column show the same instant of the simulation. Yellow stars stand for the pacing location; pink line marks the conduction block. Fibrotic and scar regions are described as in panel C. For the SCN5A model, a sustained re- entry was induced, where the cyan curved arrow represents the re-entrant wave trajectory. For the SCN5A +S10s model, no re-entry was induced, where wave propagation was represented in white arrow. Figure 9. S10s-Volt presents similar characteristics to improve sodium current density in presence of SCN5A. (A) Average I-V relationships of normalized peak sodium current in SCN5A-expressing HEK cells. (B) Peak sodium current at - 20 mV. Data are presented as mean ± SEM. Data were compared using one-way ANOVA with post-hoc Fisher’s LSD test. *p < 0.01; ns, not significant. (C) Scheme of the generation of S10s-Volt. Figure 10. S10s-Ultrashort presents similar characteristics to improve sodium current density in presence of SCN5A. (A) Scheme of the generation of S10s-Ultrashort. (B) Average I-V relationships of normalized peak sodium current in SCN5A-expressing HEK cells. Inset, voltage clamp protocol used. Note that the I-V relationships are virtually overlapping. (C) Voltage-dependency of SCN5A (in)activation with S10s and S10s-Ultrashort. Inset, voltage clamp protocol used to measure the inactivation. Note that the voltage-dependency of (in)activation curves are overlapping. Figure 11. S10s-1kb presents different characteristics and does not improve sodium current density in presence of SCN5A. (A) Scheme of the generation of S10s-1kb. (B) Sodium current-driven action potential maximal upstroke velocity (dV/dtmax) of cardiomyocytes transduced with GFP, S10s or S10s-1kb. (C) Action potential duration at 90% repolarization cardiomyocytes transduced with GFP, S10s or S10s-1kb. Data are presented as mean ± SEM. Data were compared using one-way ANOVA with post-hoc Fisher’s LSD test. **p < 0.01; ns, not significant. DETAILED DESCRIPTION OF THE INVENTION The potential of S10s and its shorter variants as a gene therapy target for cardiac conduction abnormalities and associated arrhythmias was investigated. To highlight the potential universal applicability of this gene therapy, the effect of AAV-S10s gene therapy in wild type mice was studied. These studies indicated that S10s overexpression increased INa and maximal Action Potential (AP) upstroke velocity (dV/dtmax) in isolated cardiomyocytes, and improved ventricular conduction in mouse hearts. Secondly, overexpression of S10s effectively rescued conduction slowing in an Scn5a-haploinsufficient mouse model (Man et al, 2019. Nat Commun 10: 4943). Thirdly, S10s-induced augmentation of INa in human inducible pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), and reduced susceptibility to cardiac arrhythmias using in silico human cardiac tissue and whole-heart models was established (Ten Tusscher et al., 2004. Am J Physiol 286: H1573-1589; Ten Tusscher and Panfilov, 2006. Physics Med Biol 51: 6141-6156; Zhang et al., 2023. Elife 12: RP88865). The S10s transcript was identified in the hearts of mouse and human through genetic variants associated with conduction abnormalities and BrS, and was shown to represent an endogenous mechanism to modulate Nav1.5-mediated INa in vitro and in vivo (Man et al., 2021. Circulation 144: 229-242). Although the exact mechanism of action remains unknown, it is likely based on a physical interaction of S10s and Nav1.5 proteins in vivo (Man et al., 2021. Circulation 144: 229-242). As a result, a short S10s-based transcript can be used in a single AAV vector. As it is based on an endogenously occurring gene product, it is expected to be well tolerated by its recipients. Definitions As are used herein, the singular forms "a", "an" and "the", include the plural forms as well. As is used herein, the term "or" includes any and all combinations of one or more of the associated listed items, unless the context clearly indicates otherwise. As are used herein, the terms "comprise" and "comprising", and conjugations thereof, are open language and specify the presence of stated features but do not preclude the presence or addition of one or more other features. It will be understood that when a particular step of a method is referred to as subsequent to another step, it can directly follow said other step, or one or more intermediate steps may be carried out before carrying out the particular step, unless specified otherwise. As is used herein, the term “sodium voltage-gated channel alpha subunit 10 (SCN10A)”, refers to a protein product of a SCN10A gene that in humans is located on chromosome 3p22.2. The SCN10A gene encodes a tetrodotoxin-resistant channel that mediates the voltage-dependent sodium ion permeability of excitable membranes. The protein may form a sodium-selective channel through which sodium ions may pass in accordance with their electrochemical gradient. The gene is referred to as HGNC reference number 10582, NCBI gene number 6336, and Ensembl reference number ENSG00000185313. The encoded protein of 1956 amino acid residues is known as UniProt Q9Y5Y9. As is used herein, the term “C-terminal part” in the context of a C-terminal part of a voltage-gated sodium channel alpha subunit, refers to a part of a voltage- gated sodium channel alpha subunit that comprises at least part of the fourth repeat domain of a voltage-gated sodium channel alpha subunit. Said part may be a non-natural occurring part, meaning that the part as such does not normally occur in nature. In embodiments, said non-natural part is a chimeric coding sequence, comprising parts of two or more voltage-gated sodium channel alpha subunits. As an alternative, or in addition, said part is not a natural part because the amino acid sequence has been altered, for example by the addition of a methionine at the N-terminus of the part. An altered sequence preferably comprises at most 5 amino acid alterations, such as 4 amino acid alterations, 3 amino acid alterations, 2 amino acid alterations, or 1 amino acid alteration. In embodiments, said alteration is a conservative replacement. In embodiments, said voltage-gated sodium channel alpha subunit is SCN10A, or a functional part of a voltage-gated sodium channel alpha subunit that has at least 60% identity to SEQ ID NO:1. In embodiments, a C-terminal part comprises less than 690 amino acid residues, meaning that the coding part of the construct encompasses less than 2070 base pairs (bp). As is used herein, the term “cardiomyocyte (CM)”, refers to a muscle cell of the heart (e.g., a cardiac muscle cell). The term cardiomyocyte includes any cell in the cardiac myocyte lineage that shows at least one phenotypic characteristic of a cardiac muscle cell. Such phenotypic characteristics can include expression of cardiac proteins, such as cardiac sarcomeric or myofibrillar proteins or atrial natriuretic factor, or electrophysiological characteristics. Cardiomyocyte-specific markers include, but are not limited to, cardiac troponin I, cardiac troponin-C, tropomyosin, caveolin-3, GATA-4, myosin heavy chain, myosin light chain-2a, myosin light chain-2v, ryanodine receptor, and atrial natriuretic factor. As is used herein, the term “identity”, as is used in sequence identity, refers to the overall identity between nucleic acid molecules (e.g., DNA molecules and/or RNA molecules) and/or between two or more proteins. Calculation of the percent identity of two nucleic acid sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequence for optimal alignment). The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which may be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. Suitable tools for global alignment techniques include the Needleman– Wunsch algorithm (Needleman and Wunsch, 1970. J Mol Biol 48: 443-453, and Fast Optimal Global Sequence Alignment Algorithm (Chakraborty and Bandyopadhyay, 2013. Scientific Reports 3: 1746). As is used herein, the terms “transfecting” and “transfection” refer to the process of introduction of a nucleic acid molecule, such as a DNA molecule, into a cell, preferably an eukaryotic cell such as a cardiomyocyte. The term "transfection" encompasses methods known to the person skilled in the art for introducing nucleic acid molecules into cells, for example, electroporation, lipofection, e.g., cationic lipid-and/or liposome-based, calcium phosphate precipitation, nanoparticle-based transfection, and transfection based on cationic polymers such as DEAE-dextran or polyethyleneimine, and the like. As is used herein, the term “transducing” and “transduction” refer to a process of introduction of a nucleic acid molecule, such as a DNA molecule, into a cell, preferably a eukaryotic cell such as a cardiomyocyte, which is mediated by use of a viral vector. Such a viral vector may be a lentiviral vector, adenoviral vector, adeno-associated virus vector, retroviral vector, or any combination thereof. A preferred vector is an adeno-associated virus vector. As is used herein, the term “expression construct” refers to a nucleic acid molecule that provides expression of a nucleotide sequence comprising a coding sequence for a C-terminal part of a voltage-gated sodium channel alpha subunit that is present in said construct. An expression construct preferably comprises a promoter sequence, a transcribed region, a 3' untranslated region, and/or one or more post-transcriptional regulatory elements (PRE) such as a polyadenylation signal. The promoter is coupled to the transcribed region comprising a nucleotide sequence that encodes the C-terminal part of a voltage-gated sodium channel alpha subunit. In examples, the expression construct may comprise a post-transcriptional regulatory element such as a Hepatitis B virus (HPRE), a Woodchuck Hepatitis virus (WPRE), a CW3SL, a CW3A, a CW3SA, a CW3SSA or a WPRE3 (Choi et al., 2014. Mol brain 7: 17). In embodiments, said PRE/polyadenylation signal is CW3SL, as is depicted herein below, or a sequence having at least 80% sequence identity thereto. In embodiments, the polyadenylation signal is a synthetic minimal polyadenylation signal. An expression cassette may further comprise one or more enhancer sequences, that stimulate the expression of the C-terminal part of a voltage-gated sodium channel alpha subunit in certain tissues or in certain stages of development, especially in cardiomyocytes. Said C-terminal part of a voltage-gated sodium channel alpha subunit preferably includes the C-terminal part of voltage-gated sodium channel alpha subunit 10. As is used herein, the term “promoter” refers to a genetic element that initiates transcription of the transcribed region and is therefore a primary point of control for expression of the C-terminal part of a voltage-gated sodium channel alpha subunit. Said promoter preferably is a cardiomyocyte-specific promoter, such as a cardiac troponin C promoter, cardiac troponin I promoter, a cardiac troponin T2 (TNNT2) promoter (Wu et al., 2010. Genesis 48: 63-72), a cardiomyocyte-specific Na(+)-Ca(2+) exchange promoter such as NCX1 (Agostini et al., 2013. Biomed Res Int 2013: 845816), and the cardiac myosin light chain 2 promoter (Griscelli et al., 1997. Comptes Rendus Acad Sci III 320: 103-112), a MYH6 promoter, an ACTA1 promoter, a synthetic promotor such as a SPC5-12 promoter (US patent application 2004/017572), a MHCK7 promoter (Salva et al., 2007. Mol Ther 15: 320-329), a DES promoter (Pacak et al., 2008. Genetic Vaccines Ther 6: 13) or fragments of said promoters, or a core promoter from a cardiac gene such as TNNT2 or NPPA, coupled to cardiac-specific regulatory elements, such as regions of aforementioned promoters or for example a CASQ2 regulatory element (Rincon et al., 2015. Mol Ther 23: 43–52), or a PRKAA2 regulatory element (Anderson et al., 2017. Development 44: 1235-1241). Suitable cardiomyocyte-specific promoters include CE1-TNNT2core, CE2-CCP, CCP NPPAcore, and TNNT2ddd, sequences for which are included herein. Said expression construct preferably is optimized for expression in human, preferably in a human cardiomyocyte. As is used herein, the term “pharmaceutically acceptable excipient” refers to an excipient for administration of an active substance. Said pharmaceutically acceptable excipient may comprise any substance or vehicle suitable for delivering the substance to a therapeutic target, in particularly cardiomyocytes, of the individual. The term refers to any pharmaceutical acceptable diluent, salt, stabilizer, buffering agent, and other additives such as a sugar, for example sucrose, trehalose, maltose, mannitol, sorbitol, or glycerol, and/or an amino acid such as DL-methionine, glycine, L-alanine, L-arginine, and/or L-aspartate. As is used herein, the term ‘buffering agent” refers to an agent that can resist pH change of a composition upon the addition of an acidic or basic component. Buffering agents can be salts of a weak acid and a weak base. Examples are, for example, salts of citric acid, acetic acid, aspartic acid, glutamic acid, tartaric acid, succinic acid, malic acid, fumaric acid, alpha-ketoglutaric acid, histidine, lactic acid, tromethamine (2-amino-2-(hydroxymethyl)propane-1,3-diol (TRIS), gluconic acid, and combinations thereof. In embodiments, a buffering agent is used for keeping the pH at between 6-8, preferably at about 7.0. As is used herein, a pharmaceutical composition according to the invention may comprise 1-1000 mM of one or more excipients, such as 10-750 mM, 50-500 mM, 100-400 mM, including 200 mM, 250 mM, 300 mM or 350 mM. As is used herein, the term “cardiac conduction abnormality” refers to a conduction disorder that may cause an arrhythmia, including a bundle branch block such as left bundle branch block, a heart block also termed A-V block, and a long QT Syndrome. As is used herein, the term “arrhythmia” refers to any problem in the rate or rhythm of an individual’s heartbeat. During an arrhythmia, the electrical impulses may be too fast, too slow or erratic causing an irregular heartbeat. An arrhythmia may be classified as a tachycardia, a fast heartbeat with a heart rate greater than 100 beats per minute; or as a bradycardia, a slow heartbeat with a heart rate less than 60 beats per minute. As is used herein, the term “chronic/recurrent arrhythmia” refers to a prolonged arrhythmic condition that leads to morphological remodeling of the heart, ultimately resulting in heart failure. Such chronic or recurrent arrhythmia includes atrial fibrillation and ventricular fibrillation. As is used herein, the terms “ablation” and “ablative therapy” involve the generation of lesions in the cardiac tissue to disrupt cells that provide superfluous electrical pulses. Ablation is an invasive technique using a catheter that is inserted through a blood vessel, or during heart surgery. As is used herein, the term “stereotactic radiotherapy”, also termed “stereotactic arrhythmia radioablation” (STAR), refers to a noninvasive treatment through a combination of one or more of 3D electrophysiological mapping, noninvasive myocardial scar imaging, positron emission tomography (PET) and cardiac magnetic resonance (cMR), with noninvasive delivery of ablative radiation doses. S10s expression construct Expression of a C-terminal part of a voltage-gated sodium channel alpha subunit in a cardiomyocyte may be provided by an expression construct for functional expression of said amino acid sequence in cardiomyocytes. Said cardiomyocyte-specific expression may be provided, for example, by employing a cardiomyocyte-specific promoter. In embodiments, said cardiomyocyte-specific promoter is a cardiac troponin C promoter, cardiac troponin I promoter, a cardiac troponin T2 (TNNT2) promoter (Wu et al., 2010. Genesis 48: 63-72), a cardiomyocyte-specific Na(+)-Ca(2+) exchange promoter (Agostini et al., 2013. Biomed Res Int 2013: 845816), or a cardiac myosin light chain 2 promoter (Griscelli et al., 1997. Comptes Rendus Acad Sci III 320: 103-112). Said promoter preferably is optimized for expression in human, preferably in a human cardiomyocyte. Said expression construct comprises a coding sequence for a C-terminal part of a voltage-gated sodium channel alpha subunit. An alpha subunit of an voltage- gated sodium channel comprises four repeat domains, labelled I through IV, each containing six membrane-spanning segments, labelled S1 through S6, of which the conserved S4 segment acts as a voltage sensor. In embodiments, said C-terminal part comprises at least repeat domain IV of a voltage-gated sodium channel alpha subunit. Said C-terminal part may further include the intracellular loop between S6 of repeat III and S1 of repeat IV; S6 of repeat III and the intracellular loop between S6 of repeat III and S1 of repeat IV; the extracellular loop between S5 and S6 of repeat III, S6 of repeat III and the intracellular loop between S6 of repeat III and S1 of repeat IV; S5 of repeat III, the extracellular loop between S5 and S6 of repeat III, S6 of repeat III and the intracellular loop between S6 of repeat III and S1 of repeat IV; the intracellular loop between S4 and S5 of repeat III, S5 of repeat III, the extracellular loop between S5 and S6 of repeat III, S6 of repeat III and the intracellular loop between S6 of repeat III and S1 of repeat IV; S4, the intracellular loop between S4 and D5 of repeat III, S5 of repeat III, the extracellular loop between S5 and S6 of repeat III, S6 of repeat III and the intracellular loop between S6 of repeat III and S1 of repeat IV; or at least part of the extra-cellular loop between S3 and S4 of repeat III, S4, the intracellular loop between S4 and D5 of repeat III, S5 of repeat III, the extracellular loop between S5 and S6 of repeat III, S6 of repeat III and the intracellular loop between S6 of repeat III and S1 of repeat IV; at the N-terminus of a repeat domain IV of a voltage-gated sodium channel alpha subunit. Said expression construct comprising a coding sequence for a C-terminal part of a voltage-gated sodium channel alpha subunit start at any position between amino acid 152 and amino acid 272 of SEQ ID NO:1, such as at a position between amino acid 162 and amino acid 262 of SEQ ID NO:1, between amino acid 172 and amino acid 252 of SEQ ID NO:1, between amino acid 182 and amino acid 242 of SEQ ID NO:1, between amino acid 192 and amino acid 232 of SEQ ID NO:1, between amino acid 202 and amino acid 222 of SEQ ID NO:1, such as amino acid 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, or 221 of SEQ ID NO:1. A person skilled in the art will appreciate that the first amino acid of a C-terminal part of a voltage-gated sodium channel alpha subunit will be a methionine, and will introduce an ATG triplet in front of the coding sequence for a C-terminal part of a voltage-gated sodium channel alpha subunit. A preferred coding sequence for a C-terminal part of a voltage-gated sodium channel alpha subunit encompassed amino acid residues 212-691 of SEQ ID NO:1, which is preceded by a methionine. In embodiments, said C-terminal part comprises an internal truncation such that the region spanning S3-S6 of repeat III are fused to S3-S6 of repeat IV. In embodiments, said truncated region encompasses the region between amino acids 272 and 335 of SEQ ID NO:1, such as the region between amino acids 152 and 335 of SEQ ID NO:1, the region between amino acids 160 and 330 of SEQ ID NO:1, the region between amino acids 170 and 329 of SEQ ID NO:1, the region between amino acids 180 and 328 of SEQ ID NO:1, the region between amino acids 190 and 327 of SEQ ID NO:1, including the preferred region between amino acids 198 and 327 of SEQ ID NO:1 (not including). The coding sequence for a C-terminal part of a voltage-gated sodium channel alpha subunit preferably has a transcript size of less than 2070 base pairs (bp), such as between 1400 and 2070 bp, including about 1500 bp, about 1600 bp, about 1700 bp, about 1800 bp, about 1900 bp, and about 2000 bp. Said coding sequence for a C-terminal part of a voltage-gated sodium channel alpha subunit may have at least 60% sequence identity to SEQ ID NO:1, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85% at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:1. In embodiments, said coding sequence for a C-terminal part of a voltage- gated sodium channel alpha subunit is a chimeric coding sequence, for example a chimeric sequence comprising a C-terminal part of voltage-gated sodium channel alpha subunit 1, alpha subunit (SCN1A) and voltage-gated sodium channel alpha subunit 10, alpha subunit (SCN10A), SCN2A and SCN10A, SCN3A and SCN10A, SCN4A and SCN10A, SCN5A and SCN10A, SCN8A and SCN10A, SCN9A and SCN10A, or SCN11A and SCN10A. In embodiments, a chimeric coding sequence comprises the cytosolic C-terminal region of SCN10A. For example, said chimeric coding sequence may comprise S5 of repeat III up to S6 of repeat IV of SCN1A, fused to the cytosolic C-terminal region of SCN10A. In embodiments, said coding sequence for a C-terminal part of a voltage- gated sodium channel alpha subunit is derived from, or based on, the C-terminal 690 amino acid residues of SCN10A. Said C-terminal part of a voltage-gated sodium channel alpha subunit may be selected from the amino acid sequences depicted in Table 2. Said expression construct may be provided as a nucleic acid molecule, or provided in a vector, especially a viral vector, to deliver the expression construct into cardiomyocytes of the individual. Said viral vector preferably provides long term expression of the S10s expression construct. Said viral vector preferably is a recombinant adenovirus-based vector, an adenovirus associated virus-based vector, an alphavirus-based vector such as a self-amplifying alphavirus-based replicon vector (Ljungberg and Liljeström, 2015. Expert Rev Vaccines 14: 177-194), or a herpes simplex virus-based vector. Said viral vector most preferably is a adenovirus associated virus-based vector. In embodiments, the expression construct for a C-terminal part of a voltage- gated sodium channel alpha subunit may comprise a promoter operationally linked to an upstream open reading frame (uORF) comprising a translation initiation sequence (TIS), upstream of the coding sequence coding sequence for a C-terminal part of a voltage-gated sodium channel alpha subunit. Said uORF encodes at most 8 amino acid residues, preferably 1-3 amino acid residues. Said uORF resides within the 5’ untranslated region (5’UTR) of the messenger RNA (mRNA) encoding the C-terminal part of a voltage-gated sodium channel alpha subunit. The presence of an uORF may reduce the expression of the C-terminal part of a voltage-gated sodium channel alpha subunit. In addition, said uORF may ensure a more uniform level of expression of the C-terminal part of a voltage-gated sodium channel alpha subunit, when compared to expression of the C-terminal part of a voltage-gated sodium channel alpha subunit in the absence of an uORF. Sequences that may be provided as such uORF are provided in Table 3 herein below. In embodiments, said adenovirus associated virus-based vector is an AAV6- based vector, an AAV9-based vector, or a chimeric vector that is based on AAV6 and AAV9. In embodiments, said vector is an AAV6-based variant construct with an insertion of 1-10 amino acid residues, such as 7 amino acid residues, 8 amino acid residues, or 9 amino acid residues in one or more regions chosen from the regions depicted in Table 1. A preferred region is VR-VIII (amino acids 580-595) provided by nucleotide positions 3945-3992 of the enclosed adeno-associated virus 6 full genome sequence, corresponding to GenBank accession number AF028704.1. If needed, an artificial restriction site may be generated in one or more of the indicated regions depicted in Table 1, by providing an altered coding sequence for one or more of the indicated regions. Said altered coding sequence may encoded for an identical amino acid sequence. However, one or two amino acids may be altered by the provision of the altered coding sequence, such as one or two conserved amino acid alterations. In embodiments, said vector is an AAV9-based variant construct with an insertion of 1-10 amino acid residues, such as 7 amino acid residues, 8 amino acid residues, or 9 amino acid residues in one or more regions chosen from the regions termed VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, and VR-VII of AAV9 (DiMattia et al., 2012. J Virol 86: 6947-6958). In embodiments, said insertion of nucleotide sequences encoding the 1-10 amino acid residues, such as 7 amino acid residues, is in the region provided by nucleotide positions 1741-1780 of the enclosed adeno-associated virus 9 capsid protein 1 (VP1) gene sequences, corresponding to GenBank accession number MZ668415.1. In embodiments, said insertion of nucleotide sequences encoding the 1-10 amino acid residues, such as 7 amino acid residues, is between nucleotide positions 1767-1768 of the enclosed adeno-associated virus 9 capsid protein 1 (VP1) gene sequences, corresponding to GenBank accession number MZ668415.1. If needed, an artificial restriction site may be generated in one or more of the regions termed VR- I, VR-II, VR-III, VR-IV, VR-V, VR-VI, and VR-VII of AAV9 by providing an altered coding sequence for one or more of the indicated regions. Said altered coding sequence may encode an identical amino acid sequence. However, one or two amino acids may have been altered by the provision of the altered coding sequence, such as one or two conserved amino acid alterations. In embodiments, said AAV-based variant construct that is suited for local administration into the heart includes an AAV9-based vector and derivatives thereof, such as AAV9-AERYTKY; AAV9-NRVAVRP; AAV-PDRFGRP; and AAV9- RGDFRAS in VR-VIII. In these variants, the coding sequence for the consecutive amino acid residues AQ at position 589 and 590 of UniProt accession number Q6JC40TD are interrupted by an insertion encoding the indicated amino acid residues. In embodiments, said AAV-based variant construct that is suited for local administration into the heart is an AAV6-based vector and derivatives thereof, such as AAV6-ASDKPGR; AAV6-GGSEKRG; and AAV6-EDGKKAR. In these variants, the consecutive amino acid residues TD at position 589 and 590 of UniProt accession number O56137_9VIRU, are interrupted by the indicated insertion. Packaging of a viral vector into a viral particle, or viral-like particle, is known in the art, including transfection of packaging cells that express structural and packaging genes. For example, packaging cell lines for recombinant adenovirus-based vector include Human Embryonic Kidney (HEK) 293 cells (Graham et al., 1977. J Gen Virol 36: 59–74), and derivatives thereof; 911 cells (Fallaux et al., 1996. Hum Gene Ther 7: 215–222), and PER.C6 cells (Fallaux et al., 1998. Hum Gene Ther 9: 1909– 1917). For example, AAV particles may be produced by transfection of a recombinant AAV (rAAV) vector construct into packaging cells which provide AAV derived replicase and capsid proteins and, optionally, the adenoviral E1A, E4, E2A proteins, and adenovirus viral-associated (VA) RNA. As an alternative, the adenoviral E1A, E4, E2A proteins, and adenovirus VA RNA genes may be provided on one or more helper plasmids which are co-transfected into the packaging cells, together with the rAAV vector construct, on one or more helper viruses that are infected into the packaging cells, or a combination thereof. In addition, stable packaging cell lines and producer cell lines, based on HEK293 cells, HELA cells and insect cells, are available for efficient production of rAAV (Merten, 2024. Microorganisms 12: 384). Suitable packaging cells include HEK293 cells and derivatives thereof, such as HEK293T cells (Rio et al., 1985. Science 227: 23–28). HEK293 cells have been generated by transfection of cultures of normal human embryonic kidney cells with sheared adenovirus 5 DNA (Graham et al., 1977. J Gen Virol 36: 59–74). HEK293 cells are known to express the Ad5 E1A and E1B genes. Table 1. Variable regions in AAV6 genome. Numbering is based on AAV6 VP1 (UniProt O56137_9VIRU). Variable AAV6 amino-acid residues region (VP1 numbering) V_{R-I 262-269 VR-II 326-331 VR-III 381-389 VR-IV 450-469 VR-V 488-505 VR-VI 526-542 VR-VII 545-557 VR-VIII 580-595 VR-IX 705-712} For example, packaging cell lines for production of a herpes simplex virus- based vector include African green monkey kidney cells such as Vero 2-2 cells (Fernández-Frías et al., 2020. Mol Ther Methods Clin Dev 17: 491–496), Vero76 cells and HEK293 (Saeki et al., 2001. Mol Therapy 3: 591-601). In addition, efficient HSV packaging systems have been engineered using an ICP27-deleted, oversized HSV-1 DNA in a bacterial artificial chromosome (BAC) (Saeki et al., 2001. Mol Therapy 3: 591-601). For example, alphavirus vectors can be used as recombinant particles, naked or liposome encapsulated RNA replicons, or plasmid DNA-based replicons (Lundstrom, 2020. Int J Mol Sci 21: 5130. Aside from genetically editing native binding motifs, capsid proteins on the outside of a viral particle may have been altered, for example to minimize ubiquitin–proteasome enzyme degradation of the viral particles during intracellular trafficking. In addition, tissue retargeting to redirect viral tropism affinity to desired receptors may be effected by insertion of a ligand sequence into one or more capsid proteins. For example, the RGD motif (arginylglycylaspartic acid) may be introduced into a capsid protein, allowing the virus to utilize a non- native receptor to gain entry into cells (Erickson et al., 2024. Bioconjug Chem 35: 64-71). In embodiments, a recombinant viral particle comprising the expression construct for expression of a C-terminal part of a voltage-gated sodium channel alpha subunit, such as an AAV-based viral particle, may be shielded from circulating antibodies, for example by complexing the viral particle with human serum albumin, by PEGylation (Lee et al., 2005. Biotech Bioengineering 92: 24– 34), or by encapsulating the viral particles in extracellular vesicles. Said extracellular vesicles may be obtained from the packaging cells that produces the viral particle (Li et al., 2023. Circulation 148: 405-425). In addition, engineered AAV vectors with modified protein capsids, materials tethered to the capsid surface, or fully encapsulated in a second, larger carrier have been explored, which may also aid in avoiding an immune response (Lugin et al., 2020. ACS Nano 14: 14262–14283). Pharmaceutical composition Further provided is a pharmaceutical composition comprising an expression construct for functional expression of a coding sequence for a C-terminal part of a voltage-gated sodium channel alpha subunit, such as an S10s protein, in cardiomyocytes, and a pharmacologically acceptable excipient. Said pharmaceutical composition preferably is a sterile isotonic solution. Said pharmaceutically acceptable excipient preferably is selected from diluents, binders or granulating ingredients, a carbohydrate such as starch, a starch derivative such as starch acetate and/or maltodextrin, a polyol such as xylitol, sorbitol and/or mannitol, sugars such as dextrose, dextrate and/or inulin, glidants (flow aids) and lubricants, and combinations thereof. Said excipient may further include urea, L-histidine, L-threonine, L-asparagine, L-serine, L- glutamine, polysorbate, polyethylene glycol, propylene glycol, polypropylene glycol, or a combination of two or more of the above. One or more salts and a buffering agent may also be included. Said excipients may further include sugar alcohols such as inositol. Further excipients may include a surfactant, such as a nonionic surfactant. Said pharmaceutical composition may be delivered to an individual in the presence or absence of a carrier. Said carrier preferably allows prolonged expression from the expression construct in vivo in a cardiomyocyte. Said carrier may be one or more of a cationic protein such as protamine, a protamine liposome, a polysaccharide, a cation, a cationic polymer, a cationic lipid, cholesterol, polyethylene glycol, and a dendrimer. For example, in case said expression construct is a naked DNA molecule, it may be complexed with protamine, associated with a positively charged oil-in-water cationic nanoemulsion, associated with a chemically modified dendrimer and complexed with polyethylene glycol (PEG)-lipid, complexed with protamine in a PEG-lipid nanoparticle, associated with a cationic polymer such as polyethylenimine, associated with a cationic polymer such as PEI and a lipid component, or associated with a polysaccharide such as, for example, chitosan, in a cationic lipid nanoparticle such as, for example, 1,2-dioleoyloxy-3-trimethylammoniumpropane (DOTAP) or dioleoylphosphatidylethanolamine (DOPE) lipids), complexed with cationic lipids and cholesterol, and complexed with cationic lipids, cholesterol and PEG-lipid, as is described in Pardi et al., 2018 (Pardi et al., 2018. Nature Reviews 17: 261-279). Said pharmaceutical composition may comprise a nucleic acid molecule that enables expression of the C-terminal part of a voltage-gated sodium channel alpha subunit, such as S10s, or a viral particle. Said pharmaceutical composition comprising a viral particle that comprises an expression construct for a C-terminal part of a voltage-gated sodium channel alpha subunit may comprise a known number of viral particles. Said number of viral particles may be determined, for example, by virus quantification by a cell- based assay such as a plaque assay or determination of a a 50% tissue culture infectious dose (TCID50) as a measure of infectious virus titer. In addition, a colorimetric measurement based on protein quantification, such as a BCA assay (Thermo Fisher Scientific), an enzyme-linked immunosorbent assay (ELISA) that employs a capsid-specific antibody, a quantitative amplification reaction such as a quantitative polymerase chain reaction (qPCR), and a tunable resistive pulse sensing (TRPS) method may be used to determine a number of total viral particles in a sample. A preferred method is a quantitative amplification reaction such as qPCR, to determine the number of viral genomes (vg) in a sample as a proxy for the number of viral particles. Methods of treatment An expression construct for a C-terminal part of a voltage-gated sodium channel alpha subunit may be transfection or transducted into cardiomyocyte to allow at least partial restoration of a sodium current in an individual with a cardiac arrhythmia, such as a potential lethal cardiac arrhythmia. Said expression construct may be provided as pharmaceutical composition as is indicated herein above.

Table 2 D_{escription SEQ} Amino acid sequences ID S_{10s 1 MRVVVDALVGAIPSIMNVLLVCLIFWLIFSIMGVNLFAGKFWRCINYTDGEFSLVPLSIVNNKSDCKIQNSTGSFF} WVNVKVNFDNVAMGYLALLQVATFKGWMDIMYAAVDSREVNMQPKWEDNVYMYLYFVIFIIFGGFFTLNLFVGVII DNFNQQKKKLGGQDIFMTEEQKKYYNAMKKLGSKKPQKPIPRPLNKFQGFVFDIVTRQAFDITIMVLICLNMITMM VETDDQSEEKTKILGKINQFFVAVFTGECVMKMFALRQYYFTNGWNVFDFIVVVLSIASLIFSAILKSLQSYFSPT LFRVIRLARIGRILRLIRAAKGIRTLLFALMMSLPALFNIGLLLFLVMFIYSIFGMSSFPHVRWEAGIDDMFNFQT FANSMLCLFQITTSAGWDGLLSPILNTGPPYCDPNLPNSNGTRGDCGSPAVGIIFFTTYIIISFLIVVNMYIAVIL ENFNVATEESTEPLSEDDFDMFYETWEKFDPEATQFITFSALSDFADTLSGPLRIPKPNRNILIQMDLPLVPGDKI HCLDILFAFTKNVLGESGELDSLKANMEEKFMATNLSKSSYEPIATTLRWKQEDISATVIQKAYRSYVLHRSMALS NTPCVPRAEEEAASLPDEGFVAFTANENCVLPDKSETASATSFPPSYESVTRGLSDRVNMRTSSSIQNEDEATSME LIAPGPG S_{10s-V 2 MRVVVDALVGAIPSIMNVLLVCLIFWLIFSIMGVNLFAGKFWRCINYTDGEFSLVPLSIVNNKSDCKIQNSTGSFF} WVNVKVNFDNVAMGYLALLQVATFKGWMDIMYAAVDSREVNMQPKWEDNVYMYLYFVIFIIFGGFFTLNLFVGVII DNFNQQKKKLGGQDIFMTEEQKKYYNAMKKLGSKKPQKPIPRPLNKGIRTLLFALMMSLPALFNIGLLLFLVMFIY SIFGMSSFPHVRWEAGIDDMFNFQTFANSMLCLFQITTSAGWDGLLSPILNTGPPYCDPNLPNSNGTRGDCGSPAV GIIFFTTYIIISFLIVVNMYIAVILENFNVATEESTEPLSEDDFDMFYETWEKFDPEATQFITFSALSDFADTLSG PLRIPKPNRNILIQMDLPLVPGDKIHCLDILFAFTKNVLGESGELDSLKANMEEKFMATNLSKSSYEPIATTLRWK QEDISATVIQKAYRSYVLHRSMALSNTPCVPRAEEEAASLPDEGFVAFTANENCVLPDKSETASATSFPPSYESVT RGLSDRVNMRTSSSIQNEDEATSMELIAPGP S10s- _{3 MFDITIMVLICLNMITMMVETDDQSEEKTKILGKINQFFVAVFTGECVMKMFALRQYYFTNGWNVFDFIVVVLSIA} SLIFSAILKSLQSYFSPTLFRVIRLARIGRILRLIRAAKGIRTLLFALMMSLPALFNIGLLLFLVMFIYSIFGMSS Ultrashort FPHVRWEAGIDDMFNFQTFANSMLCLFQITTSAGWDGLLSPILNTGPPYCDPNLPNSNGTRGDCGSPAVGIIFFTT YIIISFLIVVNMYIAVILENFNVATEESTEPLSEDDFDMFYETWEKFDPEATQFITFSALSDFADTLSGPLRIPKP NRNILIQMDLPLVPGDKIHCLDILFAFTKNVLGESGELDSLKANMEEKFMATNLSKSSYEPIATTLRWKQEDISAT VIQKAYRSYVLHRSMALSNTPCVPRAEEEAASLPDEGFVAFTANENCVLPDKSETASATSFPPSYESVTRGLSDRV NMRTSSSIQNEDEATSMELIAPGPG

_{S10s-1kb 4 MKGIRTLLFALMMSLPALFNIGLLLFLVMFIYSIFGMSSFPHVRWEAGIDDMFNFQTFANSMLCLFQITTSAGWDG} LLSPILNTGPPYCDPNLPNSNGTRGDCGSPAVGIIFFTTYIIISFLIVVNMYIAVILENFNVATEESTEPLSEDDF DMFYETWEKFDPEATQFITFSALSDFADTLSGPLRIPKPNRNILIQMDLPLVPGDKIHCLDILFAFTKNVLGESGE LDSLKANMEEKFMATNLSKSSYEPIATTLRWKQEDISATVIQKAYRSYVLHRSMALSNTPCVPRAEEEAASLPDEG FVAFTANENCVLPDKSETASATSFPPSYESVTRGLSDRVNMRTSSSIQNEDEATSMELIAPGPG Table 3. uORF sequences. N = A/T/G/C. Y=A/G. n = 0-6. N_{ame SEQ ID} Sequence NO K_{ozak 1 GCCATGG ACC-uORF 2 ACCATGG TCT uORF 3 TCTATGG TCT-Stop 4 TCTATGGGTTGAA ACC-Stop 5 ACCATGGGTTGAA ACC-1A 6 ACCATGTGA AAC-2A 7 ACCATGGGTTGA AAC-3A 8 ACCATGGGTGAGTGA AAC-4A 9 ACCATGGGTGAGATCTGA AAC-5A 10 ACCATGGGTGAGATCCTGTGA AAC-6A 11 ACCATGGGTGAGATCCTGTTCTGA AAC-7A 12 ACCATGGGTGAGATCCTGTTCATCTGA AAC-8A 13 ACCATGGGTGAGATCCTGTTCATCCTGTGA Consensus-short 14 NNNATGG Consensus-long 15 NNNATGGNN(NNN)nTGY(N)}

Methods for delivery of an expression construct for expression of a C-terminal part of a voltage-gated sodium channel alpha subunit such as S10s in a cardiomyocyte include administration by a parenteral route, such as intramuscular and intravenous administration. Said pharmaceutical composition comprising an expression construct is preferably administered by local administration, for example by direct injection into the myocardium, or infusion into a coronary artery or vein. For example, said pharmaceutical composition comprising an expression construct may be administered to an individual in need thereof by employing a catheter. Said injection or infusion may be accomplished by use of external pump or of a fully implantable device. Said external pump is preferably equipped with a percutaneous catheter, tunneled or not tunneled, or equipped with a subcutaneous injection port and an implanted catheter. Intramyocardial injections may be conducted either surgically, through a small incision in the chest, e.g., between the ribs (intracostal), or following a full thoracotomy, allowing precise placement of one or multiple epicardial injections, or minimally invasive using an injection catheter-based approach for endomyocardial delivery and/or epicardial delivery. Intracoronary infusion may be conducted by inserting an infusion catheter into the cardiac coronary vasculature either antegrade, e.g., via femoral or radial arterial access (Hayase et al., 2005. Am J Physiol Heart Circ Physiol 288: H2995-3000), or retrograde via the coronary sinus (Weber et al., 2014. Gene Ther 21: 131-138; Lampela et al., 2024. Sci Rep 14: 1467). These infusion approaches may be combined with temporary local occlusion of the coronary perfusion, e.g., using an inflatable balloon catheter, to allow for prolonged durations for the AAV6-based viral particles to pass the endothelial barrier. Intracoronary delivery may also be combined with cardiac recirculation to enhance transduction efficacy through prolonged local persistence of the AAV6-based viral particles in the cardiac target area (Byrne and Kaye; 2017. Methods Mol Biol 1521: 261-269; Fargnoli et al., 2013. Ann Thorac Surg 96: 586-95). Said pharmaceutical composition comprising an AAV6-based viral particle may be provided to the myocardium, or into a coronary artery or vein, at a dosage of about 10¹⁰-10¹⁴ viral genomes (vg) per injection, such as about 5x10¹⁰ vg per injection, 10¹¹ vg per injection, 5x10¹¹ vg per injection, 10¹² vg per injection, 5x10¹² vg per injection, or 10¹³ vg per injection. This is significantly lower than what is currently applied in cardiac gene therapies that target the heart systemically, which is about 6.7E¹³-1.1E¹⁴ vg/kg in a recent clinical trial (Greenberg et al, 2021. Circulation 144: A10727-A10727). An AAV6-based construct, such as AAV6-ASDKPGR; AAV6-GGSEKRG; and AAV6-EDGKKAR, or an AAV9-based construct such as AAV9-AERYTKY; AAV9- NRVAVRP; AAV-PDRFGRP, efficiently targets to the myocardium, especially cardiomyocytes. Therefore, a pharmaceutical composition comprising an AAV6- based viral particle may be systemically provided at a dosage of about 10¹⁰-10¹⁴ viral genomes (vg) per injection, such as about 5x10¹⁰ vg per injection, 10¹¹ vg per injection, 5x10¹¹ vg per injection, 10¹² vg per injection, 5x10¹² vg per injection, or 10¹³ vg per injection. In embodiments, said pharmaceutical composition comprises a DNA molecule that expresses said C-terminal part of a voltage-gated sodium channel alpha sub- unit, such as S10s, upon delivery to a cardiomyocyte. Said DNA molecule may comprise modified nucleotides, for example to increase half live of the molecule. For example, said nucleic acid molecule may be provided in a plasmid, or as linear DNA. Non-virus mediated delivery of a DNA molecule according to the invention include lipofection, microinjection, and agent-enhanced uptake of DNA. Lipofection is described in e.g., U.S. Pat. Nos.5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam™, Lipofectin™, and SAINT™). Cationic and neutral lipids that are suitable for efficient lipofection of polynucleotides include those of WO 91/17424 and WO 91/16024. Said DNA molecule may also be packaged, for example in lipid vesicles such as a virosome, a liposome, or immunoliposome, prior to delivery of said DNA molecule to an individual in need thereof. In embodiments, said pharmaceutical composition comprises a viral particle that comprises a DNA molecule that expresses said C-terminal part of a voltage- gated sodium channel alpha sub-unit, such as S10s, upon delivery to a cardiomyocyte. Said viral particle preferably is a recombinant adenoviral particle, an adenovirus associated virus-based particle, an alphavirus-based particle, or a herpes simplex virus-based particle. Said pharmaceutical composition preferably comprises an adenovirus associated virus-based particle. The provision of an expression construct for a C-terminal part of a voltage- gated sodium channel alpha subunit to at least a part of a heart of an individual in need thereof may help to at least partially restore a cardiac sodium current (INa), which is responsible for the initiation and propagation of the cardiac electrical excitation wavefront, as is implicated in both acquired and inherited arrhythmia syndromes such as Brugada syndrome (BrS), progressive cardiac conduction defects (PCCD), sick sinus syndrome (SSS), atrial fibrillation (AF) and ventricular tachycardia or fibrillation (VT/VF). The provision of an expression construct, either as such or in a pharmaceutical composition, to at least a part of an heart of an individual in need thereof may help in the treatment of an arrhythmia such as a bradyarrhythmia, such as a sinoatrial node exit block, or a tachyarrhythmia, such as associated with Brugada syndrome. In addition, said expression construct or pharmaceutical composition may help in the treatment of a conduction defect such as an atrioventricular block, a left bundle branch block (LBB), a non-specific ventricular conduction abnormality, and/or a progressive cardiac conduction disease (PCCD). In addition, said expression construct or pharmaceutical composition may help in the treatment of a ventricular tachyarrhythmia, such as an ischemia-related arrhythmia, a non-ischemia-related arrhythmia (e.g., a ventricular arrhythmia in Brugada syndrome), in non-ischemic cardiomyopathies, in fibrosis-related or scar- related arrhythmia, such as an ischemia-related and/or post-myocardial infarction- related arrhythmia, and/or cardiac conduction abnormalities and associated arrhythmias, and/or contractile dysfunction, including DCM and heart failure. Transfection or transduction of an expression construct that comprises a DNA molecule that expresses said C-terminal part of a voltage-gated sodium channel alpha sub-unit, such as S10s, may be combined with further treatment. Said further treatment may be selected from a beta-blocker, a calcium channel blocker, a cardiac glycoside such as digoxin, and/or an alkaloid such as quinidine (Furlanello et al., 2019. Pharmacol Res 144: 257-263). Preferred beta blockers, also termed adrenergic beta-antagonists, include atenolol (2-[4-[2-hydroxy-3-(propan-2-ylamino)propoxy]phenyl]acetamide), bisoprolol (1-(propan-2-ylamino)-3-[4-(2-propan-2- yloxyethoxymethyl)phenoxy]propan-2-ol), carvedilol (1-(9H-carbazol-4-yloxy)-3-[2- (2-methoxyphenoxy)ethylamino]propan-2-ol), metoprolol (1-[4-(2- methoxyethyl)phenoxy]-3-(propan-2-ylamino)propan-2-ol), nadolol ((2R,3S)-5-[3- (tert-butylamino)-2-hydroxypropoxy]-1,2,3,4-tetrahydronaphthalene-2,3-diol), propranolol (1-naphthalen-1-yloxy-3-(propan-2-ylamino)propan-2-ol), timolol ((2S)- 1-(tert-butylamino)-3-[(4-morpholin-4-yl-1,2,5-thiadiazol-3-yl)oxy]propan-2-ol) and sotalol (N-[4-[1-hydroxy-2-(propan-2-ylamino)ethyl]phenyl]methanesulfonamide). Recommended dosages for a beta blocker are between 50 and 200 mg daily, preferably about 100 mg daily, for atenolol, between 2.5 and 20 mg daily, preferably about 5 mg daily, for bisoprolol, between 1 and 25 mg twice a day, preferably about 12.5 mg twice daily, for carvedilol, between 50 and 450 mg daily, preferably about 100 mg daily, for metoprolol, between 40 and 320 mg daily, preferably about 80 mg daily, for nadolol, between 40 and 640 mg daily, preferably about 160 mg daily, for propranolol, between 5 and 30 mg twice daily, preferably about 10 mg twice daily, for timolol, and between 80 and 640 mg twice daily, preferably about 240 mg twice daily, for sotalol. A preferred cardiac glycoside is a cardenolide, preferably digoxin (3- [(3S,5R,8R,9S,10S,12R,13S,14S,17R)-3-[(2R,4S,5S,6R)-5-[(2S,4S,5S,6R)-5- [(2S,4S,5S,6R)-4,5-dihydroxy-6-methyloxan-2-yl]oxy-4-hydroxy-6-methyloxan-2- yl]oxy-4-hydroxy-6-methyloxan-2-yl]oxy-12,14-dihydroxy-10,13-dimethyl- 1,2,3,4,5,6,7,8,9,11,12,15,16,17-tetradecahydrocyclopenta[a]phenanthren-17-yl]- 2H-furan-5-one). A recommended dosage for digoxin is between 0.125 mg and 0.375 mg orally or intravenously, every two hours up to a total of 1.5 mg, or between 0.125 mg and 0.375 mg orally once daily. A preferred alkaloid is quinidine ((S)-[(2R,4S,5R)-5-ethenyl-1- azabicyclo[2.2.2]octan-2-yl]-(6-methoxyquinolin-4-yl)methanol). A recommended dosage for quinidine is between a total 200 mg and a maximum daily dose of 4 g orally, or intravenously up to a total of 800 mg. Preferred calcium channel blockers include diltiazem ([(2S,3S)-5-[2- (dimethylamino)ethyl]-2-(4-methoxyphenyl)-4-oxo-2,3-dihydro-1,5-benzothiazepin- 3-yl] acetate;hydrochloride) and verapamil (2-(3,4-dimethoxyphenyl)-5-[2-(3,4- dimethoxyphenyl)ethyl-methylamino]-2-propan-2-ylpentanenitrile). A recommended dosage for diltiazem is an initial intravenous bolus of 0.25 mg/kg, followed by infusion with 10 mg/hr to 15 mg/hr for a maximum duration of 24 hours for diltiazem, and between 80 mg and 480 mg orally once daily, preferably about 160 mg once daily, for verapamil. EXAMPLES Example 1 Materials and methods Animals Animal care and experiments conform to Directive 2010/63/EU. All animal work was approved by the Animal Experimental Committee of the Academic Medical Center, Amsterdam, and was performed in compliance with the Dutch Government guidelines. For intramyocardial injection, 8-week-old female FVB/N mice were used. For intravenous injection, 6-week old male Scn5a+/∆7bp mice and their wild type littermates were used. Vector production AAV6 vectors were produced by double transfection of HEK293T cells and AAV9 vectors by triple transfections. Low passage HEK293T cells were plated in thirty 145 mm dishes at the density of 1.5 x 107 cells per dish in DMEM-GlutaMax (ThermoFisher Scientific) containing 10% fetal bovine serum (FBS; Sigma) and 1% penicillin-streptomycin (P/S; ThermoFisher Scientific). For AAV6 vectors, cells were transfected next day with 33 µg pDP6 and 16 µg AAV transfer plasmids per dish using linear polyethylenimine (PEI; Polysciences Inc). For AAV9 vectors, cells were transfected with 13 µg Rep2Cap9 plasmid, 21 µg helper plasmid and 13 µg AAV transfer plasmid per dish. Medium was also replaced during transfection to DMEM containing 1% P/S. Three days after transfection, cells were collected by centrifugation and the medium was concentrated by tangential flow filtration using the ÄKTA flux s system (GE Healthcare) to a final volume of 20 mL. Cells and medium were then combined, frozen and thawed twice followed by DNAseI, Rnase A and Benzonase treatment. AAV vectors were purified by iodixanol density- gradient ultracentrifugation overnight. The AAV-containing fraction was then collected and concentrated to 1 mL by buffer exchange to PBS containing 0.001% Pluronic F68 using Amicon Ultra-15100 kDa centrifugal filter units (Millipore). Concentrated AAV vectors were aliquoted and stored at -80°C until use. Genomic titer was determined by qPCR. Lentiviral vectors were produced by quadruple transfection of HEK293T cells. Cells were plated and cultured as described above. One day after plating, cells were transfected with 3.2 µg envelope plasmid, 2.2 µg RSV-REV plasmid, 5.8 µg MDLG plasmid (Addgene) and 8.8 µg transfer lentivirus plasmid per dish using linear PEI. Next day medium was changed to DMEM-GlutaMax without FBS. Three days after transfection, supernatant was collected and concentrated to 500 µL by buffer exchange to PBS using Amicon Ultra-15100 kDa centrifugal filter units (Millipore). Genomic titer was determined using LV900 titration kit (Applied Biological Materials). Lentiviral transduction of hiPSC-CMs hiPSC-CMs were transduced with lentiviral vectors in culture medium at an multiplicity of infection (MOI) of 10 for 24 hours. Medium were then refreshed every other day until being used in the patch-clamp experiments. Whole-cell patch-clamp recordings Cell preparation and data acquisition. Left ventricular cardiomyocytes of mice were isolated by an enzymatic dissociation procedure (Man et al., 2021. Circulation 144:229-242) and ventricular-like hiPSC-CMs were differentiated and dissociated as described previously (Li et al., 2022. J Tissue Eng. 13: 20417314221127908) from the control hiPSC line LUMC0099iCTRL04. Single mouse cells were stored at room temperature for at least 45 min in a modified Tyrode’s solution containing (in mmol/L): NaCl 140, KCl 5.4, CaCl21.8, MgCl21.0, glucose 5.5, HEPES 5.0; pH 7.4 (NaOH). Cells were put into a recording chamber on the stage of an inverted microscope (Nikon Diaphot), and we selected for electrophysiological measurements GFP-positive quiescent single mouse cells with smooth surfaces and single regularly beating hiPSC-CMs. INa and APs were recorded with ruptured and amphotericin-perforated patch-clamp technique, respectively, using an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA, USA). Voltage control, data acquisition, and analysis were realized with custom software. Signals were low-pass-filtered with a cut-off of 5 kHz and digitized at 20 and 40 kHz for INa and APs, respectively. Potentials were corrected for the calculated liquid junction potential (Barry and Lynch, 1991. J Membr Biol 121: 101-117). Cell membrane capacitance (Cm) was estimated by dividing the time constant of the decay of the capacitive transient in response to 5 mV hyperpolarizing voltage clamp steps from –40 mV by the series resistance. For INa measurements, Cm and series resistance were compensated for by at least 80%. INa measurements INa was characterized at room temperature using a bath solution containing (in mmol/L): NaCl 7, CsCl 133, CaCl21.8, MgCl21.2, glucose 11.0, HEPES 5.0, and pH 7.4 (CsOH). Nifedipine (5 µmol/L) was added to block the L-type calcium current. Pipettes were filled with solution containing (in mmol/L): NaCl 3, CsCl 133, MgCl22.0, Na2ATP 2.0, TEA-Cl 2.0, EGTA 10.0, HEPES 5.0, and pH 7.2 (CsOH). INa amplitudes and voltage dependency of INa activation were measured by 50 ms depolarizing voltage clamp steps from a holding potential of -120 mV. A two- step protocol consisting of series of 500 ms pulses between -140 and 40 mV from a holding potential of -120 mV, followed by a second 50 ms step to -20 mV, was used to establish the voltage dependency of INa inactivation. Cycle lengths of the protocols were 5 s. INa was defined as the difference between peak current and steady-state current and INa density was calculated by dividing current amplitudes by Cm. Voltage dependence of activation and inactivation were determined by fitting a Boltzmann function (y=[1+exp{(V-V1/2)/k}]-1) to the individual plots, where V1/2 is the voltage of half-maximal (in)activation and k the slope factor (in mV). AP measurements APs were recorded at 36 ± 0.2°C using the modified Tyrode’s solution as bath solution. Pipette solution contained (in mmol/L): K-gluconate 125, KCl 20, NaCl 5.0, amphotericin-B 0.44, HEPES 10, and pH 7.2 (KOH). APs in hiPSC-CMs were measured with dynamic clamp25 to inject an in silico IK1 with a current-voltage relationship resembling Kir2.1 channels with a 2 pA/pF outward peak26, resulting in quiescent hiPSC-CMs with a RMP of −80 mV or more negative. APs were elicited at 6 and 1 Hz for murine cardiomyocytes and hiPSC-CMs, respectively, by 3-ms, ≈10–30% suprathreshold current pulses through the patch pipette. Average AP parameters were taken from 10 consecutive APs. Intramyocardial injection of AAV Animals were injected subcutaneously with buprenorphine (0.075 mg/kg) and carprofen (0.05 mg/kg) for analgesia, at least 30 min prior to surgery. Anaesthesia was induced with 4% isoflurane in 1 L/min O2. Mice were shaved, intubated and placed on a heating mat to maintain body temperature. Subsequently, an analgesic mixture consisting of lidocaine (2 mg/kg) and bupivacaine (3 mg/kg) was applied subcutaneously at the site of the incision. Anaesthesia was maintained using ventilation with 2-3% isoflurane in 1 L/min O2. Left thoracotomy was performed to expose the apex of the heart at the fourth intercostal space. To inject the vector into the apex a 10 µmol/L Hamilton syringe fitted with a 31-G needle (13 mm, point style 4) was inserted from the anterior LV towards the apex. Five µL of the viral vector solution was slowly administered at each injection site to administer a total volume of 20 µL viral vectors. The thoracotomy and skin were closed with a C-112 mm cutting needle with a 6-0 silicone coated braided silk wire (Sofsilk, Covidien). Post-surgery analgesia consisted of 4 days of ad libitum carprofen (Rimadyl Cattle, 0.06 mg/ml) in drinking water and wet food. Intravenous injection of AAV Animals were anesthetized using 4% isoflurane using an induction chamber. The animal was taken out of the chamber and restrained using the thumb and middle finger. Using the index finger skin above the eye is pulled back until the eye slightly protrudes. A 0.3 mL (30G) x 8 mm U-100 insulin needle (BD Micro-Fine) was inserted at an angle of 45° starting around the medial canthus towards the retro-orbital sinus. The construct was slowly injected in one smooth motion in a maximum volume of 100 µL after which the animal was placed on a heating pad until it fully regained consciousness. Mice were monitored 2 days post injection for any abnormalities. Mouse electrocardiograms Mice were anesthetized by inhaling 4% isoflurane and maintained in anesthesia with 2% isoflurane in 1 L/min O2. Subcutaneous recording electrodes were placed at the left armpit (L), right armpit (R) and left groin (F) and ECGs were recorded for a period of 1 min. ECG parameters (RR, PR, QRS interval) were calculated from lead II (lead II = F-R) using LabChart Pro 8 (ADInstruments). Immunofluorescence staining Cells were fixed in 4% paraformaldehyde (PFA) for 10 min at room temperature. Hearts were fixed in 4% PFA overnight and sectioned at 7 µm. Sections were deparaffinised and dehydrated by a series of ascending ethanol concentrations. For antigen retrieval, sections were boiled in unmasking solution (H3300, Vector). Cells and sections were blocked in 4% BSA and incubated with chicken anti-GFP (1:500, Aves Labs, GFP-1020), mouse anti-2A (1:1000, Novus Biologicals, NBP2-59627). DAPI (1ug/mL, Sigma, D 9542) was used as a nuclear stain. Wheat Germ Agglutinin (WGA; 1:250, Invitrogen, W32464 ) was used as a membrane stain. Fluorescence images were acquired using a Leica DM6000 fluorescence microscope or a Leica TCS SP8 confocal microscope (Leica Microsystems). RNA isolation and RT-qPCR Total RNA was isolated from cardiac tissue using NucleoSpin RNA (Macherey-Nagel) according to the manufacturer’s protocol. cDNA library was transcribed from 500-1000 ng total RNA with oligo-dT primers (125 µmol/L) and the Superscript II system (Invitrogen). Quantitative PCR was performed using the LightCycler 480 Real-Time PCR system (Roche). Relative start concentration was calculated using LinRegPCR58 and values were normalized to Hprt expression level. Optical mapping of APs in mouse hearts The mice were killed by cervical dislocation after which the heart was excised, cannulated and mounted on a Langendorff perfusion set-up. The hearts were perfused with Tyrode’s solution (37°C) containing (in mmol/L): NaCl 128, KCl 4.7, CaCl21.45, MgCl20.6, NaHCO327, NaH2PO40.4, and glucose (Nguyen et al., 2022. Nat Commun 13: 620). The solution was maintained at 7.4 pH by equilibration with a mixture of 95% O2 and 5% CO2. Hearts were incubated with 15 µmol/L di-4-ANEPPS (Bio-Techne) in 10 mL Tyrode’s solution after which they were placed in the optical mapping setup and perfused with 10 µmol/L blebbistatin (Bio-Techne) in Tyrode’s solution to reduce motion artefacts. Excitation light was provided by a 5-watt power LED (filtered 510 ± 35 nm). Fluorescence (filtered > 610 nm) was transmitted through a tandem lens system on a CMOS sensor (100 x 100 elements, sampling rate 5 kHz, MiCAM Ultima, SciMedia). Activation patterns were measured during epicardial stimulation at an interval of 120 ms. Optical APs and conduction velocity were analysed with custom software. Computer simulations of linear ventricular tissues The functional effect of a complete loss-of-function mutation in SCN5A and the conduction improving effect of S10s were assessed by computer simulations of a linear strand of cardiomyocytes, using the human left ventricular cell model (Ten Tusscher et al., 2004. Am J Physiol 286: H1573-1589) as updated by Ten Tusscher and Panfilov (Ten Tusscher and Panfilov, 2006. Physics Med Biol 51: 6141-6156) to describe individual cells. When simulating AP propagation in strands of cells, the myoplasmic resistivity was set to 150 Ω∙cm27 and the CV was computed across the middle third of the strand. Software was compiled as a 32-bit Windows application using Intel Visual Fortran Composer XE 2013 and run on an Intel Core i7 processor-based workstation. For the numerical integration of differential equations we applied a simple and efficient Euler-type integration scheme with a 1 µs time step.59 All simulations were run for a sufficiently long period to reach steady-state behaviour. Computer simulations using 3D ventricular model The biventricular heart model was reconstructed from 2D late-gadolinium cardiac magnetic resonance image. In line with previous studies modelling genetic heart diseases (Zhang et al., 2023. Elife 12: RP88865; Shade et al., 2020. Heart Rhythm 17: 408-414) we used the Otsu thresholding and standard deviation method to categorize the segmented myocardium into three distinct tissue types: non-fibrotic, fibrotic and scar tissue. Subsequently, we created finite-element tetrahedral meshes with an average resolution of 400μm, using finite-element analysis software (Mimics Innovation Suite; Materialise, Leuven, Belgium). Fibre orientations were incorporated into each element of the computational mesh through a validated rule-based method (Bayer et al., 2012. Ann Biomed Eng 40: 2243-2254). To represent the electrophysiological properties of the various tissue types, we incorporated two different cell models into the whole-heart model. For cardiomyocytes with plakophilin-2 loss-of-function mutation (PKP2+/-) in non- fibrotic regions, we used a cell model developed in our recent study (Zhang et al., 2023. Elife 12: RP88865). This PKP2+/- cell model features a 70% decrease in maximal channel conductance for sodium current and a 50% decrease in L-type calcium current as compared to normal myocytes. We also upregulated the background calcium current, representative of connexin 43 hemichannel-mediated calcium entry, by 5-fold, which was in line with the in vitro properties of PKP2- deficient cells. For cardiomyocytes in fibrotic regions, we incorporated modifications based on experimental data (Coppini et al., 2013. Circulation 127: 575-584), which have also been validated in previous studies (Shade et al., 2020. Heart Rhythm 17: 408-414; O'Hara et al., 2022. Elife 11: e73325). Scar tissue was represented as non-conductive regions. To incorporate S10s treatment into our PKP2-deficient cell model, we upregulated the residual maximal sodium channel conductance by 144%. This resulted in a PKP2+/- +S10s model with a sodium current at 43.2% of its baseline. To simulate the heart’s electrical activity, we utilized the openCARP software package (Plank et al., 2021. Comput Methods Programs Biomed 208:106223). Full details on the simulation can be found in previous publications (Zhang et al., 2023. Elife 12: RP88865; Prakosa et al., 2018. Nat Biomed Eng 2: 732-740). The model was paced sequentially from 9 uniformly distributed endocardial RV locations from base to apex using a validated rapid pacing protocol (Arevalo et al., 2016. Nat Commun 7: 11437). After inducing the re-entries, we analyzed activation maps to identify the VT circuit. Results S10s gene therapy increases INa in mouse cardiomyocytes We started our evaluation of S10s overexpression with cellular studies. In order to deliver S10s to cardiomyocytes, we cloned human S10s into a bicistronic expression cassette, which contains a self-cleaving P2A-GFP as a fluorescent marker, allowing identification and isolation of transduced cells for electrophysiological studies (Figure 1). The control vector expresses GFP alone. AAV serotype 6–S10s-P2A-GFP (AAV6-S10s) and AAV6-GFP vectors were produced (Figure 1C, top) and injected in the apex of wild type mouse hearts via intramyocardial injection, at an empirically determined dose of 1 x 10¹¹ vg/mouse. Immunofluorescence staining confirmed the successful overexpression of S10s and GFP in the injected mice (Figure 2A). We then performed whole-cell patch-clamp studies on GFP-positive ventricular cardiomyocytes isolated from AAV-injected mice two weeks post injection to measure INa. Typical INa recordings are shown in Figure 2B and the average current-voltage relationships are shown in Figure 2C. Peak INa density was significantly increased in cardiomyocytes isolated from AAV6-S10s mice, compared to those isolated from AAV6-GFP mice. For example, at -40 mV INa density was increased by 45% (-96.5 ± 7.8 pA/pF for AAV6-S10s vs. -66.7 ± 6.1 pA/pF for AAV6- GFP; p < 0.01; n = 11 and 12, respectively; Figure 2D). Moreover, overexpression of S10s did not affect voltage-dependency of activation or inactivation, as indicated by the absence of differences on the half-(in)activation voltage and slope factor (data not shown) of the Boltzmann curves that were fitted to the individual (in)activation data (Figure 2E). These findings indicated that S10s overexpression increased INa density in mouse cardiomyocytes without affecting INa gating. To further characterize the electrophysiological effect of S10s overexpression, we measured APs in isolated cardiomyocytes (typical examples shown in Figure 3A). AP measurements revealed that dV/dtmax was increased by 41% by S10s overexpression (302 ± 18 V/s for AAV6-S10s vs. 214 ± 9 V/s for AAV6-GFP; p < 0.01; n = 9 and 7, respectively; Figure 3B). Since dV/dtmax is regarded as a measure of sodium channel availability (Berecki et al., 2010. PLoS One 5: e15772), these data are in line with our findings of increased INa induced by S10s (Figure 2). S10s overexpression did not affect other AP parameters, such as resting membrane potential (RMP; Figure 3C), AP amplitude (APA, Figure 3D), and AP duration (APD) at 50% repolarization (APD50; Figure 3E) and 90% repolarization (APD90; Figure 3F). S10s gene therapy increases cardiac conduction velocity in wild type mice Given the importance of INa to cardiac impulse propagation,19 we hypothesized that S10s-driven increase in INa density would increase cardiac conduction velocity (CV) at the whole organ level. In order to achieve homogenous S10s overexpression throughout the heart, we adapted our existing AAV delivery vector in two ways: 1) the ubiquitous CMV promoter was replaced by a cardiac specific troponin T (cTnT) promoter, and 2) we made use of the AAV cardio-tropic serotype 9 (AAV9), instead of AAV6 (Figure 1C, bottom). We first administered the AAV vectors to 6-week-old wild type mice via intravenous injection at a dose of 8 x 10¹¹ vg/mouse (Figure 4A). Overexpression of S10s in left ventricular tissue of AAV9-S10s mice was detected by RT-qPCR (Figure 4B). Two weeks post injection, optical mapping on isolated hearts revealed significantly increased longitudinal CV (CVL) in the left ventricles of AAV9-S10s mice, compared to AAV9-GFP mice (92.9 ± 3.9 cm/s for AAV9-S10s vs.73.3 ± 4.6 cm/s for AAV9-GFP; p < 0.01; n = 5; Figure 4C and 4D). A trend of CVL increase was also observed in the right ventricles, although not significant (Figure 4C and 4D). Transversal CV (CVT) was not changed in both left and right ventricles (data not shown), likely due to the absence of Nav1.5 channels at the transverse-tubular system (Cohen, 1996. Circulation 94: 3083-3086; Dhar Malhotra et al., 2001. Circulation 103: 1303-1310; Westenbroek et al., 2013. J Mol Cell Cardiol 64: 69-78). Surface ECG analyses showed no significant differences in RR, PR, or QRS intervals (Figure 4E, 4F) between AAV9-S10s mice and AAV9-GFP mice. These data support the concept that S10s overexpression increases ventricular CV in wild type mice by augmenting INa. S10s gene therapy rescues conduction slowing in Scn5a-haploinsufficient mice With the ultimate goal of applying this gene therapy for the treatment of cardiac conduction abnormalities, we tested the potential of S10s overexpression in an Scn5a-haploinsufficient mouse model. To this end, we made use of the Scn5a+/∆7bp model heterozygous for a 7 bp deletion in exon 3 causing haploinsufficiency, as reflected in various conduction parameters, e.g., prolongation of the QRS interval (Man et al., 2019. Nat Commun 10: 4943). AAV9-GFP or AAV9-S10s were administrated to Scn5a+/∆7bp mice via intravenous injection at an intermediate dose of 8 x 10¹¹ vg/mouse (Figure 5A). Overexpression of S10s in left ventricular tissue of AAV9-S10s mice was detected by RT-qPCR (Figure 5B). S10s protein was not detected by immunofluorescence staining (Figure 5C), probably because the expression level was below the detection threshold of the anti-P2A antibody. We subsequently injected Scn5a+/∆7bp mice with AAV9-S10s at a higher dose of 8 x 10¹² vg/mouse (HD_AAV9-S10s; Figure 5A). S10s expression was detected in heart and liver by RT-qPCR (Figure 5B and S4A). Comparing to those from the intermediate dose group, cardiac S10s expression is 20-fold higher in animals from the high dose group (Figure 5B). Immunofluorescence staining showed clear S10s protein expression in the left ventricles (Figure 5C). Mice injected with empty vectors at the same dose were used as control (HD_AAV9-MCS; Figure 1C and 5A). This was to avoid the potential adverse effect of high GFP expression on cardiac conduction since GFP has previously been shown to negatively affect CV in neonatal rat cardiomyocytes cultures (Sekar et al., 2007. Am J Physiol 293: H2757-2770). Two weeks post injection, ECGs were recorded in vivo and hearts were isolated for optical mapping. Comparing to the wild type mice, Scn5a+/∆7bp mice injected with AAV9-GFP showed slower CVL in both left ventricles (49.1 ± 4.3 cm/s for Scn5a+/∆7bp vs. 73.3 ± 4.6 cm/s for wild type; p < 0.01; n = 5) and right ventricles (56.2 ± 1.3 cm/s for Scn5a+/∆7bp vs. 74.4 ± 4.4 cm/s for wild type; p < 0.01; n = 5). Significantly prolonged QRS interval was also observed (10.3 ± 0.2 ms for Scn5a+/∆7bp vs. 8.6 ± 0.5 ms for wild type; p < 0.01; n = 8 and 10, respectively), in line with the previously reported prolongation of QRS interval in this mouse line (Man et al, 2019. Nat Commun 10: 4943). In the intermediate dose groups, AAV9-S10s gene therapy significantly increased CVL in the left ventricles comparing to control (66.4 ± 4.2 cm/s for AAV9- S10s vs.49.1 ± 4.3 cm/s for AAV9-GFP; p < 0.01; n = 5; Figure 5D and 5E). A trend of CVL increase was observed in the right ventricles but was not statistically significant (Figure 5D and 5E). CVT was not changed (data not shown). No significant changes in RR, PR and QRS intervals were observed between the intermediate dose groups (Figure 5F). In the high dose groups, comparing to control, HD_AAV9-S10s gene therapy significantly increased CVL in both left ventricles (68.5 ± 2.9 cm/s for HD_AAV9-S10s vs. 59.1 ± 3.2 cm/s for HD_AAV9- MCS; p < 0.05; n = 5) and right ventricles (72.9 ± 3.1 cm/s for HD_AAV9-S10s vs. 64.7 ± 1.2 cm/s for HD_AAV9-MCS; p < 0.05; n = 5; Figure 5G and 5H). CVT was not changed (data not shown). Surface ECG analyses revealed significantly shortened QRS interval in HD_AAV9-S10s mice comparing to control (9.0 ± 0.3 ms for HD_AAV9-S10s vs.10.2 ± 0.3 ms for HD_AAV9-MCS; p < 0.05; n = 5 and 10, respectively; Figure 5I). No significant changes in RR and PR intervals were observed between the high dose groups (data not shown). Moreover, HD_AAV9- S10s gene therapy rescued the conduction slowing phenotype of Scn5a+/∆7bp mice. Scn5a+/∆7bp mice injected with HD_AAV9-S10s showed no significant differences in QRS interval nor CVL in both ventricles as compared to the wild type mice injected with AAV9-GFP (data not shown). These data showed that S10s gene therapy improved cardiac conduction and rescued the conduction slowing phenotype in Scn5a-haploinsufficient mice. S10s overexpression increases dV/dtmax in hiPSC-CMs In order to study the impact of S10s on electrophysiological properties of human cardiomyocytes, we made use of hiPSC-CMs. For optimal transduction efficiency, we transduced hiPSC-CMs with lentiviral vectors containing either the S10s-GFP bicistronic expression cassette (Lenti-S10s) or GFP alone (Lenti-GFP) (Figure 1C, top). Successful delivery of S10s was confirmed by immunofluorescence staining on cells (Figure 6A). Transduced hiPSC-CMs were used for AP measurements with the patch-clamp methodology, 5-7 days post transduction (Figure 6B). Dynamic clamp was used to inject a 2 pA/pF Kir2.1-like current to overcome the depolarized and spontaneous state of the hiPSC-CMs which hampers the functional availability of Nav1.5 channels (Verkerk et al., 2021. J Cardiovasc Pharmacol 77: 267-279; Wilders, 2006. J Physiol 576: 349-359; Meijer van Putten et al., 2015. Front Physiol 6: 7). Similar to our results in mouse cardiomyocytes (Figure 3B), S10s overexpression led to significantly increased dV/dtmax with a 73% increase compared to the GFP group (196 ± 29 V/s for Lenti-S10s vs. 113 ± 12 V/s for Lenti-GFP, p < 0.01; n = 13 and 14, respectively; Figure 6C). No significant differences were observed in RMP, APA, APD50, and APD90 between the groups (Figure 6D-6G). S10s overexpression increases excitability and CV in simulated human tissues Based on our results that S10s overexpression increases CV in Scn5a+/∆7bp mice, and that it increases dV/dtmax in hiPSC-CMs, we hypothesized that introduction of S10s into human ventricular tissues could increase CV in SCN5A- haploinsufficent background and prevent conduction block. We performed in silico experiments using computer-simulated one- dimensional strands of human left ventricular cardiomyocytes to test our hypothesis. Eighty cells with a myoplasmic resistivity of 150 Ω∙cm were electrically coupled through a gap junctional conductance gj and stimulated at one end of the strand with a square stimulus of 2 ms duration at a rate of 1 Hz (Figure 7A, inset) (Ten Tusscher et al., 2004. Am J Physiol 286: H1573-1589; Ten Tusscher and Panfilov, 2006. Physics Med Biol 51: 6141-6156). In this in silico tissue model, a heterozygous loss-of-function mutation in SCN5A was simulated by a 50% decrease in Nav1.5 conductance (labelled SCN5A+/−), representing an entirely non- functional SCN5A mutation. The effect of S10s was modelled by a 44.7% increase in the residual Nav1.5 conductance to 72% of the wild type Nav1.5 conductance (labelled SCN5A+/− + S10s). This 44.7% increase was chosen according to the increase in peak INa density observed in our voltage clamp experiments of isolated murine cardiomyocytes in Figure 2. As illustrated in Figure 7A, modelling heterozygosity of the complete loss-of- function mutation in SCN5A resulted in an approximately 21% decrease in CV over a wide range of gj values, which was halved to 10–11% upon the simulated application of S10s. At the same time, the mutation-induced decrease in excitability, expressed as increase in stimulus current threshold, amounted to ≈15% in absence of S10s, which was reduced to only ≈4% in presence of S10s (Figure 7B), further indicating that excitability could be largely restored by S10s. We also constructed a linear strand with multiple branches and gj set to 10 µS27 (Figure 7C) that was stimulated at rates of 50–150 beats/min (bpm). In this more demanding setting, the mutant INa elicited regular APs in the unbranched part of the strand at 50 and 100 bpm (Figure 7D, left top and middle panels, dark grey solid lines) that were successfully conducted to the branched part of the strand (Figure 7D, right top and middle panels, dark grey solid lines). However, the mutant INa appeared unable to do so at 150 bpm (Figure 7D, bottom panels, dark solid lines). At this higher rate, every other AP in the unbranched part of the strand was not full-blown, resulting in alternating long-short APs (Figure 7D, left bottom panel, dark grey solid lines). The short AP was not successfully conducted to the branched part of the strand and this partial block thus resulted in a 2:1 conduction pattern in the strand (Figure 7D, right bottom panel, dark grey solid lines). Of note, such irregularities were not observed in the unbranched strand (data not shown). Application of S10s completely restored successful conduction (Figure 7D, dark gray dashed lines), illustrating that S10s could prevent (unidirectional) conduction block at higher rates, thereby potentially preventing a principal component of reentrant arrhythmias. S10s overexpression reduces VT inducibility in a computational whole-heart model with SCN5A loss-of-function mutations Following our demonstration of S10s' capability to restore NaV1.5 and to increase CV and prevent conduction block in computer-simulated one-dimensional strands of human ventricular tissue, we proceeded to investigate the impact of S10s overexpression on a more complex and clinically relevant scenario. We hypothesized that S10s gene therapy has the potential to mitigate arrhythmia induction in a human heart affected by NaV1.5 dysfunction. To do so, we employed a three-dimensional (3D) computational whole-heart model and incorporated a cell model representing EP properties of SCN5A loss-of-function mutation (SCN5A+/-). To incorporate S10s gene therapy, we augmented the remaining INa by 80.4% as previously described, resulting in a post-treatment cell model referred to as SCN5A+/- + S10s. As compared to the SCN5A+/- model, the post-treatment SCN5A+/- + S10s model has a higher peak current density (S10s 431.98 vs Control 241.8) and a faster upstroke velocity (S10s 205.5 vs Control 167.5), as seen in Figure 8A-B. Subsequently, we incorporated both the SCN5A+/- and SCN5A+/- + S10s cell models in the whole-heart model with fibrotic tissue and dense scar distribution derived from late-gadolinium enhanced magnetic resonance images (LGE-CMR) (Figure 8C). We conducted simulations to assess the induction of ventricular tachycardia (VT) following a validated in silico rapid pacing protocol as outlined in the method. We then analysed the activation maps generated by these simulations to ascertain the occurrence of VT and to interpret reentrant circuit morphologies. Our simulation revealed a sustained reentrant circuit induced on the inferior RV base for the SCN5A+/- model (Figure 8C). Notably, no sustained reentry was induced in the SCN5A+/- + S10s model. To gain a deeper insight into the role of S10s in preventing VT induction, we conducted a detailed analysis of the reentry simulated under SCN5A+/- and SCN5A+/- +S10s conditions, as depicted in Figure 8D. For both simulations at t0, the wave fronts elicited at the basal-inferior pacing sites encounter preceding wave tails that have been slowed by a region of diffuse fibrosis. This transient conduction block causes the wave fronts to curl clockwise. In the SCN5A+/- case (Figure 8D, top row), the slower CV due to the INa alteration allows the formation of an excitable gap that provides enough time for the tissue to recover and become stimulated again by the curling wave. As a result, a reentrant circuit evolves and anchors around an adjacent region of dense scar. In contrast, the SCN5A+/-+S10 case (Figure 8D, bottom row) has a recovered INa and CV, allowing the wave front to curl fast enough to encounter still refractory tissue. This functional block of the curling wave front prevents the formation of a reentrant circuit. Example 2 In general it is desirable to deliver the smallest possible transcript to carry the desired functional effect. Therefore, we have continued to search for smaller transgenes that can correct sodium channel dysfunction. Materials and methods HEK cells stably expressing human SCN5A were used for the experiments. SCN5A-expressing HEK cells were cultured in 6-well plates and transfected with 1ug of pcDNA plasmids containing either nothing, S10s, or S10s-Volt. Two-to-three days later, transfected cells were used for patch-clamp experiments to measure the peak sodium current. Results We further developed a new version of the SCN10A-short(S10s) which is smaller in size (1.7kb), which is named S10s-Volt or S10s-V (see Figure 9C). Patch- clamp experiment was performed in a HEK transfection system to compare the effect of S10s-Volt and S10s. We found that S10s-Volt significantly increased peak sodium current (INa) comparing to control, and showed no significant difference from S10s (Figure 9). Therefore, S10s-Volt could also be used for the aforementioned applications and its smaller size makes it more versatile in combining with other transgenes in one therapeutic vector. Example 3 Materials and methods Plasmid transfection in HEK cells HEK cells stably expressing human NaV1.5 channels were cultured in 6-well plates in DMEM with Glutamax supplemented with 10% FBS, penicillin- streptomycin and 200 g/ml Zeocin in a 5% CO2 incubator at 37°C. One microgram of pcDNA plasmids expressing either S10s, or S10s-Ultrashort were transfected to HEK cells. Two-to-three days later, transfected cells were disassociated and used for patch-clamp experiments to measure the peak sodium current (INa). Vector production Lentiviral vectors were produced by quadruple transfection of HEK293T cells. Cells were plated and cultured as described above. One day after plating, cells were transfected with 3.2 µg envelope plasmid, 2.2 µg RSV-REV plasmid, 5.8 µg MDLG plasmid (Addgene) and 8.8 µg transfer lentivirus plasmid per dish using linear PEI. Transfer lentivirus plasmid contains either GFP, S10s, or S10s-1kb. Next day, the medium was changed to DMEM-GlutaMax without FBS. Three days after transfection, supernatant was collected and concentrated to 500 µL by buffer exchange to PBS using Amicon Ultra-15100 kDa centrifugal filter units (Millipore). Genomic titer was determined using LV900 titration kit (Applied Biological Materials). Lentiviral transduction in human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hiPSC-CMs) hiPSC-CMs were transduced with lentiviral vectors in culture medium at a multiplicity of infection (MOI) of 10 for 24 hours. Media were then refreshed every other day until being used in the patch-clamp experiments. Whole-cell patch-clamp recordings Ventricular-like hiPSC-CMs were differentiated and dissociated as described previously (Li et al., 2022. J Tissue Eng. 13: 20417314221127908) from the control hiPSC line LUMC0099iCTRL04. Single mouse cells were stored at room temperature for at least 45 min in a modified Tyrode’s solution containing (in mmol/L): NaCl 140, KCl 5.4, CaCl21.8, MgCl21.0, glucose 5.5, HEPES 5.0; pH 7.4 (NaOH). Cells were put into a recording chamber on the stage of an inverted microscope (Nikon Diaphot), and GFP-positive single regularly beating hiPSC-CMs were selected for electrophysiological measurements. APs were recorded with amphotericin-perforated patch-clamp technique using an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA, USA). Voltage control, data acquisition, and analysis were realized with custom software. Signals were low-pass-filtered with a cut-off of 5 kHz and digitized at 20 and 40 kHz for INa and APs, respectively. Potentials were corrected for the calculated liquid junction potential (Barry and Lynch, 1991. J Membr Biol 121: 101-117). Cell membrane capacitance (Cm) was estimated by dividing the time constant of the decay of the capacitive transient in response to 5 mV hyperpolarizing voltage clamp steps from –40 mV by the series resistance. For INa measurements, Cm and series resistance were compensated for by at least 80%. INa measurements INa was characterized at room temperature using a bath solution containing (in mmol/L): NaCl 20, CsCl 120, CaCl21.8, MgCl21.0, glucose 5.5, HEPES 5.0, pH 7.4 (CsOH). Pipettes were filled with solution containing (in mmol/L): NaF 10, CsCl 10, CsF 110, EGTA 11, CaCl21.0, MgCl21.0, Na2ATP 2.0, 10 HEPES, pH 7.2 (CsOH). The INa density and voltage dependence of activation were determined by 50- ms depolarizing pulses (between -100 and +40 mV) from a holding potential of –120 mV. Voltage-dependent inactivation was obtained by measuring the peak currents during a 50-ms test step to -20 mV, which followed a 500 ms prepulse to membrane potentials between - 140 and 0 mV to allow inactivation. The holding potential was –120 mV. All voltage clamp steps were applied with a 5-sec cycle length. Peak INa was defined as the difference between peak and steady-state current. Current density was calculated by dividing the measured currents by Cm. To determine the activation characteristics of INa, current-voltage curves were corrected for differences in driving force and normalized to maximum peak current. AP measurements APs were recorded at 36 ± 0.2°C using the modified Tyrode’s solution as bath solution. Pipette solution contained (in mmol/L): K-gluconate 125, KCl 20, NaCl 5.0, amphotericin-B 0.44, HEPES 10, and pH 7.2 (KOH). APs in hiPSC-CMs were measured with dynamic clamp to inject an in silico IK1 with a current-voltage relationship resembling Kir2.1 channels with a 2 pA/pF outward peak, resulting in quiescent hiPSC-CMs with a RMP of −80 mV or more negative. APs were elicited at 1 Hz for hiPSC-CMs by 3-ms, ≈10–30% suprathreshold current pulses through the patch pipette. Results S10s-Ultrashort increases INa in SCN5A-expressing cells We developed a further version of S10s (named S10s-Ultrashort) by removing amino acids 2-211 of S10s (Figure 10A; Table 2). Patch-clamp experiments were performed in a HEK transfection system to compare the effect of S10s-Ultrashort and S10s. We found that S10s-Ultrashort showed no significant difference from S10s in voltage-current relationship or activation and inactivation (Figure 10B and 10C). Therefore, the smaller size of S10s-Ultrashort makes it more versatile in combining it with other transgenes in one therapeutic vector. S10s-1kb does not increase INa-driven dV/dtmax in hiPSC-CMs We then studied whether another truncated version of S10s (named S10s-1kb) can increase sodium current. S10s-1kb was generated by removing amino acids 2-324 of S10s (Figure 11A). We transduced hiPSC-CMs with lentiviral vectors containing either GFP, S10s or S10s-1kb. Transduced hiPSC-CMs were used for AP measurements with the patch-clamp methodology, 5-7 days post transduction (Figure 11B and 11C). S10s overexpression led to significantly increased dV/dtmax with a 73% increase compared to the GFP group, reflecting significantly increased INa in these cells (Figure 11B). However, S10s-1kb overexpression led to no significant differences in dV/dtmax compared to GFP group (Figure 11B). Moreover, S10s-1kb overexpression significantly increased action potential duration at 90% repolarization (APD90) compared to the other two groups (Figure 11C), which might impose proarrhythmic risks.

Sequences S10s ATGCGGGTGGTGGTGGATGCCCTGGTGGGCGCCATCCCATCCATCATGAATGTCCTCCTCGTCT GCCTCATCTTCTGGCTCATCTTCAGCATCATGGGTGTGAACCTCTTCGCAGGGAAGTTTTGGAG GTGCATCAACTATACCGATGGAGAGTTTTCCCTTGTACCTTTGTCGATTGTGAATAACAAGTCT GACTGCAAGATTCAAAACTCCACTGGCAGCTTCTTCTGGGTCAATGTGAAAGTCAACTTTGATA ATGTTGCAATGGGTTACCTTGCACTTCTGCAGGTGGCAACCTTTAAAGGCTGGATGGACATTAT GTATGCAGCTGTTGATTCCCGGGAGGTCAACATGCAACCCAAGTGGGAGGACAACGTGTACATG TATTTGTACTTTGTCATCTTCATCATTTTTGGAGGCTTCTTCACACTGAATCTCTTTGTTGGGG TCATAATTGACAACTTCAATCAACAGAAAAAAAAGTTAGGGGGCCAGGACATCTTTATGACAGA GGAGCAGAAGAAATACTACAATGCCATGAAGAAGTTGGGCTCCAAGAAGCCCCAGAAGCCCATC CCACGGCCCCTGAACAAGTTCCAGGGTTTTGTCTTTGACATCGTGACCAGACAAGCTTTTGACA TCACAATCATGGTCCTCATCTGCCTCAACATGATCACCATGATGGTGGAGACTGATGACCAAAG TGAAGAAAAGACGAAAATTCTGGGCAAAATCAACCAGTTCTTTGTGGCCGTCTTCACAGGCGAA TGTGTCATGAAGATGTTCGCTTTGAGGCAGTACTACTTCACAAATGGCTGGAATGTGTTTGACT TCATTGTGGTGGTTCTCTCCATTGCGAGCCTGATTTTTTCTGCAATTCTTAAGTCACTTCAAAG TTACTTCTCCCCAACGCTCTTCAGAGTCATCCGCCTGGCCCGAATTGGCCGCATCCTCAGACTG ATCCGAGCGGCCAAGGGGATCCGCACACTGCTCTTTGCCCTCATGATGTCCCTGCCTGCCCTCT TCAACATCGGGCTGTTGCTATTCCTTGTCATGTTCATCTACTCTATCTTCGGTATGTCCAGCTT TCCCCATGTGAGGTGGGAGGCTGGCATCGACGACATGTTCAACTTCCAGACCTTCGCCAACAGC ATGCTGTGCCTCTTCCAGATTACCACGTCGGCCGGCTGGGATGGCCTCCTCAGCCCCATCCTCA ACACAGGGCCCCCCTACTGTGACCCCAATCTGCCCAACAGCAATGGCACCAGAGGGGACTGTGG GAGCCCAGCCGTAGGCATCATCTTCTTCACCACCTACATCATCATCTCCTTCCTCATCGTGGTC AACATGTACATTGCAGTGATTCTGGAGAACTTCAATGTGGCCACGGAGGAGAGCACTGAGCCCC TGAGTGAGGACGACTTTGACATGTTCTATGAGACCTGGGAGAAGTTTGACCCAGAGGCCACTCA GTTTATTACCTTTTCTGCTCTCTCGGACTTTGCAGACACTCTCTCTGGTCCCCTGAGAATCCCA AAACCCAATCGAAATATACTGATCCAGATGGACCTGCCTTTGGTCCCTGGAGATAAGATCCACT GCTTGGACATCCTTTTTGCTTTCACCAAGAATGTCCTAGGAGAATCCGGGGAGTTGGATTCTCT GAAGGCAAATATGGAGGAGAAGTTTATGGCAACTAATCTTTCAAAATCATCCTATGAACCAATA GCAACCACTCTCCGATGGAAGCAAGAAGACATTTCAGCCACTGTCATTCAAAAGGCCTATCGGA GCTATGTGCTGCACCGCTCCATGGCACTCTCTAACACCCCATGTGTGCCCAGAGCTGAGGAGGA GGCTGCATCACTCCCAGATGAAGGTTTTGTTGCATTCACAGCAAATGAAAATTGTGTACTCCCA GACAAATCTGAAACTGCTTCTGCCACATCATTCCCACCGTCCTATGAGAGTGTCACTAGAGGCC TTAGTGATAGAGTCAACATGAGGACATCTAGCTCAATACAAAATGAAGATGAAGCCACCAGTAT GGAGCTGATTGCCCCTGGGCCCGGG S10s-V ATGCGGGTGGTGGTGGATGCCCTGGTGGGCGCCATCCCATCCATCATGAATGTCCTCCTCGTCT GCCTCATCTTCTGGCTCATCTTCAGCATCATGGGTGTGAACCTCTTCGCAGGGAAGTTTTGGAG GTGCATCAACTATACCGATGGAGAGTTTTCCCTTGTACCTTTGTCGATTGTGAATAACAAGTCT GACTGCAAGATTCAAAACTCCACTGGCAGCTTCTTCTGGGTCAATGTGAAAGTCAACTTTGATA ATGTTGCAATGGGTTACCTTGCACTTCTGCAGGTGGCAACCTTTAAAGGCTGGATGGACATTAT GTATGCAGCTGTTGATTCCCGGGAGGTCAACATGCAACCCAAGTGGGAGGACAACGTGTACATG TATTTGTACTTTGTCATCTTCATCATTTTTGGAGGCTTCTTCACACTGAATCTCTTTGTTGGGG TCATAATTGACAACTTCAATCAACAGAAAAAAAAGTTAGGGGGCCAGGACATCTTTATGACAGA GGAGCAGAAGAAATACTACAATGCCATGAAGAAGTTGGGCTCCAAGAAGCCCCAGAAGCCCATC CCACGGCCCCTGAACAAGGGGATCCGCACACTGCTCTTTGCCCTCATGATGTCCCTGCCTGCCC TCTTCAACATCGGGCTGTTGCTATTCCTTGTCATGTTCATCTACTCTATCTTCGGTATGTCCAG CTTTCCCCATGTGAGGTGGGAGGCTGGCATCGACGACATGTTCAACTTCCAGACCTTCGCCAAC AGCATGCTGTGCCTCTTCCAGATTACCACGTCGGCCGGCTGGGATGGCCTCCTCAGCCCCATCC TCAACACAGGGCCCCCCTACTGTGACCCCAATCTGCCCAACAGCAATGGCACCAGAGGGGACTG TGGGAGCCCAGCCGTAGGCATCATCTTCTTCACCACCTACATCATCATCTCCTTCCTCATCGTG GTCAACATGTACATTGCAGTGATTCTGGAGAACTTCAATGTGGCCACGGAGGAGAGCACTGAGC CCCTGAGTGAGGACGACTTTGACATGTTCTATGAGACCTGGGAGAAGTTTGACCCAGAGGCCAC TCAGTTTATTACCTTTTCTGCTCTCTCGGACTTTGCAGACACTCTCTCTGGTCCCCTGAGAATC CCAAAACCCAATCGAAATATACTGATCCAGATGGACCTGCCTTTGGTCCCTGGAGATAAGATCC ACTGCTTGGACATCCTTTTTGCTTTCACCAAGAATGTCCTAGGAGAATCCGGGGAGTTGGATTC TCTGAAGGCAAATATGGAGGAGAAGTTTATGGCAACTAATCTTTCAAAATCATCCTATGAACCA ATAGCAACCACTCTCCGATGGAAGCAAGAAGACATTTCAGCCACTGTCATTCAAAAGGCCTATC GGAGCTATGTGCTGCACCGCTCCATGGCACTCTCTAACACCCCATGTGTGCCCAGAGCTGAGGA GGAGGCTGCATCACTCCCAGATGAAGGTTTTGTTGCATTCACAGCAAATGAAAATTGTGTACTC CCAGACAAATCTGAAACTGCTTCTGCCACATCATTCCCACCGTCCTATGAGAGTGTCACTAGAG GCCTTAGTGATAGAGTCAACATGAGGACATCTAGCTCAATACAAAATGAAGATGAAGCCACCAG TATGGAGCTGATTGCCCCTGGGCCCGG cTNT promoter GCAGTCTGGGCTTTCACAAGACAGCATCTGGGGCTGCGGCAGAGGGTCGGGTCCGAAGCGCTGC CTTATCAGCGTCCCCAGCCCTGGGAGGTGACAGCTGGCTGGCTTGTGTCAGCCCCTCGGGCACT CACGTATCTCCGTCCGACGGGTTTAAAATAGCAAAACTCTGAGGCCACACAATAGCTTGGGCTT ATATGGGCTCCTGTGGGGGAAGGGGGAGCACGGAGGGGGCCGGGGCCGCTGCTGCCAAAATAGC AGCTCACAAGTGTTGCATTCCTCTCTGGGCGCCGGGCACATTCCTGCTGGCTCTGCCCGCCCCG GGGTGGGCGCCGGGGGGACCTTAAAGCCTCTGCCCCCCAAGGAGCCCTTCCCAGACAGCCGCCG GCACCCACCGCTCCGTGGGACCT CMV promoter CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACG TCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGG AGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCC TATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGAC TTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGC AGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGA CGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCC GCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCT CW3SL CGCTCGAGATAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTAT GTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCC GTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTAGTTCTTGCCACGGCGGAACTCAT CGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTG TTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATCTAGCTTTATTTGTGAAATT TGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATT GCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTTTAAAGC CE1-TNNT2core CGGTACCGGCGCGCCAGTAGAAAAACAGCCAAGCTAGGGAGGCTGGGAGGCCAAGCCCCAGATA CCTTACATAGCTCTGCTCAGCCTCTGTCTCATTAGGAACTCCATTTTTAGGATGCAGTTGTTTC AGGCTAAAAATAAATCATGCAATGAATAAAAAAGTTAGATACGACACTGTAGAGGGATTCGCTG ATACAGTCTGTCCGAACGCGTGGTACCACATGCCTGCTTAAAGCCCTCTCCATCCTCTGCCTCA CCCAGTCCCCGCTGAGACTGAGCAGACGCCTCCAGGATCTGTCGGCAG CE2-CCP Ctcagtccattaggagccagtagcctggaagatgtctttacccccagcatcagttcaagtggag cagcacataactcttgccctctgccttccaagattctggtgctgagacttatggagtgtcttgg aggttgccttctgccccccaaccctgctcccagctggccctcccaggcctgggttgctggcctc tgctttatcaggattctcaagagggacagctggtttatgttgcatgactgttccctgcatatct gctctggttttaaatagcttatctgagcagctggaggaccacatgggcttatatggcgtggggt acatgttcctgtagccttgtccctggcacctgccaaaatagcagccaacaccccccacccccac cgccatccccctgccccacccgtcccctgtcgcacattcctccctccgcagggctggctcacca ggccccagcccgggctataaaaagaggcggcactgggcagctgggagacagggacagacgtagg ccaagagaggggaaccagagag CCP NPPAcore Gggctataaaaagaggcggcactgggcagctgggagacagggacagacgtaggccaagagaggg gaaccagagag TNNT2ddd TCCCAGCTGGCCCTCCCAGGCCTGGGTTGCTGGCCTCTGCTTTATCAGGATTCTCAAGAGGGAC AGCTGGTTTATGTTGCATGACTGTTCCCTGCATATCTGCTCTGGTTTTAAATAGCTTATCTGAG CAGCTGGAGGACCACATGGGCTTATATGGCGTGGGGTACATGTTCCTGTAGCCTTGTCCCTGGC ACCTGCCAAAATAGCAGCCAACACCTCCCCTGTCGCACATTCCTCCCTCCGCAGGGCTGGCTCA CCAGGCCCCAGCCCACATGCCTGCTTAAAGCCCTCTCCATCCTCTGCCTCACCCAGTCCCCGCT GAGACTGAGCAGACGCCTCCAGGATCTGTCGGCAG

Claims

Claims 1. An expression construct, comprising a coding sequence for a C-terminal part of a voltage-gated sodium channel alpha subunit, under control of a promoter that directs expression of the C-terminal part of a voltage-gated sodium channel alpha subunit in cardiomyocytes.

2. The expression construct of claim 1, wherein said voltage-gated sodium channel alpha subunit is SCN10A.

3. The expression construct of claim 1 or claim 2, wherein said coding sequence for a C-terminal part of a voltage-gated sodium channel alpha subunit has at least 60% identity to SEQ ID NO:1.

4. The expression construct of any one of claims 1-3, wherein the transcript size of the C-terminal part of a voltage-gated sodium channel alpha subunit is less than 2070 base pairs (bp).

5. The expression construct of any one of claims 1-4, wherein said coding sequence for a C-terminal part of a voltage-gated sodium channel alpha subunit is selected from the amino acid sequences depicted in Table 2.

6. The expression construct of any one of claims 1-5, wherein said expression construct is a viral construct, preferably a recombinant adeno-associated virus (rAAV) construct.

7. The expression construct of any one of claims 1-6, wherein said expression construct is a recombinant AAV6 or an AAV6 variant construct.

8. The expression construct of any one of claims 1-7, wherein the promoter is selected from cardiac troponin C, cardiac troponin I, and cardiac troponin T (cTnT).

9. A pharmaceutical composition, comprising the expression construct of any one of claims 1-8, and a pharmacologically acceptable excipient.

10. The pharmaceutical composition of claim 9, for use in a method of treatment of an individual suffering from a cardiac conduction abnormality and/or an arrhythmia, such as a chronic/recurrent arrhythmia.

11. The pharmaceutical composition for use according to claim 10, wherein the arrhythmia is a bradyarrhythmia, such as a sinoatrial node exit block, a conduction defect such as an atrioventricular block, a left bundle branch block (LBB), a non- specific ventricular conduction abnormality, a progressive cardiac conduction disease (PCCD), and/or a tachyarrhythmia, such as an ischemia-related arrhythmia, a non-ischemia-related arrhythmia (such as associated with Brugada syndrome), a non-ischemic cardiomyopathy and/or a fibrosis-related or scar-related arrhythmia, such as post-myocardial infarction-related arrhythmia.

12. The pharmaceutical composition for use according to claim 10 or 11, wherein the arrhythmia is a malignant ventricular arrhythmia.

13. The pharmaceutical composition for use according to any one of claims 10-12, wherein the individual has previously been treated with an antiarrhythmic drug, by ablation, by stereotactic radiotherapy, or a combination thereof.

14. The pharmaceutical composition for use according to any one of claims 10-13, wherein the pharmaceutical composition is injected or infused into the myocardium, preferably by intramyocardial injection.

15. The pharmaceutical composition for use according to any one of claims 10-14, wherein the individual is further treated with an antiarrhythmic drug.