De novo designed peptide for use in reversing sodium channel dysfunction linked to cardiac arrhythmias and epilepsy

Abstract

Provided are synthetic modulators that precisely tune voltage-gated sodium (NaV) channels. Voltage-gated sodium (NaV) channels initiate the action potential, and whose dysfunction causes late sodium current (I NaL), a commonality that underlies various human diseases including cardiac arrhythmias and epilepsy. Here, using a computational protein design platform, Applicants engineered a de novo peptide modulator, ELIXIR, that binds NaV channels with submicromolar affinity. Functional analysis revealed an unexpected selectivity in inhibiting 'pathogenic' I NaL and confirmed its effectiveness in reversing NaV dysfunction linked to both cardiac arrhythmias and epilepsy in cellular and murine models.

Description

Attorney Docket 44010.196WO-PCT / / CU24196DE NOVO DESIGNED PEPTIDE FOR USE IN REVERSING SODIUM CHANNEL DYSFUNCTION LINKED TO CARDIAC ARRHYTHMIAS AND EPILEPSYRELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 780,040, filed March 28, 2025, to The Trustees of Columbia University, titled “DE NOVO DESIGN OF A PEPTIDE MODULATOR TO REVERSE SODIUM CHANNEL DYSFUNCTION LINKED TO CARDIAC ARRHYTHMIAS AND EPILEPSY,” the entirety of the disclosure of which is hereby incorporated by this reference. The entire contents of the above-identified application are hereby fully incorporated herein by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under OD026389, HL163576, HL141084, and HL142282 awarded by the National Institutes of Health. The government has certain rights in the invention.INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY FILED

[0003] The official copy of the sequence listing is submitted electronically in an.xml file format having the file name “196WO-PCT.xml” created on March 26, 2026, and having a size of 10,838 bytes, and is filed concurrently with the specification. The Sequence Listing is part of the specification and is herein incorporated by reference in its entirety.TECHNICAL FIELD

[0004] The subject matter disclosed herein is generally directed to synthetic peptides capable of binding to the CTD of voltage gated sodium channels and inhibiting late sodium current (INaL) arising from impaired voltage-gated sodium (Nav) channel inactivation.BACKGROUND

[0005] Ion channels are pore-forming proteins that serve as nature’s transistor1. Unlike their silicon counterparts, ion channel activity is dynamic and tightly regulated, allowing cells to adapt their electrical properties to match the complex requirements of life. Indeed, ion channel dysfunction underlies various life-threatening human diseases including cardiac arrhythmias2-4, myotonia5, epilepsy6, migraines, and intellectual disability7. For many voltagegated ion channels, pathophysiological changes in channel function result in altered channelAttorney Docket 44010.196WO-PCT / / CU24196dynamics that disrupt overall electrical signaling. As such, strategies to precisely tune ion channel function are highly desired; yet current approaches, including small-molecule pharmacology, are limited and often have off-target effects8’9. In nature, the functional plasticity of ion channels, a defining feature of these proteins, is often attained through association with diverse auxiliary subunits or regulatory proteins that have evolved to fine tune specific channel properties10. Structural studies over the past several decades have furnished an unprecedented atomistic view of such interactions and shed light onto mechanisms of regulation11'14. Yet, rational engineering of de novo or synthetic ion channel modulators with high specificity has remained intractable.

[0006] A prominent example of this deficiency involves the family of voltage-gated sodium channels (Navi.1-1.9) which are responsible for the generation and propagation of action potentials (APs) in excitable cells including cardiomyocytes and neurons. A hallmark of Nav channel function is the fast activation and inactivation that drives the rapid depolarization of many excitable cells, including neurons and muscle cells. Disruption of Nav channel function is linked to a broad spectrum of clinical phenotypes including cardiac arrhythmias, congenital myotonia, epilepsy, neurodevelopmental disorders and painful neuropathies. Despite their distinct etiology, these disorders are linked to deficits in channel inactivation that can contribute to sustained Na+influx, referred to as either late or persistent Na+current (INaL). Two prominent examples involve SCN5A gene which encodes the Navi.5 in the heart, and SCN8A gene which encodes Navi.6 in the nervous system. Specifically, gain-of-function human variants in Nav1.5 that upregulate INaLare linked to arrhythmogenic long-QT syndrome 3 (LQT3)15, and atrial fibrillation2. Similarly, pathological remodeling associated with heart failure (HF)3or following myocardial ischemia16, have been shown to modify Navi.5 function and elevate ZNaL, resulting in increased risk of cardiac arrhythmias. Increased / \ai. causes a proarrhythmic prolongation of the AP4’17’18(FIG. 1A) and disrupts Na+and Ca2+homeostasis, which sets off a vicious cycle involving CaMKII, ROS, and RyR19. Similarly, in the nervous system, gain-of-function Navi.6 variants are linked to severe forms of epilepsy and sudden unexpected death from epilepsy (SUDEP)20. These mutations disrupt sodium channel inactivation and alter neuronal action potential properties, to ultimately disrupt excitationinhibition balance. Beyond this, increased INaLin related skeletal muscle and neuronal channels are associated with congenital myotonia5, epilepsy, and pain disorders21, pointing to the potential utility of INaLinhibitors. Despite advances in the understanding of pathophysiological mechanisms and the availability of high-resolution structures22’23, devising effective strategies to reverse pathophysiological changes in Nav function has been limited. The prevailingAttorney Docket 44010.196WO-PCT / / CU24196approach has been small molecule INaLblockers such as Ranolazine and GS967 which show promise. However, these drugs largely target a common anti arrhythmic drug binding site in the transmembrane region which may variably inhibit peak Na+current (ZNap) or block other ion channels8. In addition, these small molecules may also have reduced affinity for disease-linked mutant channels, limiting their potential utility. Recently, certain auxiliary proteins such as the intracellular fibroblast growth factor homologous factors (iFGF / FHF) have been shown to inhibit ZNaL with minimal effects on ZNaP24’25. Additionally, peptides derived from iFGF / FHF have been shown to be sufficient to recapitulate these effects. Even still, despite optimization, these peptides are comparatively large and show reduced efficacy for certain channel variants. To this end, major advances in machine-learning based computational protein design have increased the accuracy of de novo protein design while simultaneously reducing computational time26-28. These emerging methods may provide an alternative strategy to rationally design peptide modulators that precisely modify Nav function. If substantiated, these approaches may be generalized to engineer a toolkit of synthetic ion channel regulators.

[0007] Citation or identification of any document in this application is not an admission that such a document is available as prior art to the present invention.SUMMARY

[0008] In one aspect, the present invention provides for a synthetic peptide comprising a sequence of MSPRREALYRGFRA[X]YDVLRH (SEQ ID NO. 1) or a variant thereof with at least 90% sequence identity, wherein X is cysteine or serine. In certain embodiments, the peptide comprises a substitution in a residue other than L8, F12, and Y16. In certain embodiments, the dissociation constant (Kd) of the peptide for the CTD of Nav1.5 is 0.89 ± 0.25 μM. In certain embodiments, the peptide comprises a sequence of MSPRREALYRGFRACYDVLRH (SEQ ID NO. 2). In certain embodiments, the peptide comprises a sequence of MSPRREALYRGFRASYDVLRH (SEQ ID NO. 3). In certain embodiments, the synthetic peptide further comprises a cell penetrating peptide (CPP) sequence that is fused to SEQ ID NO. 1. In certain embodiments, the CPP is a viral tat sequence, for example, YGRKKRRQRRRGSG (SEQ ID NO. 5). In certain embodiments, the peptide comprises a sequence of YGRKKRRQRRRGSGMSPRREALYRGFRASYDVLRH (SEQ ID NO. 4). In certain embodiments, the peptide is modified to increase stability, reduce immunogenicity, and / or optimize cellular uptake. In certain embodiments, the peptide comprises an N-terminal acetyl group and / or a C-terminal amide. In certain embodiments, the peptide is conjugated to a carrier. In certain embodiments, the peptide is conjugated to a label.Attorney Docket 44010.196WO-PCT / / CU24196In certain embodiments, the amino acid sequence of the synthetic peptide is set forth in SEQ ID NO. 2, SEQ ID NO. 3, or SEQ ID NO. 4.

[0009] In another aspect, the present invention provides for a pharmaceutical composition comprising the peptide of any of the preceding claims and a pharmaceutically acceptable carrier.

[0010] In another aspect, the present invention provides for a polynucleotide encoding the synthetic peptide of any embodiment herein.

[0011] In another aspect, the present invention provides for a vector comprising the polynucleotide of any embodiment herein, wherein the vector is selected from a viral vector, a non-viral plasmid, an mRNA delivery construct, a lipid nanoparticle formulation, an exosome-based construct, or a protein nanocarrier. In certain embodiments, the vector is a viral vector selected from the group consisting of an adeno-associated virus (AAV) vector, a lentiviral vector, and an adenoviral vector. In certain embodiments, the viral vector is an AAV vector.

[0012] In another aspect, the present invention provides for a method of treating a disease associated with pathological late sodium current (INaL) arising from impaired voltage-gated sodium (NaV) channel inactivation comprising administering to a subject in need thereof an effective amount of the synthetic peptide or vector of any one of claims 1 to 18, wherein the synthetic peptide selectively inhibits INaL. In certain embodiments, the subject exhibits increased late sodium current (INaL) relative to a wild-type baseline level. In certain embodiments, the subject expresses a disease-linked nonsense or truncation variant of a Nav channel carboxyl-terminal domain. In certain embodiments, the disease is caused by a mutation that reduces the propensity of the inactivation gate to occupy the pore-proximal site of the sodium channel. In certain embodiments, the disease is caused by a mutation of the inactivation gate of a sodium channel. In certain embodiments, the disease is caused by hyperactivation of protein kinase A (PKA) or Ca2+ / calmodulin-dependent protein kinase II (CaMKII). In certain embodiments, the disease is selected from the group consisting of arrhythmogenic long-QT syndrome type 3, cardiac arrhythmia, atrial fibrillation, pathological cardiac remodeling related to heart failure or myocardial ischemia, congenital myotonia, myotonic syndromes, epilepsy, migraines, intellectual disability, painful neuropathies, and chronic pain conditions In certain embodiments, the synthetic peptide or vector is administered to skeletal muscle cells, neurons, or cardiomyocytes. In certain embodiments, the peptide is administered systemically. In certain embodiments, the peptide is delivered by viral vector, mRNA vector, lipid nanoparticle, protein nanocarrier, exosome, or a cell-penetrating peptide.Attorney Docket 44010.196WO-PCT / / CU24196

[0013] In another aspect, the present invention provides for a method of identifying a modulator of binding to a voltage-gated sodium channel carboxyl-terminal domain (CTD), the method comprising: contacting a fluorescently labeled peptide that binds the CTD with a CTD polypeptide under conditions suitable for CTD-peptide complex formation; measuring fluorescence anisotropy of the mixture; contacting the mixture with a test agent; measuring fluorescence anisotropy following contact with the test agent; and identifying the test agent as a modulator of CTD-peptide binding when the fluorescence anisotropy differs from that measured in the absence of the test agent. In certain embodiments, the fluorescently labeled peptide comprises a sequence of SEQ ID NO. 2 or SEQ ID NO. 3. In certain embodiments, a decrease in fluorescence anisotropy indicates that the test agent inhibits binding of the peptide to the CTD. In certain embodiments, an increase in fluorescence anisotropy indicates that the test agent stabilizes or enhances binding of the peptide to the CTD. In certain embodiments, the CTD polypeptide comprises a CTD of Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, Nav1.8, or Nav1.9. In certain embodiments, the CTD polypeptide comprises a disease-associated mutant or truncation variant. In certain embodiments, the test agent comprises a small molecule, peptide, peptidomimetic, antibody fragment, or protein.

[0014] These and other aspects, objects, features, and advantages of the example embodiments will become apparent to those having ordinary skill in the art upon consideration of the following detailed description of example embodiments.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0016] An understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention may be utilized, and the accompanying drawings of which:FIGs. 1A-1H – De novo design of a peptide modulator of Nav1.5. (FIG. 1A) Increased NaL prolongs AP duration and increases the risk of cardiac arrhythmias. (FIG. 1B) Conceptual scheme for rational design of an INaLinhibitor. Left, following channel activation, the inactivation gate (IG) binds to a pore-proximal site to inactivate Nav1.5 (black / gray; IG, orange; EFL, dark red). Middle, in pathophysiology, IG has reduced efficacy for binding the pore-proximal site and remains associated to CTD. Competitive displacement of IG from EFLAttorney Docket 44010.196WO-PCT / / CU24196by the designed peptide named ELIXIR may promote inactivation and inhibit / NaL. (FIG. 1C) Schematic shows the peptide design framework. The initial coordinates of the Nav1.5 CTD (PDBID: 4DCK) served as input into AfDesign to hallucinate a peptide binder (rainbow helix). Structural predictions of ELIXIR-Nav1.5 CTD complex were compared to assess consensus.(FIG. 1D) Top, helical model of ELIXIR along with protein sequence. Residues L8, F12, and Y16 are shown as ball and stick in the helical model. Bottom, ColabFold predicted model of the Nav1.5 CTD - ELIXIR complex. (FIG. 1E) Detailed view of the ColabFold predicted Nav1.5 CTD EFL bound to ELIXIR. ELIXIR residues L8, F12, and Y16 are shown as ball and stick. Vacuum electrostatic surface (positive; blue, neutral, white; negative, red). (FIG. 1F) Equilibrium titration of ELIXIR with the Nav1.5 CTD show saturable binding. Average KDand standard deviation were obtained from 3 replicate titrations. (FIG. 1G) FRET efficiency between Venus-tagged Nav1.5 CTD and Cerulean-tagged ELIXIR (black) or ELIXIR F12E mutant plotted against the concentration of free Venus-tagged Nav1.5 CTD, a.u. is arbitrary units. Compared to wild-type ELIXIR, F12E mutant displays weak FRET. The solid line depicts the fit of a 1: 1 binding isotherm. (FIG. 1H) Bar graph summary of KI,EFF, the relative dissociation constant for ELIXIR-Nav1.5 CTD interaction obtained from FRET 2-hybrid experiments. ELIXIR mutations variably disrupt binding.

[0017] FIGs. 2A-2M – Functional inhibition of INaLby ELIXIR. (FIG. 2A) Schematic shows ELIXIR interaction with the CTD of wild-type Nav1.5. (FIG. 2B) Exemplar multichannel recordings of wild-type Nav1.5 in HEK293 cells in the absence (left) and presence of ELIXIR (right). Channel openings are downward deflections from the zero line (gray). Insets show the late phase enlarged for better visualization. (FIG. 2C) Bar graph summary shows changes in INaLquantified as Rpersist. Wild-type Nav1.5 without (n = 17; 1069 sweeps) and with ELIXIR (n = 9; 924 sweeps). Nav1.5 Δ1864 without ELIXIR (n = 17; 907 sweeps), with ELIXIR (n = 11; 948 sweeps), or with 10 μM ranolazine (n = 10; 848 sweeps). Each bar, mean± SEM. **** / ? <0.001 compared to wild-type, *p = 0.0147 compared to NavL 5 A18641 + ELIXIR by a Kruskal -Wallis test followed by Dunn’s multiple comparison test.(FIGs. 2D-2F) ELIXIR inhibits INaLof Nav1.5 Δ1864. Schematic (FIG. 2D), Exemplar multichannel recordings of Nav1.5 Δ1864 mutant in the absence and presence of ELIXIR (FIG.2E), and with Ranolazine (FIG. 2F). Format as in Panels A-C. (FIG. 2G) Schematic shows weak association of ELIXIR mutant F12E to Nav1.5 Δ1864 mutant. (FIG. 2H) Exemplar multichannel recordings of Nav1.5 Δ1864 mutant with ELIXIR F12E mutant. (FIG. 2I) Bar graph summary of changes in Rpersistof Nav1.5 Δ1864 when expressed with ELIXIR L8A (n =Attorney Docket 44010.196WO-PCT / / CU241969, 852 sweeps), ELIXIR F12E (n = 10, 885 sweeps), or ELIXIR Y16A (n = 8, 671 sweeps). The grey and light blue dashed lines show the average Rpersist of NavL5 Al 864 alone and coexpressed with ELIXIR, respectively. Each bar, mean± SEM. ***p = 0.0008 (+ELIXIR F12E), compared to Nav1.5 Δ1864 in the presence of WT ELIXIR by a Kruskal-Wallis test followed by Dunn’s multiple comparison test. (FIG. 2J) The relative affinity of Venus-tagged Nav1.5 CTD for Cerulean-tagged WT and mutant ELIXIR sequences plotted against the Rpersistof Nav1.5 Δ1864 when co-expressed with the corresponding ELIXIR construct. The trendline between the data points is shown in solid black. (FIG. 2K) Schematic shows Nav1.5 Δ1810 mutant that lacks the ELIXIR-interacting CTD. (FIG. 2L) Exemplar multichannel recordings of Nav1.5 Δ1810 mutant with ELIXIR F12E mutant. (FIG. 2M) Bar graph summary of changes in Rpersistof Nav1.5 Δ1810 when expressed with ELIXIR.

[0018] FIGs. 3A-3F – Generality of late Na+current inhibition by ELIXIR. (FIG. 3A) Schematic showing that either mutations in or phosphorylation of Nav1.5 can lead to increased INaL. Stars designate mutations and blue circles phosphorylation. (FIGs. 3B-3D) Exemplar multichannel recordings of Nav1.5 ΔKPQ (FIG. 3B), Nav1.5 F1759A (FIG. 3C) and WT Nav1.5 with PKAcat (FIG. 3D) in the absence (top) and presence of ELIXIR (bottom). Insets show the late phase enlarged for better visualization. (FIG. 3E) Bar graph summarizes effect of ELIXIR on ZxaL resulting from various mutations and phosphorylation, quantified as / / persist. AKPQ without (n = 17, sweeps = 995) and with ELIXIR (n = 10; 936 sweeps); F1759A without (n = 17; 1083 sweeps) and with ELIXIR (n = 7; 725 sweeps); IQ / AA without (n = 15; 926 sweeps) and with ELIXIR (n = 10; 977 sweeps), WT NavL5 + PKAcat without (n = 10; 710 sweeps) and with ELIXIR (n = 10; 841 sweeps); NavL5 + CaMKILmei) without (n = 11; 1033 sweeps) and with +ELIXIR (n = 10; 1029 sweeps). Each bar, mean ± SEM. ****p < 0.0001 (AKPQ, F1759A), ** / ? = 0.0015 (IQ / AA), * / ? = 0.0101 (+PKACat), *p = 0.0101 (CaMKIfeseo) by a two-tailed Mann-Whitney U-test. (FIG. 3F) Schematic shows ELIXIR modified with a cell-penetrating peptide to facilitate intracellular delivery (ELIXIR-cpp).of Nav1.5 ΔKPQ following treatment with different concentrations of ELIXIR-cpp. The IC50 and n values were obtained through a global fit of the data with a Hill equation. O.E. shows the INaLwhen ELIXIR is overexpressed.

[0019] FIGs. 4A-4K – Selectivity of ELIXIR for pathogenic late Na+current. (FIG.4A) Phylogenetic tree of the nine human Nav CTD sequences along with gain of function phenotypes. (FIG. 4B) ColabFold model of the Nav1.5 CTD+ELIXIR complex. Nav1.5 CTD cartoon thickness and color correspond to the average identity to the Nav1.5 sequence at eachAttorney Docket 44010.196WO-PCT / / CU24196position. ELIXIR is shown as a gray cylinder. (FIGs. 4C and 4D) Exemplar multichannel recordings show effect of ELIXIR on wild-type Nav1.1 (FIG. 4C), Nav1.7 (FIG. 4D) channels. Top, without ELIXIR; Bottom, with ELIXIR. Insets show the channel openings in the late phase enlarged for better visualization. (FIG. 4E) Bar graph summary of effect of ELIXIR on wild-type Nav1.1 and Nav1.7. Each bar, mean ± SEM, Nav1.1 without (n = 9; 912 sweeps) and with ELIXIR (n = 10; 880 sweeps); Nav1.7 without (n = 9; 833 sweeps) and with ELIXIR (n = 8; 765 sweeps). (FIGs. 4F and 4G) Exemplar multichannel recordings show effect of ELIXIR on wild-type (FIG. 4F) and F1698I mutant (FIG. 4G) Nav1.4 channels. Top, without ELIXIR; Bottom, with ELIXIR. Insets show the channel openings in the late phase enlarged for better visualization. (FIG. 4H) Bar graph summarizes effect of ELIXIR on wildtype and mutant NavL4 channels. Each bar, mean ± SEM. NavL4 without (n = 10, 953 sweeps), and with ELIXIR (n = 9, 812 sweeps); NavL4 F1698I mutant (n = 20; 1647 sweeps) and with ELIXIR (n = 10; 956 sweeps). § / ? = 0.0101 (wild-type NavL4 and NavL4 F1698I), *p = 0.0118 (NavL4 F1698I mutant with and without ELIXIR) by a one-way ANOVA followed by a Tukey’s multiple comparisons test. (FIGs. 4I and 4J) Exemplar multichannel recordings show effect of ELIXIR on wild-type (FIG. 4I) and N1768D mutant (FIG. 4J) Nav1.6 channels. Top, without ELIXIR; Bottom, with ELIXIR. Insets show the channel openings in the late phase enlarged for better visualization. (FIG. 4K) Bar graph summarizes effect of ELIXIR on wild-type and mutant NavL6 channels. Each bar, mean ± SEM. NavL6 without (n = 10; 973 sweeps) and with ELIXIR (n = 9; 885 sweeps); NavL6 N1768D mutant without (n = 7; 876 sweeps) and with ELIXIR (n = 10; 1162 sweeps). § / ? = 0.0009 (wild-type NavL6 andNavL6N1768D), *p = 0.0008 (NavL6N1768D mutant with and without ELIXIR) by a one-way ANOVA followed by a Tukey’s multiple comparisons test.

[0020] FIGs. 5A-5G – ELIXIR rescues Nav channelopathy-induced AP abnormalities in mouse brain. (FIG. 5A) AAV9-mediated expression of ELIXIR in neurons of the cerebral cortex, marked with mCherry fluorescence. (FIG. 5B) Exemplar AP recordings of control (black trace) and ELIXIR expressing (blue) neurons from WT Nav1.6 (left) and Nav1.6 N1768D (right) mice. (FIG. 5C) Bar graph summarizing the effect of ELIXIR on the APD90 of control and ELIXIR expressing WT NavL6 or NavL6 N1768D neurons. ***p<0.001; ****pO.0001 by Kruskal-Wallis test followed by Dunn’s multiple comparison test. (FIGs.5D and 5E) Exemplar recordings of APs elicited from either WT (FIG. 5D) or N1768D+ / +(FIG. 5E) with or without ELIXIR in response to 300 pA (top), 600pA (middle), and 900 pA (bottom) current injection. (FIG. 5F) AP rate of WT neurons in the presence or absence ofAttorney Docket 44010.196WO-PCT / / CU24196ELIXIR as a function of applied current. (FIG. 5G) AP rate of Nav1.6 N1768D+ / +neurons in the presence or absence of ELIXIR as a function of applied current.

[0021] FIGs. 6A-6L – ELIXIR inhibits INaLand reduces APD90 in murine ventricular cardiomyocytes. (FIG. 6A) Schematic shows design of cell penetrating ELIXIR (ELIXIR-cpp). We attached a viral tat sequence to the amino terminus of ELIXIR as well as a FITC fluorophore to monitor cellular uptake. (FIG. 6B) Epifluorescence images show uptake of ELIXIR-cpp into freshly dissociated ventricular myocytes from adult mice. Scale bar, 1 μm.(FIG. 6C) Flow cytometric analysis shows concentration dependent uptake of ELIXIR-cpp into aMVMs. (FIG. 6D) Exemplar whole cell recordings show INaLin freshly dissociated aMVM from non-transgenic myocytes (nTG) at baseline (black trace) and following treatment with 3 μM ELIXIR-cpp (blue trace). (FIG. 6E) Exemplar whole cell recordings show INaLinhibition in aMVM from a CaMKIIδc,OE transgenic mice that develops heart failure. Format as in FIG. 6D. (FIG. 6F) Bar graph summarizes the effect of ELIXIR on ZxaL in MVM from nTG versus CaMKIIδC,OEmice. Each bar, mean ± SEM. nTG without (n = 19 from 6 mice) or with ELIXIR-cpp (n = 19 from 6 mice); CaMKII6c OE without (n — 16 from 5 mice) or with ELIXIR-cpp (n = 12 from N = 5 mice). *p = 0.034, ***p < 0.001 by a two-way ANOVA followed by a Tukey’s multiple comparisons test. (FIGs. 6G and 6H) Exemplar AP recordings from nTG (FIG. 6G) and CaMKIIδc,OE (FIG. 6H) aMVMs. Black trace, without ELIXIR; Blue trace, with ELIXIR. (FIG. 61) Bar graph summarizes ELIXIR effect on APD90. Each bar, mean ± SEM. nTG without (n = 25 from 6 mice) or with ELIXIR-cpp (n = 17 from 6 mice) and CaMKIISc., OE without (n = 19 from 5 mice) or with ELIXIR-cpp (n = 9 from 4 mice) **p = 0.004 by two-way ANOVA followed by Tukey’s multiple comparisons test. (FIGs. 6J and 6K) Diary plot shows variability in action potential durations (APD90) for non-transgenic (FIG. 6J) or CaMKIIδc,OE (FIG. 6K) transgenic mice in the presence and absence of ELIXIR.(FIG. 6L) Bar graph summarizes changes in short term variability (STV) of APDs.

[0022] FIGs. 7A-7I – ELIXIR reverses Nav1.5 dysfunction in LQT3 hiPSC-CMs and in F1759Atgmice. (FIG. 7A) Schematic shows AAV9 mediated expression of ELIXIR into cultured LQT3 hiPSC-CMs and F1759Atgmice to determine changes in INaLor electrocardiogram. (FIGs. 7B and 7C) Exemplar multichannel recordings of iPSC-CMs differentiated from a healthy donor (FIG. 7B), or from an LQT3 patient carrying a heterozygous AKPQ mutation (FIG. 7C) transduced with an adenovirus containing GFP (top) or ELIXIR (bottom). Insets show channel openings in the late phase enlarged for better visualization. (FIG. 7D) Bar graph summarizes effect of ELIXIR on INaLin iPSC-CMs. EachAttorney Docket 44010.196WO-PCT / / CU24196bar, mean ± SEM. HD iPSC with GFP (n = 9; 781 sweeps from N = 2 differentiations) or ELIXIR (n = 9; 803 sweeps from N = 2 differentiations); LQT3(AKPQ) iPSC-CMs with GFP (n = 21; 1361 sweeps from N = 2 differentiations) or ELIXIR (n = 11; 885 sweeps from N = 2 differentiations). **** / ?<0.0001 by a two-tailed Mann-Whitney U-test. (FIG. 7E) Confocal image shows mCherry expression as a marker of ELIXIR production by ventricular myocytes freshly isolated from adult Nav1.5 F1759A mice. Scale bar, 50 μm. (FIG. 7F) Exemplar multichannel recordings from ventricular myocytes isolated from NavL5 F1759A mice infected with an AAV containing GFP (top) or ELIXIR (bottom). Insets show channel openings in the late phase enlarged for better visualization. (FIG. 7G) Bar graph summarizes effect of ELIXIR on AjaL of NavL5 F1759A aMVMs. Each bar, mean ± SEM. NavL5 F1759A mice injected with GFP AAV (n = 31; 2,181 sweeps from N = 6 mice) or ELIXIR AAV (n = 33; 2,396 sweeps from N = 6 mice). **** / ?<0.0001 by a two-tailed Mann-Whitney U-test. (FIG.7H) Exemplar limb-lead surface ECGs of isoflurane-anesthetized F1759A mice infected with an AAV containing GFP (top) or ELIXIR (bottom). (FIG.71) Bar graph comparison of the RR (FIG. 7H), and QRS and QTc (FIG. 71) duration of isoflurane-anesthetized F1759A mice infected with an AAV containing GFP (N = 7 mice) or ELIXIR (N = 5 mice). **** / ?<().0001 by nested t-test.

[0023] FIGs. 8A-8C – Conceptual schematic of the mechanism of action of the designed peptide. (FIG. 8A) Following channel activation, the inactivation gate (IG) dissociates from the EF-hand like region (EFL, dark red) allowing it to interact with a pore-proximal site and inactivate Nav1.5 (black / gray). (FIG. 8B) In pathophysiology, IG has reduced efficacy for binding the pore-proximal site and remains associated to CTD, which allows for the continued flow of Na+into the cell. (FIG. 8C) By competitively displacing the IG from the EFL, the designed peptide (ELIXIR) may promote channel inactivation and inhibit INaL.

[0024] FIGs. 9A-9J – Models of the ELIXIR-Nav1.5 CTD complex. (FIG. 9A) Comparison of a high-resolution structure of the Nav1.5 CTD (PDB ID: 4DCK) to computational models of the Nav1.5 CTD-ELIXIR complex generated with ColabFold, ESMFold, OmegaFold, and RoseTTAFold. The Nav1.5 CTD is shown as gray cylinders and the ELIXIR peptide is shown in rainbow (blue, N-terminus; red, C-terminus). The models were aligned using residues 1786 - 1892 of the model generated with ColabFold. (FIGs. 9B, 9D, 9F, and 9H) Contacts of Structural Units analysis of the ELIXIR-Nav1.5 CTD models generated with ColabFold (FIG. 9B), ESMFold (FIG. 9D), OmegaFold (FIG. 9F) andAttorney Docket 44010.196WO-PCT / / CU24196RoseTTAFold (FIG. 9H). Nav1.5 residues within 6 Å (within 4.5 Å colored blue) of the ELIXIR peptide are listed. (FIGs. 9C, 9E, 9G, and 91) Residues of the Navi.5 CTD (black sticks) within 6 Å of the ELIXIR peptide shown in the ELIXIR-Nav1.5 CTD complexes generated with ColabFold (FIG. 9C), ESMFold (FIG. 9E) OmegaFold (FIG. 9G), RoseTTAFold (FIG. 9I). The Nav1.5 CTD is shown as gray cylinders and ELIXIR as a light blue (actinium) α-helix. Models were aligned as in A. (FIG. 9J) ColabFold generated models of the NavL5 CTD + NavL5 IG + calmodulin (CaM) complex without (left) and with (right) ELIXIR. CaM is shown as pale green cylinders and the NavL5 IG as a light orange cylinder, the Nav1.5 CTD and ELIXIR are colored as in FIG. 9C. Models were aligned as in FIG. 9A.

[0025] FIGs. 10A-10F - ELIXIR sequence alignment and effect on whole-cell properties. (FIG. 10A) alignment of the human Nav1.5 IG (top, hNav1.5 IG), Nannochloropsis gaditana 50s ribosome (middle, nc50s Rib) and Leptonychotes weddellii DNA replication factor CDT1 (bottom, IwCDTl) sequences with ELIXIR. Similar residues are shown in bold, and identical residues are colored red and shown in bold. (FIGs. 10B and 10C) FRET efficiency between Cerulean-tagged ELIXIR L8A (FIG. 10B) or ELIXIR Y16A (FIG. 10C) and Venus-tagged Nav1.5CTD plotted against the concentration of free Venus-tagged Nav1.5CTD, a.u. is arbitrary units. The solid lines depict the fit of a 1: 1 binding isotherm. (FIG. 10D) Peak current density of wild-type Nav1.5 in the absence (gray) or presence of ELIXIR (light blue). Data are shown as the mean ± SEM, with the number of replicate experiments (n) listed in parentheses. Exemplar currents are for wild-type Nav1.5 alone (gray) and with ELIXIR (light blue) are shown above the plot. (FIG. 10E) Steady state inactivation relationships for wild-type Nav1.5 in the absence (gray) and presence of ELIXIR (light blue). Data are shown as the mean ± SEM, with the number of replicate experiments (n) shown in parentheses. Exemplar currents are for wild-type Nav1.5 alone (gray) and with ELIXIR (light blue) are shown above the plot. (FIG. 10F) Fractional recovery of wild-type Nav1.5 from inactivation in the absence (gray) and presence of ELIXIR (light blue). Data are shown as the mean ± SEM, with the number of replicate experiments (n) listed. Exemplar currents are for wild-type NavL5 alone (gray) and with ELIXIR (light blue) are shown above the plot.

[0026] FIGs. 11A-11J - Effect of Nav p-subunits and mutations on ELIXIR. (FIGs.11A and 11B) Comparison of the ensemble average NPoof Nav1.5 wild-type (FIG. 11A) and Δ1864 (FIG. 11B) in the absence (black) or presence (light blue) of ELIXIR. The lower panel shows an expanded view near the baseline. (FIG. 11C) Schematic of Nav1.5 in the open state with ELIXIROriginal(light red) and ELIXIRfinal(light blue) (top). Exemplar multichannelAttorney Docket 44010.196WO-PCT / / CU24196recording of Nav1.5 Δ1864 co-expressed with the initial ELIXIR peptide sequence (ELIXIROriginal; MSPRREALYRGFRACYDVLRH, SEQ ID NO. 2) (bottom). Inset shows the late phase enlarged for better visualization. (FIG. 11D) Comparison of the ensemble average NPo of Navi.5 Al 864 in the absence (black) and presence of ELIXIRoriginai (light red). The lower panel shows an expanded view near the baseline. (FIG. 11E) Bar graph summarizing the effect of ELIXIROriginalon the INaLof Nav1.5 Δ1864 (n = 9, 632 sweeps). Data are represented as mean ± SEM. The gray and light blue circles show the average (± SEM) Rpersist of the channel alone or with ELIXIR, respectively. (FIGs. 11F and 11G) Exemplar multichannel recordings of Nav1.5 wild-type (FIG. 11F) and Δ1864 (FIG. 11G) in the absence (top) or presence (bottom) of ELIXIR. Insets show the late phase enlarged for better visualization. (FIGs. 11H and 11I) Comparison of the ensemble average NPoof Nav1.5 wildtype (FIG. 11H) or Δ1864 (FIG. 11I) co-expressed with β1 and β4 in the absence (black) or presence (light blue) of ELIXIR. The lower panel shows an expanded view near the baseline.(FIG. 11J) Bar graph summarizes effect of β1 / β4-subunits and ELIXIR on INaLof Nav1.5 wildtype and Δ1864. Each bar, mean ± SEM. Nav1.5 with β1 / β4 alone (n = 10, 953 sweeps) or with β1 / β4 and ELIXIR (n = 11; 921 sweeps), Nav Δ1864 with β1 / β4 alone (n = 9, 775 sweeps) or with β1 / β4 and ELIXIR (n = 10; 960 sweeps). **p = 0.0021 (Nav1.5 Δ1864+β1 / β4+ELIXIR) compared to Nav1.5 Δ1864+β1 / β4 by a Brown-Forsythe test followed by Dunn’s multiple comparison test.

[0027] FIGs. 12A-12G - Mutations in ELIXIR reduce INaLinhibition. (FIG. 12A) Comparison of the ensemble average NPoof Nav1.5 Δ1864 in the absence (black) and presence (pink) of 10 pM ranolazine. The lower panel shows an expanded view near the baseline. (FIGs.12B and 12C) Top: vacuum electrostatic surface (positive; blue, neutral, white; negative, red) of the Nav1.5 CTD EFL bound to ELIXIR with L8 (FIG. 12B) or Y16 (FIG. 12C) shown as ball-and-stick (top). Bottom: exemplar multichannel recordings of Nav1.5 Δ1864 in presence of ELIXIR L8A (FIG. 12B) or ELIXIR Y16A (FIG. 12C). Insets show the late phase enlarged for better visualization. (FIGs. 12D and 12F) Comparison of the ensemble average NPoof Nav1.5 Δ1864 in the absence (black) and presence of ELIXIR L8A (FIG. 12D, cyan), ELIXIR F12E (FIG. 12E, green) or ELIXIR Y16A (FIG. 12F, orange). The lower panel shows an expanded view near the baseline. (FIG. 12G) Comparison of the ensemble average NPoof Nav1.5 Δ1810 in the absence (black) and presence of ELIXIR (light blue). The lower panel shows an expanded view near the baseline.Attorney Docket 44010.196WO-PCT / / CU24196

[0028] FIG. 13A-13K - General inhibition of INaLby ELIXIR. (FIG. 13A - FIG. 13B) Comparison of the ensemble average NPoof Nav1.5 ΔKPQ (FIG. 13A) and Nav1.5 F1759A (FIG. 13B) in the absence (black) and presence of ELIXIR (light blue). The lower panel shows an expanded view near the baseline. (FIG. 13C) Exemplar multichannel recordings of Nav1.5 IQ / AA in the absence (top) and presence (bottom) of ELIXIR. Insets show the late phase enlarged for better visualization. (FIG. 13D - FIG. 13E) Comparison of the ensemble average NPoof Nav1.5 IQ / AA (FIG. 13D) and WT Nav1.5+PKACat(FIG. 13E) in the absence (black) and presence of ELIXIR (light blue). The lower panel shows an expanded view near the baseline. (FIG. 13F) Exemplar multichannel recordings of WT Nav1.5+CaMKIIT286Din the absence (top) and presence (bottom) of ELIXIR. Insets show the late phase enlarged for better visualization. (FIG. 13G) Comparison of the ensemble average NPoof WT Nav1.5+CaMKIIT286D(FIG. 13G) in the absence (black) and presence of ELIXIR (light blue). The lower panel shows an expanded view near the baseline. (FIG. 13H - FIG. 13K) Exemplar multichannel recordings (top) and comparison of the ensemble average NPo (bottom) of Nav1.5 ΔKPQ following treatment with 0.01 μM ELIXIR-cpp (FIG. 13H), 0.1 μM ELIXIR-cpp (FIG. 13I), 1 μM ELIXIR-cpp (J) and 10 μM ELIXIR-cpp (FIG. 13K). In each panel the ensemble average NPoof Nav1.5 ΔKPQ in the absence of ELIXIR is shown in black, with ELIXIR overexpressed in gray and following treating with the designated concentration of ELIXIR-cpp in light blue. In the upper panel the insets show the late phase enlarged for better visualization. The bottom panel shows an expanded view, near the baseline, of the ensemble average NPo.

[0029] FIGs. 14A-14N - ColabFold models of ELIXIR-Nav CTD complexes. (FIGs.14A-14H) ColabFold generated models of ELIXIR (light blue) bound to the CTD (gray cylinders) of Nav1.1 (FIG. 14A), Nav1.2 (FIG. 14B), Nav1.3 (FIG. 14C), Nav1.4 (FIG. 14D), Nav1.6 (FIG. 14E), Nav1.7 (FIG. 14F), Nav1.8 (FIG. 14G), and Nav1.9 (FIG. 14H). ELIXIR residues L8, F12, and Y16 are shown as ball and stick. The structures were aligned to residues 1788 - 1893 of Nav1.5. For clarity only the EFL region (residues 1788-1893 of Nav1.5) of each CTD is shown. (FIGs. 14I-14N) Comparison of the ensemble average NPoof WT Nav1.1 (FIG. 14I), WT Nav1.7 (FIG. 14J), WT Nav1.4 (FIG. 14K), Nav1.4 F1698I (FIG. 14L), WT Nav1.6 (FIG. 14M) and Nav1.6 N1768D (FIG. 14N) in the absence (black) and presence of ELIXIR (light blue). The lower panel shows an expanded view near the baseline.

[0030] FIGs. 15A-15F - Effect of ELIXIR on WT Nav1.6 and Nav1.6 N1768D neuronal AP properties. (FIG. 15A) Exemplar AP recordings of control neurons from WT Nav1.6 (black) and Nav1.6 N1768D (red) mice. (FIG. 15B) Bar graph summarizing the effect of ELIXIR on the AP half width of control and ELIXIR expressing neurons from WT NavL6 (control n = 8, N = 3 mice; ELIXIR n = 13, N = 3 mice) or NavL6 N1768D (control n = 8, N = 2 mice; ELIXIR n = 7, N = 2 mice) mice. Each bar, mean ± SEM. ****p < 0.0001 (WT NavL6 control), *p = 0.0330 (NavL6 N1768D ELIXIR) compared toNavL6 N1768D control by a two-way ANOVA followed by a Tukey’s multiple comparisons test. (FIG. 15C) Bar graph summarizing the effect of ELIXIR on the maximum rise rate of APs from control and ELIXIR expressing neurons from WT Nav1.6 (control n = 8, N = 3 mice; ELIXIR n = 13, N = 3 mice) and Nav1.6 N1768D (control n = 8, N = 2 mice; ELIXIR n = 7, N = 2 mice) mice. Each bar, mean ± SEM. *p = 0.0296 (WT Nav1.6 control) compared to Nav1.6 N1768D control by a two-way ANOVA followed by a Tukey’s multiple comparisons test. (FIG. 15D) Bar graph summarizing the effect of ELIXIR on the maximum fall rate of APs from control and ELIXIR expressing neurons from WT NavL6 (control n = 8, N = 3 mice; ELIXIR n = 13, N = 3 mice) and NavL6 N1768D (control n = 8, N = 2 mice; ELIXIR n = 7, N = 2 mice) mice. Each bar, mean ± SEM. ****p < 0.0001 (WT NavL6 control), *p = 0.0320 (NavL6 N1768D ELIXIR) compared to NavL6 N1768D control by a two-way ANOVA followed by a Tukey’s multiple comparisons test. (FIG. 15E) Bar graph summarizing the effect of ELIXIR on the number of APs fired at 1 nA from control and ELIXIR expressing neurons from WT NavL6 (control n = 8, N = 3 mice; ELIXIR n = 13, N = 3 mice) and NavL6 N1768D (control n = 8, N = 2 mice; ELIXIR n = 7, N = 2 mice) mice. Each bar, mean± SEM. ***p < 0.0004 (WTNavL6 control), *p = 0.0104 (NavL6 N1768D ELIXIR) compared to NavL6 N1768D control by a two-way ANOVA followed by a Tukey’s multiple comparisons test. (FIG. 15F) Bar graph summarizing the effect of ELIXIR on the depolarization block threshold of control and ELIXIR expressing neurons from WT NavL6 (control n = 8, N = 3 mice; ELIXIR n = 13, N = 3 mice) and NavL6 N1768D (control n = 8, N = 2 mice; ELIXIR n = 7, N = 2 mice) mice. Each bar, mean ± SEM. *p < 0.01884 (WT NaV1.6 control), *p = 0.0289 (NaV1.6 N1768D ELIXIR) compared to NavL6 N1768D control by a two-way ANOVA followed by a Tukey’s multiple comparisons test.

[0031] FIGs. 16A-16H - Comparison of nTG and CaMKIIδC,OEmurine heart morphology. (FIGs. 16A-16C) bar graph comparison of the body weight (FIG. 16A), heart weight (FIG. 16B), and heart weight body weight ratio (FIG. 16C) of nTG (N = 6 mice) and CaMKIIδC,OE(N = 5 mice) mice. Data are shown as mean ± SEM. **p = 0.004 for both heart weight and heart weight / body weight ratio by a Mann-Whiteny U test. (FIG. 16D) bar graphAttorney Docket 44010.196WO-PCT / / CU24196comparison of the membrane capacitance of cardiomyocytes isolated from nTG (n = 80 cells, N = 6 mice) and CaMKIIδC,OE(red, n = 56 cells, N = 5 mice) mice. *** p < 0.001 by a Mann-Whiteny U test. (FIGs. 16E and 16F), Comparison of the dv / dt (FIG. 16E) and resting membrane potential (FIG. 16F) of cardiomyocytes isolated from nTG mice in the absence (n = 25 cells, N = 6 mice) and presence (n = 17 cells, N = 6 mice) of ELIXIR-cpp or from CaMKIIδC,OEmice in the absence (n = 19 cells, N = 5 mice) or presence (n = 9 cells, N = 4 mice) of +ELIXIR-cpp. (FIGs. 16G and 16H) Comparison of the ensemble average NP0of Healthy Donor (FIG. 16G), LQT3 (AKPQ) (FIG. 16H) iPSC-CMs, infected with adenovirus containing GFP (black) or ELIXIR (light blue). The lower panel shows an expanded view near the baseline.

[0032] The figures herein are for illustrative purposes only and are not necessarily drawn to scale.DETAILED DESCRIPTION OF THE EXAMPLE EMBODIMENTSGeneral Definitions

[0033] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Definitions of common terms and techniques in molecular biology may be found in Molecular Cloning: A Laboratory Manual, 2ndedition (1989) (Sambrook, Fritsch, and Maniatis); Molecular Cloning: A Laboratory Manual, 4thedition (2012) (Green and Sambrook); Current Protocols in Molecular Biology (1987) (F.M. Ausubel et al. eds.); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (1995) (M.J. MacPherson, B.D. Hames, and G.R. Taylor eds.): Antibodies, A Laboratory Manual (1988) (Harlow and Lane, eds.): Antibodies A Laboratory Manual, 2ndedition 2013 (E.A. Greenfield ed.); Animal Cell Culture (1987) (R. I. Freshney, ed.); Benjamin Lewin, Genes IX, published by Jones and Bartlet, 2008 (ISBN 0763752223); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0632021829); Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 9780471185710); Singleton etal., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N. Y. 1994), March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, N. Y. 1992); Marten H. Hofker and Jan van Deursen, Transgenic Mouse Methods and Protocols, 2ndedition (2011);Attorney Docket 44010.196WO-PCT / / CU24196and Yeast Genetics: Methods and Protocols (Jeffrey S. Smith & Daniel J. Burke, eds., Methods in Molecular Biology, vol. 1205, Humana Press / Springer, 2014).

[0034] As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise.

[0035] The term “optional” or “optionally” means that the subsequent described event, circumstance or substituent may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0036] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

[0037] The terms “about” or “approximately” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of + / -10% or less, + / -5% or less, + / -1% or less, and + / -0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” or “approximately” refers is itself also specifically, and preferably, disclosed.

[0038] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.

[0039] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. These terms also include proteins that are post-translationally modified through reactions that include glycosylation, acetylation and phosphorylation. The term “at least a portion” of a polypeptide means a portion having the minimal size characteristics of such sequences, or any larger fragment of the full length molecule, up to and including the full length molecule. For example, a portion of a polypeptide may be 4 to 25 amino acids, or may be 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, and so on, up to a full length polypeptide.

[0040] The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified. Unnatural amino acids areAttorney Docket 44010.196WO-PCT / / CU24196not encoded by the genetic code and can, but do not necessarily have the same basic structure as a naturally occurring amino acid. “Amino acid analogs” refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs may have modified R groups (e.g., norleucine) or modified peptide backbones but retain the same basic chemical structure as a naturally occurring amino acid. “Amino acid mimetics” refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

[0041] Amino acids may be referred to by either the three letter symbols or by the one-letter symbols recommended by the IUPAC, the IUAPC letter code are as follows: G = Glycine; A = Alanine; L = Leucine; M = Methionine; F = Phenylalanine; W = Tryptophan; K = Lysine;Q = Glutamine; E = Glutamic Acid; S = Serine; P = Proline; V = Valine; I = Isoleucine; C = Cysteine; Y = Tyrosine; H = Histidine; R = Arginine; N = Asparagine; D = Aspartic Acid; T = Threonine.

[0042] As used herein, the term “conservative substitution” refers to the replacement of one amino acid residue with another amino acid residue having similar physicochemical properties, such that the substitution does not substantially alter the structure, stability, binding properties, or biological activity of the peptide. In various embodiments, conservative substitutions are those in which an amino acid is replaced by another amino acid within the same functional or structural class, including but not limited to substitutions that preserve side-chain charge, polarity, hydrophobicity, size, or hydrogen-bonding capacity. Such substitutions are well recognized in the art as being less likely to disrupt protein folding or functional interactions. In some embodiments, a conservative substitution preserves key interaction surfaces involved in binding to a target protein, such as a voltage-gated sodium channel carboxyl-terminal domain (CTD). In example embodiments, conservative substitutions may be introduced at positions that are not essential for binding or activity, while residues identified as critical for target engagement are preserved (e.g., F12). For example, substitutions may be excluded at positions corresponding to residues that directly contact the target protein or contribute substantially to binding affinity. In some embodiments, conservative substitutions include substitutions between cysteine and serine, particularly where maintenance of side-chain size and polarity is desired while reducing susceptibility to oxidation or disulfide bond formation. In other embodiments, conservative substitutions include alanine scanning substitutions used to probe structure-function relationships, provided that biologicalAttorney Docket 44010.196WO-PCT / / CU24196activity is retained. Accordingly, unless otherwise indicated, reference to a peptide sequence includes variants comprising one or more conservative amino acid substitutions that do not materially affect the peptide’ s ability to bind its target or modulate sodium channel inactivation.

[0043] Various embodiments are described hereinafter. It should be noted that the specific embodiments are not intended as an exhaustive description or as a limitation to the broader aspects discussed herein. One aspect described in conjunction with a particular embodiment is not necessarily limited to that embodiment and can be practiced with any other embodiment(s). Reference throughout this specification to “one embodiment”, “an embodiment,” “an example embodiment,” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment,” “in an embodiment,” or “an example embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment - but they may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention. For example, in the appended claims, any of the claimed embodiments can be used in any combination.

[0044] Reference is made to Mahling R, Hegyi B, Cullen ER, Cho TM, Rodriques AR, Fossier L, Yehya M, Yang L, Chen BX, Katchman AN, Chakouri N, Ji R, Wan EY, Kushner J, Marx SO, Ovchinnikov S, Makinson CD, Bers DM, Ben-Johny M. De novo design of a peptide modulator to reverse sodium channel dysfunction linked to cardiac arrhythmias and epilepsy. Cell. 2025 Oct 30;188(22):6170-6185.el9.

[0045] All publications, published patent documents, and patent applications cited herein are hereby incorporated by reference to the same extent as though each individual publication, published patent document, or patent application was specifically and individually indicated as being incorporated by reference.OVERVIEW

[0046] Embodiments disclosed herein provide synthetic peptides capable of binding to the CTD of voltage gated sodium channels and inhibiting late sodium current (INaL) arising from impaired voltage-gated sodium (Nav) channel inactivation.Attorney Docket 44010.196WO-PCT / / CU24196

[0047] Ion channels orchestrate electrical signaling in excitable cells. In nature, ion channel function is customized by modulatory proteins that have evolved to fulfill distinct physiological needs. Yet, engineering synthetic modulators that precisely tune ion channel function is challenging. One example involves the voltage-gated sodium (Nav) channel that initiates the action potential, and whose dysfunction causes late sodium current (INaL), a commonality that underlies various human diseases including cardiac arrhythmias and epilepsy. Here, using a computational protein design platform, we engineered a de novo peptide modulator, ELIXIR, that binds Nav channels with submicromolar affinity. Functional analysis revealed an unexpected selectivity in inhibiting ‘pathogenic’ ZNaL and confirmed its effectiveness in reversing Nav dysfunction linked to both cardiac arrhythmias and epilepsy in cellular and murine models. These findings exemplify the efficacy of de novo protein design for engineering synthetic ion channel modulators and sets the stage for rational design of future therapeutic approaches.

[0048] Specifically, focusing on cardiac Navi.5, we here used de novo protein design to generate a peptide modulator that selectively inhibits pathogenic INaLby enhancing a native mechanism of channel inactivation. To do so, we implemented a protocol for peptide hallucination within the ColabDesign framework29’30and leveraged this to generate a short 21-aa peptide targeting the Nav 1.5 carboxy -terminal domain (CTD), an intracellular channel domain that serves as regulatory hub. In vitro binding analysis confirmed interaction of candidate modulatory peptide with the Navi.5 CTD. In depth electrophysiological analysis confirmed the functionality of the de novo designed peptide and revealed an unexpected selectivity of the designed peptide for pathogenic INaLof multiple members of the Nav channel family. We further demonstrate the effectiveness of this peptide in both neuronal and cardiac settings. Our findings highlight the utility of de novo protein design in the development of synthetic ion channel modulators that tune specific aspects of ion channel function in physiology and pathophysiology.Nav channel carboxyl-terminal domain (CTD) binding peptides

[0049] In example embodiments, synthetic peptides are provided that bind to the CTD of voltage gated sodium channels (Nav) (e.g., ELIXIR). The inactivation gate (IG) of a Nav binds to a pore-proximal site to inactivate the channel. The IG also binds an acidic EF-hand like region (EFL) in the C-terminal domain (CTD) of the channel. In example embodiments, a Al 865 truncation of the channel keeps the EFL, but loses the rest of the CTD that is essential for stabilizing and positioning the inactivation gate. Without these additional structuralAttorney Docket 44010.196WO-PCT / / CU24196elements, the channel cannot complete the normal inactivation process, leading to pathological late sodium current (INaL). In example embodiments, the peptide binds to the EFL allowing proper binding of the IG to the pore-proximal site. In example embodiments, the peptide inhibits inactivation-gate release from a pore-proximal site to inhibit INaLwith minimal effects on INaP. In example embodiments, the peptide comprises a sequence of MSPRREALYRGFRAXYDVLRH (SEQ ID NO. 1) or a variant thereof with at least 90% sequence identity, wherein X is cysteine or serine. In example embodiments, the peptide comprises a substitution in a residue other than F12. In example embodiments, the peptide comprises a substitution in a residue other than L8. In example embodiments, the peptide comprises a substitution in a residue other than Y16. In preferred embodiments, any substitution is a conservative substitution. In example embodiments, the peptide comprises a sequence of MSPRREALYRGFRACYDVLRH (SEQ ID NO. 2). In example embodiments, the peptide comprises a sequence of MSPRREALYRGFRASYDVLRH (SEQ ID NO. 3).

[0050] In example embodiments, the peptide may include addition residues at the N- or C-terminus that do not interfere with binding to the EFL. The peptide may also include modifications that do not interfere with binding to the EFL. In example embodiments, the peptide is modified to increase stability, reduce immunogenicity, and / or optimize cellular uptake.

[0051] Proteins or peptides may be made by any technique known to those of skill in the art, including the expression of proteins, polypeptides or peptides through standard molecular biological techniques, in vitro translation, or the chemical synthesis of proteins or peptides. Peptides can be readily synthesized chemically utilizing reagents that are free of contaminating bacterial or animal substances (Merrifield RB: Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J. Am. Chem. Soc. 85:2149-54, 1963). Alternatively, a nucleic acid (e.g., a polynucleotide) encoding a peptide of the invention may be used to produce the peptide in vitro. Many exemplary systems exist that one skilled in the art could utilize (e.g., Retie Lysate IVT Kit, Life Technologies, Waltham, MA). An expression vector capable of expressing a polypeptide can also be prepared. Expression vectors for different cell types are well known in the art and can be selected without undue experimentation.

[0052] The present invention further contemplates the use of nucleic acid molecules as vehicles for producing therapeutic peptides for administration to a subject in vivo. In example embodiments, a polynucleotide encoding a peptide described herein is administered in the formAttorney Docket 44010.196WO-PCT / / CU24196of a DNA vector, RNA vector, viral vector, or messenger RNA (mRNA) construct that directs intracellular expression of the peptide in target cells. Such nucleic acid-based delivery systems are useful for intracellularly acting peptides and protein replacement therapies and may be formulated to promote transient or sustained peptide expression.Delivery of peptides

[0053] The present invention further contemplates delivery of therapeutic peptides, including ELIXIR peptides and variants thereof, by a variety of peptide- and nucleic-acid-based delivery modalities that enable intracellular access to target tissues. Such delivery modalities include direct administration of the peptide, delivery using cell-penetrating peptides (CPPs), and administration of nucleic acids encoding the peptide, including DNA, RNA, and messenger RNA (mRNA) constructs. The delivery modalities described herein are suitable for administration to cardiomyocytes, skeletal muscle cells, neurons, and peripheral sensory neurons, and may be adapted based on the disease indication, target tissue, and desired duration of therapeutic effect.

[0054] In various embodiments, the ELIXIR peptide, ELIXIR-derived variants, functional fragments, or related sodium-channel-modulating peptides are administered to a subject using any suitable route or delivery modality that achieves a therapeutically effective intracellular concentration in target tissues. Because ELIXIR acts intracellularly on the Nav channel carboxyl-terminal domain, administration strategies may be selected to optimize tissue exposure, cellular uptake, pharmacokinetic properties, or biodistribution, depending on the disorder being treated.Direct Peptide Delivery

[0055] In example embodiments, the peptide is administered directly in a pharmaceutically acceptable carrier by parenteral, local, or systemic routes, including intravenous, intramuscular, subcutaneous, intrathecal, intracardiac, intramyocardial, or perineural administration. The peptide may be chemically modified, for example by N-terminal acetylation, C-terminal amidation, cyclization, lipidation, or PEGylation, to enhance stability, bioavailability, or cellular uptake.

[0056] In example embodiments, the peptide is administered directly to a subject in a pharmaceutically acceptable carrier. Suitable formulations include aqueous buffers, isotonic saline, sterile water, or biocompatible excipients designed to preserve peptide stability andAttorney Docket 44010.196WO-PCT / / CU24196solubility. In some embodiments, lyophilized formulations are reconstituted immediately prior to use.

[0057] In some embodiments, the peptide is delivered by parenteral injection, including but not limited to intravenous (IV) bolus or slow-push injection, IV infusion, including continuous or intermittent infusion, intra-arterial injection, intramuscular (IM) injection, subcutaneous (SC) injection, intraperitoneal (IP) injection, or intracardiac or intramyocardial injection.

[0058] In example embodiments, controlled-rate infusion pumps are used to maintain a therapeutic steady-state concentration. In other embodiments, depot-forming SC or IM formulations are used to extend peptide half-life.

[0059] In some embodiments, the peptide comprises an N-terminal acetyl group, a C-terminal amide, or both. Terminal acetylation and amidation are well-established peptide modifications used to enhance the biochemical and biophysical properties of therapeutic polypeptides. N-terminal acetylation blocks the free primary amine at the first residue, thereby preventing amino-terminal proteolysis, reducing positive charge, and improving metabolic stability. C-terminal amidation neutralizes the terminal carboxyl moiety and protects the peptide from degradation by carboxypeptidases, while also increasing structural stability and, in example cases, enhancing binding affinity for intracellular or extracellular targets.

[0060] In example embodiments, peptide termini are engineered with N-terminal acetylation and / or C-terminal amidation to increase stability without altering the core activity. Incorporation of these modifications may increase the in vivo half-life of the peptide, improve resistance to enzymatic degradation in serum or cellular environments, and reduce immunogenicity by mimicking naturally occurring post-translational modifications found in endogenous human peptides. These terminal caps may be introduced during solid-phase peptide synthesis or through standard chemical modification techniques and do not alter the core amino acid sequence responsible for the peptide’s biological activity. Accordingly, N-terminal acetylation and C-terminal amidation provide optional, pharmaceutically advantageous embodiments of the peptides disclosed herein.Cell-Penetrating Peptides (CPPs)

[0061] In other embodiments, the peptide is delivered as a fusion or conjugate with a cellpenetrating peptide (CPP). CPPs facilitate translocation of peptide cargo across cellular membranes and may include arginine-rich peptides, amphipathic peptides, penetratin, transportan, TAT-derived peptides, or engineered CPP variants. CPP-mediated delivery ofAttorney Docket 44010.196WO-PCT / / CU24196therapeutic peptides and CPP-peptide fusion constructs for intracellular delivery is described, for example, in US20240052005A1, WO2022129926 Al, and WO2019018898 Al.

[0062] In example embodiments, the peptide is fused to a protein to increase cellular uptake. In example embodiments, the peptides described herein are delivered to target cells using cell-penetrating peptides (CPPs). CPPs are short, generally cationic or amphipathic peptide sequences that facilitate the transport of conjugated cargos across cellular membranes through energy-dependent and energy-independent mechanisms. CPPs may enhance intracellular uptake of ELIXIR or an ELIXIR-derived peptide by promoting efficient membrane translocation, overcoming endosomal sequestration, and increasing cytosolic delivery in tissues that express voltage-gated sodium channel isoforms relevant to the disease indication.

[0063] In some embodiments, ELIXIR or an ELIXIR variant is directly fused to a CPP to generate a single polypeptide construct capable of entering cells without requiring additional delivery vehicles. Suitable CPPs include naturally occurring CPPs, engineered CPPs, arginine-rich CPPs, amphipathic CPPs, or chimeric peptides designed to maximize cellular uptake while minimizing cytotoxicity. Representative CPP sequences include TAT, penetratin, transportan, poly-arginine sequences, and other well-characterized membrane-translocating domains. The CPP may be positioned at the N-terminus, C-terminus, or within an internal region of the polypeptide, provided that the resulting fusion protein retains the ability to modulate sodium channel inactivation and inhibit pathological late sodium current.

[0064] In other embodiments, CPPs are non-covalently associated with the ELIXIR peptide through electrostatic interactions, hydrophobic interactions, or reversible chemical linkages. Such formulations may allow controlled release of the therapeutic peptide once inside the cell or within endosomal compartments. CPP -mediated delivery may be used alone or in combination with other intracellular delivery systems, such as viral vectors, mRNA constructs, lipid nanoparticles, protein nanocarriers, or exosome-based formulations.

[0065] CPP fusions may also enhance delivery to tissues that are difficult to target with viral or lipid-based systems, including cardiomyocytes, skeletal muscle fibers, dorsal root ganglion neurons, and central nervous system neurons. In example embodiments, the CPP is selected based on its capacity to translocate into the specific cell type of interest, such as cardiac-targeting CPPs, muscle-penetrating CPPs, or neuron-penetrating CPPs. The use of CPPs may permit systemic administration of ELIXIR peptides, as CPP fusion constructs can distribute throughout the body and selectively enter cell types displaying elevated late sodium current without requiring tissue-specific promoter control.Attorney Docket 44010.196WO-PCT / / CU24196

[0066] In further embodiments, CPPs may be engineered to include modifications that increase stability, reduce immunogenicity, or optimize cellular uptake. Such modifications may include cyclization, lipidation, PEGylation, incorporation of non-natural amino acids, or terminal capping such as N-terminal acetylation and C-terminal amidation. These enhancements can improve pharmacokinetics, extend circulating half-life, and promote intracellular accumulation of ELIXIR peptides to therapeutically relevant levels.

[0067] Accordingly, CPP-mediated delivery constitutes a versatile platform for enabling intracellular delivery of ELIXIR or related peptides in a broad range of tissues, providing an alternative or complementary strategy to viral vector-mediated, mRNA-based, or nanoparticle-based delivery systems. In example embodiments, the use of CPPs expands the therapeutic applicability of ELIXIR peptides by enabling efficient cytosolic delivery across cell types implicated in sodium channelopathies, including cardiac, skeletal muscle, neuronal, and sensory tissues.Exosomes

[0068] In additional embodiments, exosome-based delivery systems may be used. Exosomes and other extracellular vesicles can be engineered to selectively load peptides through peptide-sorting domains or fusion tags. Due to their endogenous origin, exosomes offer favorable biocompatibility and may enable targeted delivery to cardiomyocytes, neurons, or other excitable cells relevant to Nav channelopathies.

[0069] In example embodiments, the peptide is delivered using extracellular vesicles or exosome-based carriers. Exosomes may be loaded with therapeutic peptides or proteins and optionally modified with targeting peptides or cationic polypeptides to enhance tissue specificity and intracellular uptake. Exosome-mediated delivery of peptide and protein therapeutics is described, for example, in in US11103586B2, W02013084001A1, US20140348904A1, and WO2024039896A1.Nucleic acid administration

[0070] In example embodiments, the peptides disclosed herein are delivered by nucleic acid-based expression systems, including viral and non-viral vectors, mRNA constructs, and nanoparticle formulations. Such vectors comprise a polynucleotide encoding ELIXIR or an ELIXIR-derived peptide operably linked to a promoter, optionally in combination with regulatory elements such as enhancers, introns, polyadenylation signals, and untranslated region (UTR) elements that enhance transcript stability and translation (e.g., expressionAttorney Docket 44010.196WO-PCT / / CU24196cassette). In particular embodiments, the vector is formulated or selected to achieve expression in cells that endogenously express a target voltage-gated sodium channel (Nav) isoform implicated in the subject’s disease. In further embodiments, the peptide is delivered by administration of a nucleic acid encoding the peptide, thereby enabling intracellular expression of the peptide in target cells. The nucleic acid may be delivered as plasmid DNA, viral vectors, or RNA constructs, including mRNA formulated for in vivo delivery.

[0071] In example embodiments, an expression cassette may encode a cytosolic ELIXIR peptide, a secreted ELIXIR peptide bearing a secretion signal sequence, and / or an ELIXIR fusion to a cell-penetrating peptide to facilitate uptake by neighboring cells following secretion. For AAV formats, single-stranded or self-complementary designs may be used; for mRNA formats, optimized UTRs, modified nucleotides, and cap structures may enhance translation and durability. In some embodiments, miRNA response elements are included to detarget the liver, hematopoietic cells, or other off-target tissues.

[0072] In additional embodiments, the tropism of a vector may be modified through capsid engineering, peptide display, DNA shuffling, directed evolution, rational design, or computational design to create novel vectors capable of preferential transduction of tissues expressing a particular Nav isoform. Engineered AAV capsids may incorporate mutations, insertions, or ligand-binding motifs that enhance cellular uptake or tissue specificity.

[0073] Vector tropism may further be refined at the transcriptional level. In some embodiments, a tissue-specific promoter is combined with a tropism-enhanced capsid to restrict expression to disease-relevant tissues while allowing systemic administration. Conversely, detargeting elements such as microRNA response elements may be included to suppress expression in off-target tissues despite vector entry. These strategies permit high-precision delivery of ELIXIR peptides or related modulators to those tissues exhibiting pathological late sodium current while minimizing effects in tissues that express wild-type sodium channels with normal inactivation properties.

[0074] In all embodiments, the selected vector and capsid combination provides targeted, durable, and efficient delivery of the peptide to cells expressing the sodium channel isoforms implicated in the subject's disease. The encoded peptide may be expressed intracellularly, secreted, or fused to a cell-penetrating peptide to facilitate uptake by neighboring cells, thereby enabling modulation of I\ai. in a tissue-specific manner.

[0075] Vectors may be administered systemically or locally according to the target tissue. Exemplary routes include intravenous, intramuscular, intracoronary, intrathecal, or perineural delivery. Dosing may be adjusted to achieve a therapeutically effective level of peptideAttorney Docket 44010.196WO-PCT / / CU24196expression sufficient to reduce pathological I\ i. without substantially affecting peak sodium current (Ixap). For inducible systems, dose timing and inducer concentration provide additional control over therapeutic expression windows.Nanoparticles

[0076] In example embodiments, the nucleic acid encoding the peptide is delivered using lipid nanoparticle (LNP) formulations. LNP-encapsulated mRNA encoding a therapeutic peptide or protein enables transient intracellular expression without genomic integration. Lipid nanoparticle compositions and methods for delivery of mRNA encoding polypeptides are described, for example, in US10507249B2, US20180000953 Al, WO2022251953A1, and US20240374754A1.

[0077] In other embodiments, peptide-assisted or peptide-containing nanoparticle systems are used to deliver mRNA encoding the therapeutic peptide. Such systems may include lipidpeptide nanocomplexes or peptide-containing nanoparticles that enhance cellular uptake and endosomal escape of mRNA. Exemplary peptide-assisted mRNA delivery systems are described in US20200323964A1 and WO2019079215 Al.Viral vectors

[0078] In additional embodiments, the nucleic acid encoding the peptide is delivered using viral vectors, including adeno-associated virus (AAV), lentiviral, or adenoviral vectors. AAV vectors may be selected or engineered for tissue-specific tropism, including cardiotropic, neurotropic, or myotropic serotypes, and may employ tissue-specific promoters to restrict expression. Modular and tissue-targeted AAV systems for delivery of therapeutic proteins or peptides are described, for example, in US20200407751A1, US20240139340A1, and W02024016003 A3.

[0079] In example embodiments, the peptides described herein are delivered using viral vectors having natural or engineered tropism for tissues that express the targeted voltage-gated sodium channel isoforms. Adeno-associated virus (AAV) vectors are particularly useful due to their safety profile, ability to transduce non-dividing cells, and capacity for long-term expression. In some embodiments, the vector comprises an AAV capsid serotype selected or engineered to preferentially transduce cardiomyocytes, skeletal muscle fibers, central nervous system neurons, or peripheral sensory neurons, thereby enabling isoform-specific delivery aligned with the disease indication.Attorney Docket 44010.196WO-PCT / / CU24196Combination delivery

[0080] In example embodiments, delivery approaches may be combined. For example, direct peptide administration may be used for acute treatment, followed by nucleic-acid-based delivery for sustained intracellular expression. Alternatively, CPP-mediated peptide delivery may be combined with viral or non-viral nucleic acid delivery to enhance therapeutic efficacy.Other Emerging Protein Delivery Approaches

[0081] In addition to viral vectors and cell-penetrating peptide (CPP) fusions, a number of emerging strategies may enable efficient intracellular delivery of ELIXIR or related de novo peptides. These approaches are designed to overcome current limitations associated with endosomal trapping, immunogenicity, and restricted biodistribution.

[0082] In example embodiments, the synthetic peptide may be delivered using engineered protein nanocarriers, including but not limited to self-assembling protein cages, nanoparticle-binding peptide scaffolds, or de novo designed carriers capable of encapsulating short polypeptides. Such carriers can be modified to enhance membrane translocation, modulate release kinetics, or target specific tissues through conjugation to affinity ligands or antibodies.

[0083] In some embodiments, lipid-based nanoparticles (LNPs) may be adapted for polypeptide cargo. Although LNPs are traditionally optimized for nucleic acid delivery, recent work has enabled direct loading of peptides or peptide-lipid conjugates into LNP formulations, facilitating cytosolic release following endosomal escape.

[0084] In further embodiments, engineered bacterial toxin-derived delivery platforms may be utilized. Several bacterial proteins contain natural translocation domains capable of transporting folded peptides into the cytosol. Modified, non-pathogenic variants of these systems may permit efficient intracellular delivery while minimizing toxicity.

[0085] Alternative embodiments may employ stimuli-responsive delivery materials, such as pH-sensitive or redox-responsive polymers that release peptide cargo selectively within intracellular compartments. These systems can be tuned to enhance intracellular bioavailability and reduce off-target exposure.

[0086] Finally, advances in protein stapling (see, e.g., Moiola M, Memeo MG, Quadrelli P. Stapled Peptides-A Useful Improvement for Peptide-Based Drugs. Molecules.2019;24(20):3654), lipidation, PEGylation, and backbone engineering may increase the inherent membrane permeability and stability of ELIXIR-derived peptides, thereby enabling direct peptide uptake without auxiliary carriers.Attorney Docket 44010.196WO-PCT / / CU24196

[0087] Collectively, these emerging platforms may provide additional routes for efficient and tissue-specific intracellular delivery of ELIXIR or related synthetic Nav modulators and may be used alone or in combination with cell-penetrating peptides or viral vectors described herein.Pharmaceutical Compositions

[0088] The present invention is also directed to pharmaceutical compositions comprising an effective amount of peptides according to the present invention (including a pharmaceutically acceptable salt, thereof), optionally in combination with a pharmaceutically acceptable carrier, excipient or additive.

[0089] The pharmaceutical compositions can be processed in accordance with conventional methods of pharmacy to produce medicinal agents for administration to patients in need thereof, including humans and other mammals. For example, the peptides can include a pharmaceutically acceptable carrier and / or excipient. In example embodiments, the pharmaceutically acceptable carrier comprises water. In example embodiments, the pharmaceutically acceptable carrier further comprises dextrose. In example embodiments, the pharmaceutically acceptable carrier further comprises dimethyl sulfoxide. A carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., ocular, oral, topical or parenteral, including gels, creams ointments, lotions and time released implantable preparations, among numerous others. Solutions or suspensions used for ocular, parenteral, intradermal, subcutaneous, or topical application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose. Liposomal suspensions may also be pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art.

[0090] The invention provides for pharmaceutical compositions containing peptides as described herein. In embodiments, the pharmaceutical compositions contain a pharmaceutically acceptable carrier, excipient, or diluent, which includes any pharmaceutical agent that does not itself induce the production of an immune response harmful to a subject receiving the composition, and which may be administered without undue toxicity. As used herein, the term “pharmaceutically acceptable” means being approved by a regulatory agencyAttorney Docket 44010.196WO-PCT / / CU24196of the Federal or a state government or listed in the U. S. Pharmacopeia, European Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans. These compositions can be useful for treating and / or preventing viral infection and / or autoimmune disease.

[0091] A thorough discussion of pharmaceutically acceptable carriers, diluents, and other excipients is presented in Remington's Pharmaceutical Sciences (17th ed. 1975, Mack Publishing Company) and Remington: The Science and Practice of Pharmacy (21st ed. 2005, Lippincott Williams & Wilkins), which are hereby incorporated by reference. The formulation of the pharmaceutical composition should suit the mode of administration. In embodiments, the pharmaceutical composition is suitable for administration to humans, and can be sterile, non-particulate and / or non-pyrogenic. Pharmaceutically acceptable carriers, excipients, or diluents include, but are not limited, to saline, buffered saline, dextrose, water, glycerol, ethanol, sterile isotonic aqueous buffer, and combinations thereof.Methods of TreatmentDiseases Treated

[0092] In various embodiments, ELIXIR peptides, ELIXIR variants, and related modulators of sodium channel inactivation are used to treat disorders characterized by pathological late sodium current (INaL) arising from impaired voltage-gated sodium (Nav) channel inactivation. As demonstrated herein, increased UaL represents a convergent pathogenic mechanism across cardiac, neuronal, and skeletal muscle tissues and arises from diverse perturbations including inherited mutations, post-translational modifications, pathological remodeling, and truncations of the Nav channel carboxyl-terminal domain (CTD). ELIXIR peptides selectively suppress pathogenic, but not physiological, UaL by promoting release of the inactivation gate (IG) from the CTD and enhancing occupancy of the pore-proximal inactivation site without significantly altering peak sodium current or voltage-dependent gating of wild-type channels.Cardiac Disorders

[0093] In example embodiments, ELIXIR is used to treat cardiac diseases in which pathological I\ i. carried by NavL5 contributes to electrical instability. These conditions include long-QT syndrome type 3 (LQT3) caused by gain-of-function mutations such as AKPQ, atrial fibrillation associated with altered NavL5 regulation, and heart failure (HF) or myocardial ischemia, in which phosphorylation or remodeling of NavL5 increases NaL. AsAttorney Docket 44010.196WO-PCT / / CU24196shown herein, ELIXIR markedly reduces NaL in multiple Navi.5 disease mutants, including AKPQ, F1759A, IQ / AA, and truncation mutants, as well as in heart failure-associated CaMKII-dependent phosphorylation states. ELIXIR also shortens pathological action potential prolongation (APD90) and normalizes beat-to-beat variability in adult ventricular cardiomyocytes from CaMKIIδC,OEmice, and reduces INBL in LQT3 patient-derived human iPSC-cardiomyocytes. Because wild-type NavL5 channels maintain minimal I\ai. under physiological conditions, ELIXIR treatment is expected to spare normal cardiac excitability.

[0094] In example embodiments, heart failure, myocardial ischemia, P-adrenergic stress, oxidative injury, and atrial or ventricular arrhythmias is treated. In various embodiments, the invention recognizes that specific cardiac disease conditions activate intracellular kinase pathways, including protein kinase A (PKA) and Ca2+ / calmodulin-dependent protein kinase II (CaMKII), which in turn modulate voltage-gated sodium (Nav) channel function through targeted phosphorylation. These pathological signaling states arise in the context of heart failure, myocardial ischemia, P-adrenergic stress, oxidative injury, and atrial or ventricular arrhythmias, all of which are associated with enhanced kinase activity within cardiomyocytes.

[0095] Under physiological conditions, the Nav channel inactivation gate (IG), located within the intracellular III-IV linker, rapidly engages a pore-proximal receptor site following channel opening, thereby terminating sodium influx within milliseconds. This mechanism ensures brief action potential upstrokes and protects against sustained sodium entry.

[0096] However, in example disease states, hyperactivation of PKA and CaMKII leads to phosphorylation of defined regulatory residues within the NavL5 C-terminal domain (CTD) and adjacent modulatory elements. These post-translational modifications destabilize fast inactivation, alter the energetic landscape of the IG docking step, and reduce the likelihood that the IG will successfully bind the pore-proximal site. As a result, a subset of channels remains non-inactivated or reopens aberrantly, giving rise to persistent or late sodium current (iNa ).

[0097] In example embodiments, CaMKII activation, driven by elevated intracellular Ca2+levels characteristic of heart failure, ischemia, or stress-induced Ca2+dysregulation, induces phosphorylation of sites within the CTD known to influence inactivation-gate engagement. The CaMKII-dependent modifications weaken the interaction between the CTD’s EF-hand-like region (EFL) and the inactivation machinery. Consequently, the IG remains ectopically associated with the CTD rather than transitioning to its pore-proximal receptor, thereby permitting continued Na+influx late during depolarization.Attorney Docket 44010.196WO-PCT / / CU24196

[0098] In other embodiments, PKA hyperactivation, such as that occurring during heightened β-adrenergic stimulation, chronic sympathetic drive, or atrial fibrillation, further contributes to dysregulation of Nav1.5 gating. PKA-mediated phosphorylation enhances channel availability and shifts inactivation dynamics in a manner that reduces the probability of complete IG engagement, thereby increasing the number of channels that reenter a conducting state during the action potential plateau.

[0099] In example embodiments, these kinase-dependent modifications exert synergistic effects, as PKA-driven signaling increases calcium loading and membrane excitability, which in turn stimulates additional CaMKII activation. This feed-forward loop results in further phosphorylation of the Nav channel and an escalating disruption of inactivation-gate behavior. In this pathological setting, Nav channels increasingly fail to transition into their non-conducting state, leading to prolonged action potentials, electrical instability, and heightened arrhythmia susceptibility.

[0100] Accordingly, the invention provides compositions and methods for treating diseases in which cardiac-condition-induced activation of PKA and CaMKII leads to phosphorylation-dependent impairment of IG-pore engagement, culminating in pathological iNaL. By counteracting these defects, for example, through administration of ELIXIR peptides that restore proper IG release and promote inactivation, normal Nav channel function and electrical stability may be reestablished.

[0101] For treatment of cardiac disorders associated with pathological late sodium current in Navi.5, including long-QT syndrome type 3, atrial fibrillation, myocardial ischemia, and heart failure-related remodeling, expression constructs are configured for selective transduction and expression in cardiomyocytes. In example embodiments, the promoter is a cardiac-specific promoter that restricts expression to the myocardium. Exemplary promoters include cardiac troponin T (cTnT) promoters, myosin heavy chain promoters, myosin light chain 2 ventricular promoters, or natriuretic peptide promoters. In some embodiments, the vector is a cardiotropic AAV-based construct or another delivery system exhibiting enhanced myocardial transduction. In further embodiments, microRNA response elements are incorporated to detarget expression from non-cardiac tissues, thereby increasing the therapeutic index. The expressed peptide suppresses pathological NaL in Navl.5-expressing cells while sparing peak sodium current.

[0102] For cardiac applications involving modulation of Navi.5, the vector may comprise a cardiotropic AAV serotype, such as an AAV9, AAVrh74, or an engineered AAV variant optimized for myocardial delivery. Such vectors exhibit efficient transduction of ventricularAttorney Docket 44010.196WO-PCT / / CU24196and atrial cardiomyocytes following systemic or intracoronary administration. In some embodiments, capsid variants with enhanced cardiac tropism allow lower vector doses while achieving high levels of expression of the encoded peptide within the myocardium.

[0103] In example embodiments, intramyocardial injection is used during catheter-based cardiac procedures.Skeletal Muscle Disorders

[0104] In other embodiments, ELIXIR is used to treat neuromuscular disorders involving aberrant INaL in Nav1.4. Example forms of congenital myotonia and cold-aggravated myotonia arise from Navi.4 variants such as F1698I that impair fast inactivation. As demonstrated herein, ELIXIR selectively reduces the elevated NaL in such pathogenic Nav1.4 variants while leaving wild-type Nav1.4 function largely unaltered. Consequently, ELIXIR is expected to normalize muscle membrane repolarization and reduce myotonic discharges without suppressing normal muscle excitability.

[0105] For skeletal muscle disorders such as congenital myotonia, expression constructs drive peptide production in Nav1.4-expressing fibers. In particular embodiments, a muscle-specific promoter is used, such as a muscle creatine kinase promoter, an alpha-skeletal actin promoter, or a desmin promoter. The vector may be formulated for intramuscular administration or for systemic delivery with enhanced muscle tropism. In example embodiments, the cassette further comprises elements that enhance expression in fast-twitch fibers or restrict expression away from cardiac tissue. Expression of ELIXIR or an ELIXIR variant stabilizes inactivation of Nav1.4, thereby reducing excessive late sodium influx and normalizing membrane excitability.

[0106] For skeletal muscle disorders associated with Nav1.4 dysfunction, the vector may be an AAV serotype with high affinity for muscle tissue, including AAV1, AAV6, AAV8, AAV9, or engineered capsids developed for robust transduction of both fast-twitch and slow-twitch muscle fibers. These vectors can be administered intramuscularly or systemically depending on the extent of tissue involvement. In example embodiments, tropism-enhanced capsids enable broad muscle delivery while minimizing off-target expression in non-muscle tissues.

[0107] In example embodiments, intramuscular microinjection is used for myotonic disorders.Attorney Docket 44010.196WO-PCT / / CU24196Neurological and Neurodevelopmental Disorders

[0108] In further embodiments, ELIXIR may be administered to treat neurological disorders arising from impaired inactivation of neuronal Nav channels, including epilepsy, migraine, and neurodevelopmental disorders involving Nav1.1, Nav1.2, Nav1.3, or Nav1.6. As shown with the Nav1.6 N1768D epilepsy-associated variant, ELIXIR reverses excessive INaL, rescues prolonged action potentials, restores spike fidelity, and mitigates depolarization block in cortical neurons expressing pathogenic channels. These results demonstrate that ELIXIR can normalize neuronal firing patterns across neuron subtypes affected by Nav channelopathy -related hyperexcitability.

[0109] For neurological indications, including epilepsy, migraine, and neurodevelopmental disorders linked to Nav1.1, Nav1.2, Nav1.3, or Nav1.6, vectors are configured for neuronal expression within the central nervous system. In example embodiments, the promoter is neuron-specific. Exemplary promoters include synapsin promoters, CaMKIIa promoters for excitatory neurons, neuron-specific enolase promoters, or inhibitory neuron-biased promoters (e.g., GABAergic interneuron drivers). In further embodiments, regulatory elements are used to achieve regional or cell-type-biased expression profiles suited to the disease substrate. Inducible or drug-responsive promoters may be employed to enable temporal control, thereby allowing titration of peptide expression to patient response. The resulting expression of ELIXIR corrects impaired inactivation dynamics and reduces neuronal hyperexcitability associated with pathological NaL.

[0110] For neurological conditions involving Nav1.1, Nav1.2, Nav1.3, or Nav1.6, the vector may comprise an AAV serotype or engineered capsid with central nervous system tropism. In some embodiments, the vector is selected from AAV9, AAVrhlO, AAV-PHP. B, AAV-PHP.eB, or other CNS-penetrant capsids capable of crossing the blood-brain barrier when administered systemically. Alternatively, administration may occur via intrathecal, intracerebroventricular, or parenchymal injection to enhance delivery to relevant neuronal populations. Engineered capsids may further be selected to preferentially target excitatory neurons, inhibitory interneurons, cortical neurons, or specific neuronal circuits relevant to the targeted sodium channel isoform.[OHl] For neurological disorders involving Nav channel dysfunction, the peptide may be administered using delivery routes that permit access to the central nervous system. These include intrathecal injection, intracerebroventricular (ICV) injection, epidural administration, cistema magna administration, or convection-enhanced delivery (CED).Attorney Docket 44010.196WO-PCT / / CU24196

[0112] In example embodiments, perineural administration is used for sensory neuron channelopathies.Pain Disorders and Peripheral Sensory Channelopathies

[0113] In some embodiments, ELIXIR is used to treat chronic pain conditions associated with increased late sodium current in Navi.7, Navi.8, or Navi.9. Pathological enhancement of NaL in these sensory neuron channels contributes to painful neuropathies, chronic nociceptive hypersensitivity, and inherited pain syndromes. Because ELIXIR exhibits preferential inhibition of NaL in mutant or pathologically modified channels while sparing wild-type channels, it may normalize nociceptor excitability without blocking normal sensory function.

[0114] For pain disorders involving peripheral sensory neurons, including painful neuropathies associated with NavL7, NavL8, or NavL9, expression constructs restrict peptide production to nociceptor populations. In example embodiments, promoters with peripheral sensory neuron selectivity are used, including peripherin or advillin promoters, as well as regulatory regions derived from genes encoding Nav1.8 (SCN10A) or Nav1.9 (SCN11A). In related embodiments, dorsal root ganglion-biased enhancer elements are incorporated to increase expression in nociceptors while reducing off-target expression in central or autonomic neurons. The expressed peptide modulates late sodium current in targeted sensory neurons, attenuating pathological excitability while preserving normal sensory transmission.

[0115] For pain disorders associated with Nav1.7, Nav1.8, or Nav1.9 dysfunction in peripheral sensory neurons, the vector may comprise an AAV serotype or engineered capsid with tropism for dorsal root ganglion (DRG) neurons or peripheral nociceptors. In example embodiments, AAVrhlO, AAV6, AAV9, orDRG-tropic engineered capsids are used to achieve efficient delivery to small-diameter sensory neurons following intrathecal or perineural administration. These vectors permit selective targeting of nociceptors without substantial delivery to central neurons or cardiomyocytes.

[0116] In example embodiments, topical or intradermal delivery for localized neuropathic pain.Truncation Variants and CTD-Disrupting Mutations

[0117] In example embodiments, subjects who benefit from ELIXIR include individuals harboring CTD nonsense or truncation mutations that impair IG-CTD interactions, such as Nav1.5 Δ1864, which exhibits dramatically elevated aL. As demonstrated herein, ELIXIR strongly suppresses pathological INaL in Δ1864 but does not inhibit INaL in the Δ1810 variant,Attorney Docket 44010.196WO-PCT / / CU24196which lacks the majority of the EFL region and therefore the ELIXIR binding site. Accordingly, ELIXIR is particularly suited for treating diseases involving preserved but dysregulated CTD-IG interfaces, rather than variants that structurally eliminate the binding site itself. Subjects with mutations that decrease IG affinity for the pore-proximal site, whether through phosphorylation, structural destabilization, or impaired CTD scaffolding, are suitable candidates for treatment.General Applicability Across Nay Isoforms and Disease States

[0118] Because the EFL region is conserved across Nav isoforms and ELIXIR preferentially inhibits pathogenic INaL while sparing wild-type channels, ELIXIR peptides may be used to treat a broad class of Nav channel opathies. These include conditions arising from: inherited gain-of-function mutations across Nav1.1-Nav1.9; phosphorylation-dependent or signaling-dependent enhancement of late sodium current; oxidative or metabolic stress; altered binding of auxiliary proteins; and pathophysiological remodeling that weakens inactivation-gate engagement. By enhancing inactivation through displacement of the IG from the CTD and promoting occupancy of the pore-proximal site, ELIXIR peptides restore normal sodium channel behavior across multiple tissues and disease contexts, providing a unified therapeutic platform for disorders rooted in defective Nav inactivation.Screening for modulators using CTD binding peptides

[0119] In example embodiments, the invention provides methods for identifying peptides, peptidomimetics, proteins, small molecules, or other candidate agents that modulate binding to the Nav channel carboxyl-terminal domain (CTD). In example embodiments, the assay utilizes fluorescence anisotropy to monitor displacement or binding of a fluorescently labeled peptide, such as ELIXIR or a functional fragment thereof, to the Nav 1.5 CTD or to CTDs of other Nav isoforms.

[0120] Fluorescence anisotropy offers a sensitive and solution-based readout that reflects changes in the rotational mobility of a fluorophore-conjugated peptide upon binding to a larger macromolecular target. When the labeled peptide is free in solution, it rotates rapidly, yielding low anisotropy values. Upon binding to the CTD, the rotational correlation time increases, producing higher anisotropy. Candidate molecules that compete with, disrupt, stabilize, or otherwise alter CTD-peptide binding induce measurable changes in anisotropy, permitting quantification of binding affinity and inhibition potency.Attorney Docket 44010.196WO-PCT / / CU24196

[0121] In one embodiment, the method comprises incubating a fluorescently labeled ELIXIR peptide (e.g., N-terminal fluorescein-ELIXIR, or a variant thereof) with purified Nav CTD protein under defined buffer conditions. Fluorescence polarization or anisotropy is measured before and after addition of a test agent. A decrease in anisotropy relative to control indicates that the test agent reduces peptide-CTD binding, for example by competing with ELIXIR for the CTD interaction site. Conversely, an increase in anisotropy may indicate that the test agent stabilizes the CTD-peptide complex or promotes a conformational change that restricts dye rotation.

[0122] In example embodiments, the assay is performed in 96-well or 384-well plate format, enabling high-throughput or ultra-high-throughput screening. In additional embodiments, mixtures containing multiple candidate compounds may be tested in pooled fashion, followed by deconvolution to identify active components. In some embodiments, anisotropy values are fit to a binding isotherm to determine an apparent dissociation constant (KD) or inhibition constant (IC50), which may be used to rank or triage candidate agents for subsequent validation.

[0123] In specific embodiments, the method further comprises measuring anisotropy in the presence of known binders or reference inhibitors to establish assay windows or positive and negative controls. In further embodiments, the assay is performed using mutated CTDs, truncated CTDs, or CTDs corresponding to disease-linked Nav isoforms to identify allele-specific or isoform-specific modulators.

[0124] The anisotropy assay described herein provides a robust platform for discovering molecules that regulate Nav CTD interactions, including agents that mimic or antagonize the action of ELIXIR. Because the fluorescence anisotropy signal reflects direct target engagement in solution, the assay is particularly advantageous for identifying modulators that bind transiently or weakly, or that would be difficult to detect using immobilization-based methods.

[0125] Further embodiments are illustrated in the following Examples which are given for illustrative purposes only and are not intended to limit the scope of the invention.EXAMPLESExample 1 - De novo Design of a Peptide Modulator to Reverse Sodium Channel Dysfunction Linked to Cardiac Arrhythmias and EpilepsyAttorney Docket 44010.196WO-PCT / / CU24196RESULTSA rational approach to developing an INaL inhibitor with submicromolar affinity.

[0126] Fast inactivation of Navi.5 is a multistep process involving distinct channel elements. Following depolarization, movement of voltage-sensing domains (VSDs) I-III couples to pore opening, while that of the domain IV VSD initiates conformational changes that result in inactivation31–33. Of particular importance for the latter is the inactivation gate (IG, linker between domains III and IV) which contains the IFM motif that binds to a site near the channel pore to halt Na+influx34’35(FIG. IB). In addition to this pore-proximal site, the IG also binds an acidic EF-hand like region (EFL) in the C-terminal domain (CTD) of the channel36–39, suggesting that the IG may first need to be released from this EFL site to interact with the pore-proximal site and prevent pore opening (FIG. 8A). In cardiac pathologies, this process is disrupted resulting in decreased Navi.5 inactivation, and increased ZxaL, suggesting reduced occupancy of the pore-proximal site by the IG (FIG. 8B). Given this, we reasoned that dislodging the IG from its CTD interaction site would promote Nav inactivation, and in so doing, reduce INaL and AP duration (APD, FIGs. IB and 8C). To do so, we used a ColabDesign30based protocol to generate a peptide targeting the EFL of the Navi.5 CTD as depicted in FIG. 1C. This process yielded a small (21 aa) peptide with features reminiscent of the Navi.5 IG (hydrophobic surface flanked by basic residues (FIG. ID), consistent with it being able to bind the hydrophobic cleft in the EFL of the Navi.5 CTD (FIGs. ID and IE).Given its putative mechanism, we termed this peptide, Engineered Late-current Inhibitor X by Inactivation-gate Release (ELIXIR). As an initial means of testing our designed sequence we compared models of the ELIXIR-Nav1.5 CTD (aa 1773-1940, Navl.5cTo) complex generated with several structure prediction algorithms26’40'42(FIG. 9A). In all of the models, the Navi.5 EFL and IQ motif adopted a nearly identical architecture to that observed in crystallographic structures43–45(FIG. 9A), lending credence to the predicted structures. In each, ELIXIR binds to the Nav 1.5 EFL in an identical orientation (FIG. 9A). The interface between ELIXIR and Navi.5 EFL was highly similar across the models, with peptide residues L8, F12, and Y16 making numerous close contacts to residues in the hydrophobic cleft of the EFL (FIGs. 9B-91). In addition to this, ColabFold models of the Nav1.5 CTD-CaM-Nav1.5 IG complex in the absence and presence of ELIXIR show ELIXIR bound to the EFL in place of the IG (FIG.9J), suggesting it can displace the IG from this site. Comparison of our designed peptide sequence to that of the Navi.5 IG revealed that they are only -24% identical (FIG. 10A top), indicating that our designed sequence was not merely recapitulating the native ligand of the Navi.5 EFL. Indeed, when the sequence was searched against the BLAST database, the closest homologAttorney Docket 44010.196WO-PCT / / CU24196was the 50S ribosome of the algae Nannochloropsis gaditana which showed only -52% identity (FIG. 10A middle). The closest mammalian homolog, having -33% identity, was DNA replication factor CDT1 of the Weddell seal (Leptonychotes weddellii, FIG. 10A bottom). A single conservative substitution was made to the sequence (C15S) to simplify in vitro applications of ELIXIR. Having confirmed that the ELIXIR sequence was both de novo and foreign to Navi.5, we examined whether it binds directly to the channel CTD. Equilibrium titrations of a fluorescein-tagged ELIXIR with the Nav1.5CTD, monitored through fluorescence anisotropy, displayed saturable binding (FIG. IF) and revealed that ELIXIR binds the Navl.5cTD in vitro with a submicromolar affinity (KD = 0.89 ± 0.25 μM, FIG. IF). To ensure that ELIXIR can associate with the Navl.5cTD in the complex milieu of living cells, we undertook a flow-cytometry based FRET 2-hybrid assay. Co-expression of Cerulean-tagged ELIXIR, a FRET donor, and Venus-tagged Navl.5cTD, a FRET acceptor, resulted in robust FRET confirming that the Nav1.5CTDbinds ELIXIR in mammalian cells (FIG. IF). We sought to further validate the interface between ELIXIR and Navi.5 CTD. In each of the predicted structures, residues L8, Fl 2, and Y16 of ELIXIR make many close contacts with residues in the hydrophobic cleft of the Navl.5cTD EFL (FIGs. IE and 9B-9I). Therefore, we reasoned that substitution of these aliphatic residues with alanine (L8 and Y16) or glutamate (Fl 2) would disrupt the ELIXIR-Navl.5 CTD interaction. Consistent with this expectation, FRET 2-hybrid analysis showed that each mutant diminished binding, albeit to different degrees (FIGs. 1G, 1H, 10B, and 10C). Collectively, these results demonstrate that de novo designed ELIXIR peptide interacts with Navi.5 CTD and may be positioned to tune channel function.ELIXIR decreases INaL of Nav1.5 channelopathic variants

[0127] Thus affirmed, we sought to determine functional consequences of ELIXIR interaction on Nav1.5 channel function. Whole-cell patch-clamp recordings demonstrated that co-expression of ELIXIR did not significantly alter channel activation, inactivation or recovery from inactivation of wild-type Navi.5 (FIGs. 10D-10F). To probe whether ELIXIR inhibits INaL, we conducted cell-attached multichannel recordings of HEK293 cells heterologously expressing either wild-type Navi.5 or a disease-linked non-sense variant that truncates the channel CTD (Al 864). As the channel unitary current is larger than instrument noise, multichannel recordings allow us to quantify peak current and simultaneously detect rare late channel openings in the context of an intact cell, as established previously. We quantified INaL as Φpersist, the ratio of average open probability (Po) following 50 ms of depolarization normalized to the peak Po. As in previous studies24’25’46, wild-type Navi.5 exhibited a lowAttorney Docket 44010.196WO-PCT / / CU24196baseline level of INaL, which was minimally perturbed by co-expression of ELIXIR (FIG. 2A-2C and 11 A). By contrast, the Al 864 mutant showed greatly (~40-fold) elevated INaL at baseline, consistent with the pathogenicity of this variant (FIG. 2C-2E and 11B). Strikingly, ELIXIR co-expression evoked a greater than 12-fold reduction in INaL of the mutant channel (FIGs. 2C-2E and 11B). To ensure that the C15S substitution we introduced into ELIXIR did not affect its efficacy as an INaL inhibitor, we also probed the unmodified sequence (FIGs. 11C and 11D). Both peptides caused a nearly identical reduction in the INaL of Nav1.5 Δ1864 (FIG. HE). In native tissue, Nav channels function as multi-subunit complexes that include auxiliary P-subunits which have been reported to modulate INaL. As such, we investigated whether ELIXIR was able to inhibit INOL in the presence of P-subunits. In the presence of pi and P4, ELIXIR had minimal effects on the INaL of wild-type Nav1.5 (FIGs. HF, 11H and 11 J) However, expression of pi and P4 with the Al 864 mutant yielded a baseline reduction in INaL, which was further reduced by the addition of ELIXIR (FIG. 11G, 11I, and 11J). Ranolazine, is a well-established clinically used small molecule INaL inhibitor that interacts with Nav1.5 through the antiarrhythmic drug (AAD) binding site within the transmembrane segments23.. As such, we next compared the effect of ELIXIR on Nav1.5 Δ1864 to that of ranolazine. While incubation with 10 pM ranolazine resulted in a decrease in INaL of the Δ1864 mutant (FIGs.2C, 2F, and 12A), this reduction was more modest than that elicited by ELIXIR. These findings establish the biological activity of the computationally designed ELIXIR peptide and highlight its effectiveness.

[0128] Having confirmed its functionality, we sought to further investigate whether the change in INaL observed with ELIXIR results from its bona fide interaction with Nav1.5 CTD. To do so, we first examined how mutations of key of ELIXIR residues that weaken binding to the Nav1.5 CTD alter functional inhibition of INaL (FIG. 2G). We found that F12E strongly diminished INaL inhibition, while Y16A and L8A evoked partial effects (FIGs. 2H, 2I, and 12B-12F). Quantitative analysis revealed a correlation between the level of INaL (Φpersist) and the relative dissociation constant determined by FRET analysis (FIG. 2J), pointing to the functional importance of the ELIXIR-Nav1.5 CTD interaction. Second, we reasoned that deletion of the ELIXIR binding site (i.e. the EFL) would abolish INaL inhibition by ELIXIR. To test this, we considered the channelopathic Nav1.5 Δ1810 variant which encodes only the first helix of the EFL (FIG. 2K). As with the A1864 mutant, Nav1.5 Δ1810 showed a markedly increased basal INaL relative to the wild-type channel (FIGs. 2L, 2M, and 12G). However, unlike Nav 1.5 Al 864, co-expression of ELIXIR had virtually no effect on INaL (FIGs. 2L, 2M,Attorney Docket 44010.196WO-PCT / / CU24196and 12G). Together, the reduced efficacy of ELIXIR mutants in inhibiting AjaL, and the inability of ELIXIR to inhibit INaL of Nav1.5 Δ1810 suggest that ELIXIR interacts with holo-Nav1.5 via an interface similar to that predicted in the models of the ELIXIR-Nav1.5CTD complex.ELIXIR is a general Inhibitor of Nav1.5 INaL

[0129] At the molecular level, a variety of perturbations to Nav1.5 contribute to pathogenic increases in INaL. These include mutations distributed throughout the channel sequence and post-translational modifications, such as phosphorylation, that are associated with cardiac pathologies including heart failure47. Our results with Nav1.5 Δ1864 suggest that ELIXIR is a potent inhibitor of INaL; here, we sought to determine its generality in tuning INaL triggered by distinct mechanisms. Accordingly, we assessed the efficacy of ELIXIR in inhibiting INaL of several Nav1.5 mutations located in distinct channel domains. These included: (1) the AKPQ mutation linked to LQT3, (2) a mutation (F1759A) in the classic AAD binding pocket linked to atrial fibrillation2, and (3) a structure-guided mutation (IQ / AA) engineered to block binding of the auxiliary protein calmodulin (FIG.3A). These mutations were selected as they represent disparate structural mechanisms that can contribute to increased Z^L in cardiac pathologies. Consistent with our previous results25, when expressed alone, each of these Nav1.5 mutants displayed an elevated INaL (21-fold F1759A; 13-fold ΔKPQ; 10-fold IQ / AA) relative to the wild-type channel (FIGs. 3B, 3C, 3E, and 12A-12D). For each NavL5 mutant, co-expression of ELIXIR resulted in a statistically significant decrease in INaL, with reductions ranging from 12-fold (F1759A) to 3-fold (IQ / AA) (FIGs. 3B, 3C, 3E, and 12A-12D).

[0130] Post-translational modification of NavL5 also contributes to elevated INaLand is associated with increased risk of arrhythmia in both heart failure47’48and myocardial ischemia49’50. To determine whether ELIXIR inhibits phosphorylation-dependent INaL, we coexpressed wild-type NavL5 with the PKA catalytic subunit (PKAcat) or a constitutively active Ca2+ / CaM-dependent kinase II mutant (CaMKIIT286D). In both cases, we observed an increase in basal INaL (~5-fold with PKAcat or ~2-fold with CaMKIIT286D) (FIGs. 3D, 3E, and 12E-12G). Co-expression of ELIXIR sufficed to reverse the PKAcat and CaMKIIT286D-dependent enhancement of INaL (FIGs. 3D, 3E, and 12E-12G). The ability of ELIXIR to inhibit INaL stemming from a wide range of molecular alterations (i.e. mutations in distinct channel domains and post-translational modifications) indicates that it may be generalizable and further highlights its potential utility.

[0131] We next examined the dose-dependence of / \ai. inhibition by ELIXIR. To facilitate intracellular delivery of ELIXIR, we synthesized it as a peptide fused to the cell-penetratingAttorney Docket 44010.196WO-PCT / / CU24196region of the transduction domain of HIV- 1 trans activator of transcription and an N-terminal fluorescein (ELIXIR-cpp). We incubated HEK293 cells transfected with Navi.5 AKPQ with various concentrations of ELIXIR-cpp for 4 hours and measured ZxaL (FIGs.3F and 12H-12K).We found that ELIXIR decreased the ZxaL of Nav1.5 ΔKPQ in a dose-dependent manner with an IC50 (~ 330 ± 200nM, FIG. 3H), similar to the in vitro affinity of ELIXIR for the Nav1.5CTD.ELIXIR preferentially tunes “pathological” Zxai,

[0132] The nine Nav isoforms (1.1-1.9) are well known to have distinct inactivation properties that are critical for their physiological functions51. Gain-of-function mutations in various Nav isoforms are linked to diverse clinical phenotypes, including epilepsy, myotonia, neurodevelopmental disorders, and chronic pain (FIG. 4A). The high homology of the CTD (FIG. 4B) suggests that ELIXIR may interact with each Nav isoform. Consistent with this, in ColabFold40generated models ELIXIR was predicted to adopt a highly similar binding pose with the EFL of Nav1.1 - 1.9 (FIGs. 14A-14H). Accordingly, we sought to determine the effect of ELIXIR on ZxaL of selected wild-type Nav channels expressed in distinct tissues: (1) NavL 1 in neurons, (2) NavL4 in skeletal muscle, (3) NavL6 in neurons, and (4) NavL7 in peripheral nervous system neurons. When expressed in HEK293 cells, these channels exhibited varying amplitudes of INaL, with Rpersistvalues ranging from -0.5% (Nav1.4) - 1.6% (Nav1.1). Coexpression of ELIXIR failed to evoke a statistically significant change in the basal INaLof any of the wild-type Nav isoforms examined (FIGs. 4C-4E, 4F, 4H, 4I, 4K, 14I-14K, and 14M), consistent with the minimal effect of ELIXIR on wild-type NavL5.

[0133] Although ELIXIR minimally perturbed various wild-type Nav channels, it is possible that this peptide may still modulate / \ai. of mutant channels that have deficits in inactivation (i.e. if the IG has reduced propensity for occupying the pore-proximal site). In this regard, increased / \ai. of NavL4 is thought to be a relevant pathophysiological mechanism for congenital myotonia. To probe this possibility, we investigated the effect of ELIXIR on the / \ai. of an Nav 1.4 variant (F1698I) associated with cold-aggravated myotonia in humans52’53. When expressed alone NavL4 F1698I had a small (~4-fold) but statistically significant increase in persist compared to wild-type NavL4 (FIGs. 4F-4H and 14K-14L). Co-expression of ELIXIR reversed / \ai. of the mutant channel to near wild-type levels (FIGs. 4F-4H and 14L), despite the wild-type channel being unperturbed by ELIXIR.

[0134] Gene variants in voltage-gated sodium channels are a leading cause of monogenic epilepsies1. Variants in SCN8A, encoding NavL6, often increase neuronal excitability byAttorney Docket 44010.196WO-PCT / / CU24196impairing inactivation and increasing INaL2-4Consistent with previous reports, analysis of an Navi.6 variant (N1768D) linked to epilepsy and seizure disorders showed a markedly elevated / NaL as compared to wild-type Navi.6 (FIGs.4I-4K, 14M, and 14N). Strikingly, we found that co-expression of ELIXIR reduced the / \ar (~10-fold) of Navi.6 N1768D to near wild-type levels (FIGs. 4I-4K, 14M, and 14N). These findings suggest that although ELIXIR may bind multiple Nav isoforms, its inhibition of / NaL is most prominent only when there are defects in the inactivation process. This feature may allow ELIXIR to preferentially modulate mutant or pathologically modified channels which could be advantageous.ELIXIR reverses altered neuronal excitability linked to Navi.6 channelopathy.

[0135] As ELIXIR potently inhibited the / NaL of the NavL6 N1768D variant, we next considered whether ELIXIR was able to rectify the altered in neuronal AP properties associated with this variant. To do so, we tested the effect of ELIXIR in a mouse model of neurodevelopmental disorder expressing the NavL6 N1768D variant5. Previous studies have shown that these mice develop seizures and present with SUDEP, consistent with clinical phenotypes. Moreover, these mice display aberrant neuronal AP spike properties, including AP prolongation, spike frequency adaptation, and depolarization block, reflecting an increased / NaL in various neuronal subtypes6’7. For this, we packaged ELIXIR, and an mCherry expression reporter, into an AAV9 vector and used intracerebroventricular injection to deliver this AAV to cortical neurons of P0 wild-type and N1768D homozygous knockin mice (N1768D+ / +). We then targeted layer V pyramidal neurons for whole cell patch clamp electrophysiology recordings from brain slices. Fluorescence imaging confirmed expression of ELIXIR (FIG.5A). Comparison of wild-type and N1768D+ / +neurons confirmed a prolongation of the AP duration (FIGs. 5B, 15A,and 15B), due to a slowing of the repolarizing phase (FIGs. 15C and 15D)2’6’7. ELIXIR evoked a significant reduction in the AP duration of N 1768D+ / +, but not wildtype neurons (FIGs. 5B, and 5C, and 15B). Analysis of AP spike properties showed a minimal change in AP firing rate in neurons from wild-type mice upon ELIXIR expression (FIGs. 5D, 5F, and 15E). By contrast, neurons from N1768D+ / +mice displayed premature depolarization block, which may contribute to hyperexcitability via impairment of inhibitory neurons8, that was significantly attenuated by ELIXIR expression (FIGs. 5E, 5G, 15E, and 15F). These results reveal that expression of ELIXIR is effective at reversing the altered neuronal AP spike properties observed in NavL6 channelopathies linked to epilepsy.Attorney Docket 44010.196WO-PCT / / CU24196ELIXIR Reduces and APD90 of CaMKIIδC,OEVentricular Myocytes

[0136] In HF, phosphorylation of Navi.5 by CaMKII results in elevated / NaL which contributes to increased risk of cardiac arrhythmias47’48’55. As such, we examined whether ELIXIR may reverse pathological INaLin adult mouse ventricular myocytes (aMVMs) isolated from a transgenic mouse model that overexpress the cardiac CaMKII isoform (CaMKIIδC,OE)56. Previous studies have shown that overexpression of CaMKII6c sufficed to induce HF in these mice56’57. Indeed, we observed morphological and electrophysiological changes in the hearts and cardiomyocytes of the CaMKIIδC,OEmice (FIGs. 16A-16F) that were indicative of hypertrophy, and consistent with HF. We utilized ELIXIR-cpp for short-term intracellular delivery of ELIXIR into aMVMs (FIG. 6A, ELIXIR-cpp). Flow cytometric analysis of non-transgenic (nTG) aMVMs treated with ELIXIR-cpp showed a concentration-dependent increase in FITC fluorescence intensity (FIGs. 6B and 6C), confirming uptake of ELIXIR-cpp by the myocytes. As in previous studies, / \ai. was quantified through whole-cell patch-clamp recordings as the magnitude of Na+current following 500 ms of depolarization normalized to the whole cell capacitance. In line with previous studies55, the CaMKIIδC,OEaMVMs displayed a 2-fold increase in / \ai. relative to aMVMs from nTG animals (FIGs. 6D-6F). Application of ELIXIR-cpp caused a small but statistically significant reduction in the / \ai. of nTG myocytes (FIGs. 6D and 6F). By comparison, incubation with ELIXIR-cpp had a much stronger effect on the / \ai. of CaMKIIδC,OEaMVMs, which was reduced to near wild-type levels (FIGs. 6E and 6F).

[0137] To further probe the potential functional consequences of ELIXIR-cpp on cardiomyocyte function, we undertook current clamp recordings to quantify changes in AP morphology. Consistent with the increase in INaL, the AP duration (APD) of CaMKIIδC,OEmyocytes was significantly prolonged relative to that of nTG myocytes (FIGs. 6G-6I). In addition to this, myocytes from the CaMKIIδC,OEmice had greater variability in the APD, quantified as short-term variability (STV, Figure 6J - 6I), than those from nTG animals. Remarkably, application of ELIXIR-cpp yielded a significant reduction in the APD (FIGs. 6H and 61) and STV (FIGs. 6K and 6L) of aMVMs from CaMKIIδC,OEmice, reversing both to near nTG levels. By comparison, application of ELIXIR-cpp caused essentially no change in APD or STV of nTG aMVMs. Collectively, these findings illustrate the ability of ELIXIR, applied extracellularly as a peptide, to tune native channel function.Attorney Docket 44010.196WO-PCT / / CU24196ELIXIR Inhibits Zxai, in LQT3 patient derived iPSC-CMs

[0138] While ELIXIR-cpp allows for short-term modification of Nav function in cardiomyocytes, constitutive expression of ELIXIR may be advantageous in pathophysiological settings. As such we used AAV9 to express ELIXIR in two models of cardiac diseases: (1) human cardiomyocytes differentiated from induced pluripotent stem cells (hiPSC-CMs) and (2) transgenic mice expressing a mutant Navi.5 (F1759Atg) (FIG. 7A).

[0139] First, we considered hiPSC-CMs derived from (1) an LQT3 patient heterozygous for the Nav1.5 ΔKPQ mutation25, and (2) from a healthy donor (HD)54. Following differentiation and maturation, spontaneous contractions were observed in both the HD and AKPQ hiPSC-CMs. Multichannel recordings of the HD hiPSC-CMs revealed low baseline levels of Z aL, that were minimally altered by AAV9-mediated expression of ELIXIR (FIGs.7B and 16G). By contrast, the AKPQ hiPSC-CMs exhibited markedly increased (~60-fold) / \ai.(FIGs. 7C, 7D, and 16H). Following transduction with ELIXIR, a ~7-fold reduction in / \ai. was observed in the AKPQ hiPSC-CM (FIGs.7C, 7D, and 16H). This underscores the efficacy of ELIXIR in diminishing / \ai. linked to LQT3 mutations in a more pathophysiologically relevant setting.

[0140] Second, we considered transgenic mouse model that inducibly expresses the lidocaine-resistant Navi.5 F1759A mutant channel. These mice have been previously reported to have an elevated / \ai. and prolonged QT interval. Subcutaneous injection was used to deliver an AAV9 encoding ELIXIR or GFP to P4 mice. Eight weeks after the injection of the virus, ventricular cardiomyocytes were isolated, and ELIXIR expression was confirmed by confocal imaging of mCherry fluorescence (FIG.7E). Multichannel recordings of cardiomyocytes from GFP-injected mice revealed the presence of late channel openings (FIG. 7F), with an ZxaL that was consistent with previous reports. By comparison, cardiomyocytes from ELIXIR-injected animals showed a significantly attenuated / \ai. as compared to those from the GFP control (FIGs. 7F and 7G). As increased / \ai. from Navi.5 manifests as a prolonged QT interval on the ECG and is the basis for LQT3 in humans, we obtained limb-lead surface electrocardiographic (ECG) recordings from both ELIXIR injected mice and GFP-control littermates (FIG. 7H). Comparison of the QT interval, corrected for heart rate using Bazett’s method (QTc), showed it was significantly shorter in the ELIXIR expressing mice than in the GFP expressing control animals (FIG. 71). We observed no significant differences in the RR or QRS intervals (FIG. 71). Collectively, these results confirm the effectiveness of long-termAttorney Docket 44010.196WO-PCT / / CU24196ELIXIR expression in tuning endogenous / \ai. in both human and murine models of Navi.5 dysfunction.DISCUSSION

[0141] Strategies to precisely manipulate ion channel function are highly sought after both as tools for unraveling their physiological functions and for developing new therapies. A prominent example of this concerns the family of Navi.1-1.9 channels, where gain-of-function variants are often associated with a broad range of human diseases including cardiac arrhythmia, myotonia, and epilepsy. Despite their varied clinical presentations, many of these diseases stem from a common deficit in Nav channel inactivation that leads to sustained Na influx known as / NaL. By leveraging structural insights and through a computational protein design framework, we developed a peptide modulator of Nav channels called ELIXIR programmed to promote inactivation. In depth analysis showed that ELIXIR binds to the Nav1.5CTDwith submicromolar affinity and can modulate “pathological” / \ai. of multiple Nav isoforms. We further demonstrate the efficacy of this synthetic ZxaL modulator to reverse pathophysiological changes in multiple disease models linked to Nav channel dysfunction. These findings suggest that ELIXIR could serve as a template for the development of a new class of rationally designed Nav channel modulators and further highlight the potential utility of de novo protein design for custom engineering of ion channel modulators in health and disease.

[0142] Many ion channels are large membrane-spanning multi-subunit complexes, the gating of which is controlled by intricate conformational dynamics. Consequently, engineering of synthetic ion channel modulators with desired functional effects is often challenging. To date, the prevailing strategy has been largely empirical, and focused on repurposing known ion channel modulators25’58or developing synthetic nanobodies that interact with key channel domains59. Recently, major advances in computational protein design, particularly deep learning approaches, have improved accuracy while also reducing the required computational time26'28’42’60, suggesting that these approaches may provide alternate avenues for developing ion channel modulators. Furthermore, the availability of open source and easy-to-use platforms have increased accessibility of these methods to the broader scientific community. While promising, experimental validation of de novo sequences generated by these algorithms and establishing their functionality in native biological settings has been challenging. For the in silico design of de novo protein-binders, only a small number of targets have been tested in vitro21’2and even fewer in situ or in vivo61. Here, we focused on NavL5 channels owing toAttorney Docket 44010.196WO-PCT / / CU24196their high pathophysiological relevance. We targeted the Nav1.5CTD, as this region has emerged as a regulatory hub that interacts with multiple auxiliary proteins to fine tune channel function. Furthermore, the Nav1.5CTDalso contains a large hydrophobic pocket that provides an attractive interface for binder design. With the use of the design protocol described here, we were able to generate a de novo peptide sequence that binds the Nav1.5CTDand tunes a specific aspect of Navi.5 function (i.e. / Na ) in both heterologous expression systems and primary cardiomyocytes. Our results highlight the applicability of these methods for developing novel ion channel modulators and lend further support to the general utility of currently available algorithms for targeting a wide array of proteins.

[0143] In the heart, multiple pathophysiological mechanisms have been linked to increased ZxaL. These include: (1) human Navi.5 mutations distributed across various channel domains62, (2) channel phosphorylation by CaMKII or PKA47’48, (3) reactive oxygen species (ROS)48or cellular metabolites63, (4) altered binding of channel interacting proteins46’64'66, and (5) diverse cell signaling mechanisms67'69. As such, considerable effort has been devoted to developing selective ZxaL inhibitors. In this regard, class Ib antiarrhythmics (e.g. lidocaine and mexiletine), which bind with rapid kinetics to a transmembrane site in Navi.5, have been shown to exhibit some preference for inhibiting / \ai8. Ranolazine, structurally similar to lidocaine, has been found to exhibit an even greater preference for inhibiting INaL. High-resolution structures have been solved of Navi.5 bound to several of these antiarrhythmics22’23and in each the drug is bound to a nearly identical site in the channel pore. Importantly, this shared binding site likely limits the selectivity of these molecules for late versus peak Na+current as their interaction obstructs ion flux through the pore. Instead of targeting the pore domain, our approach was to leverage the natural inactivation mechanism of Nav channels. Specifically, we sought to promote inactivation by enhancing the release of the IG from the Navi.5 CTD through the application of a competitive binder to this site (ELIXIR). This strategy has two advantages. First, by not targeting the channel pore or the voltage-sensing domains, ELIXIR is unlikely to significantly perturb channel activation. Experimentally, we found that ELIXIR had minimal effect on peak current density and voltage-dependence of activation (FIGs. 11A-11C). Second, ELIXIR is unlikely to exert substantial effects on channels that have no baseline defect in inactivation. Specifically, for wild-type channels, the DIV VSD movement is considered a rate limiting step for both the development of and recovery from inactivation33. Without the movement of DIV VSD, it is unlikely that the pore-proximal site is available for interaction with the IFM motif. In this case, displacement of the IG by ELIXIR may not promote inactivation. One interesting possibility hinted by our results here is that in pathophysiologicalAttorney Docket 44010.196WO-PCT / / CU24196settings, the release of IG from the CTD may become a limit step in addition to DIV VSD movement. As a result, promoting IG release by ELIXIR would be expected to boost inactivation. This aspect is potentially important as it could lend ELIXIR selectivity for tuning pathogenic 7NaL. Experimentally, we found that ELIXIR had minimal effects on / \ai. of unmodified wild-type Nav. channels. Nevertheless, ELIXIR was still effective at inhibiting / \ar resulting from (1) diverse NavL5 mutations (FIGs. 4A-4E) and (2) channel phosphorylation by PKA or CaMKII (FIGs. 4F and 4G). A corollary is that the generality of ELIXIR suggests that these seemingly distinct perturbations all converge on a common endpoint, diminished ability of the IG to bind the pore-proximal site. For some NavL5 perturbations examined, ELIXIR did not fully reverse the increase in ZxaL. This partial inhibition could stem from incomplete disruption of the IG-CTD interaction by ELIXIR or may reflect other structural defects caused by these perturbations. Further optimization of ELIXIR through affinity maturation or the production of mutation-specific binders could help address this shortcoming. Of note, we recently found the endogenous Nav channel interacting protein Fibroblast growth factor homologous factor 1A (FHF1A) inhibits / \ai25. We were able to engineer a 39-amino acid FHF1A fragment (FHF-inhibiting X-region, FixR) as an ZxaL inhibitor. However, the molecular mechanism of FixR is not fully established. It is likely that FHFs utilize a distinct mechanism as compared to ELIXIR as FHFs, unlike ELIXIR, promote inactivation of the A1810 mutant that lacks the CTD-IG interface. It is possible that the two peptides may be beneficial in different settings depending on the specific perturbations in channel function. Also, as A-type FHFs are natively present in various tissues, the ultimate utility of FixR may be limited. By contrast, ELIXIR as a de novo peptide could be more broadly applicable.

[0144] Beyond cardiac maladies, Nav channel malfunction has been implicated in a variety of diseases including epilepsy syndromes70, myotonia5, and chronic pain71. The high degree of sequence similarity in the Nav EFL (FIG. 4A) suggests that ELIXIR may function as an ZxaL inhibitor for channelopathies across the Nav family, an attractive possibility given the range of diseases associated with dysfunction of these channels. Consistent with this, we found that coexpression of ELIXIR was sufficient to reverse the increase in INOL observed with both NavL4 mutation F1698I variant linked to a cold-aggravated myotonia as well as the well-studied NaV1.6 N1768D variant linked to epilepsy. Moreover, slice electrophysiological recordings showed key changes in AP properties of layer V cortical neurons in the homozygous N1768D mice including slower repolarization of the AP and increased presence depolarization block, consistent with previous studies. Viral expression of ELIXIR was sufficient to partially reverse these changes in neuronal function, pointing to the potential effectiveness of ELIXIR in theAttorney Docket 44010.196WO-PCT / / CU24196nervous system. More broadly, the Nav CTD has an architecture similar to the C-termini of other ion channels, including the voltage-gated calcium channels14and the sodium leak channel NALCN13. In high-resolution structures of these channels, the EFL region of the CTD is bound to the domain III - domain IV linker13,14, hinting at conserved modulatory mechanisms. If so, our design strategy may be broadly effective for developing selective actuators of this physiologically important channel superfamily.

[0145] Three key limitations merit further attention. First, high resolution atomic structures of ELIXIR bound to Nav channels are not available. Nevertheless, structural predictions of ELIXIR / CTD complex by various algorithms all converge to a nearly identical interface. Experimentally, mutation analysis of ELIXIR residues is consistent with the predicted interface. Second, therapeutic application of ELIXIR may be impaired by the requirement for intracellular delivery. Our studies utilized two possible approaches, namely fusion with a cell penetrating peptide and viral delivery of ELIXIR. Both approaches were effective for robust delivery and reversal of INaL. Nevertheless, these strategies have potential limitations for in vivo applications including immunogenicity. Other emerging protein delivery approaches may also be beneficial for ELIXIR. Future studies will address these possibilities and assess the effectiveness of potential intracellular delivery mechanisms. Alternatively, it may be possible to identify small molecules that may interact with the IG interacting pocket of Navi CTD and mimic the action of ELIXIR. In this regard, the change in fluorescence anisotropy with ELIXIR may be advantageous for establishing in vitro high-throughput screens. Third, with viral expression of ELIXIR, we were unable to estimate a concentration as the mCherry marker is bicistronically expressed. It is possible that the concentration of ELIXIR may be supramaximal, which may cause off-target effects. Future in vivo studies will consider dose-dependence of ELIXIR expression.

[0146] Although ion channel dysfunction has been long associated with multiple lifethreatening diseases, these proteins have often been considered challenging targets for drug development72. A key difficulty is that in pathophysiology, the functional change often involves alterations in dynamic channel behavior that disrupts the cellular electrical phenomenon. As such, simply turning an ion channel on or off is often insufficient. Our results demonstrate the effectiveness of computational protein design for the development of synthetic proteins / peptides that tune channel dynamics. This ability could turbocharge the creation of innovative strategies to reverse pathophysiology of ion channelopathies and enable precision engineering of nature’s transistors.Attorney Docket 44010.196WO-PCT / / CU24196MATERIALS AND METHODSMolecular Biology

[0147] The human Navi.5 sequence used in this work corresponds to GenBank accession number M77235.1 and was expressed from a pGW vector, as described previously25. The IQ / AA mutation in the channel CTD and AKPQ mutation in the inactivation gate were generated using overlap-extension PCR followed by ligation into the NruI and XbaI, and KpnI and XbaI restriction sites, respectively. The Al 864 truncation was made by PCR amplifying the relevant segment using a forward primer upstream of the Kpnl site and reverse primer that truncated at position 1864, which included an Xbal site for ligation into the Navl.5 / pGW construct. A gene fragment containing the DNA sequence of the designed peptide (MSPRREALYRGFRACYDVLRH, SEQ ID NO. 2) was purchased from Synbio technologies and subcloned into a pIRES2-eGFP vector using the NheI and BamHI restriction sites. For FRET experiments the Venus-tagged NavE5cTD (Navi.5 residues 1771-2016) and Cerulean-tagged ELIXIR (WT, L8A, F12E, and Y16A) were expressed from a pCDNA3 vector. The PKA catalytic domain and CaMKIIT286Dwere expressed from a pCDNA3.1 plasmid as previously reported73. The WTNavl.l, Navi.4, Navi.6, and Navi.7 were expressed from pIR-CMV-IRES-mScarlet, pCDNA3.1, pCDNA4TO-IRES-mScarlet, and pCMV plasmids, respectively. For bacterial overexpression and purification, the Nav1.5CTD(Nav1.5 residues 1773-1940) was expressed as a GST-fusion inserted between the BamHI and XhoI sites of a pGEX-6P vector. Calmodulin was cloned into a pRSF plasmid using the NcoI and XhoI sites. QuickChange mutagenesis was used to substitute the single cysteine residue (C15) with a serine to generate the final ELIXIR peptide sequence (MSPRREALYRGFRASYDVLRH, SEQ ID NO. 3), to produce the binding-deficient ELIXIR mutants (L8A, F12E, Y16A) and the Navi.6 N1768D mutant channel.Peptide Design

[0148] Peptide design was done using the AfDesign algorithm implemented in ColabDesign30. The coordinates for the Navl.5cTD were taken from an x-ray crystallographic of the Navl.5cTD bound to calmodulin and FHF2u (PDB ID: 4DCK)43. For the first round of design, residues 1786-1802 and 1850-1887 of Navi.5 were used as a hotspot, the binder length was set to 21 residues, and the number of recycles, and models used were 1 and 2, respectively. The design protocol was then run with a semi-greedy optimizer (pssm semigreedy) and the default gradient decent settings. The pssm semigreedy protocol starts by optimizing, via gradient descent, a softmax representation of the sequence. This is done for 120 steps. ThisAttorney Docket 44010.196WO-PCT / / CU24196results in a vector of shape binder length (21) that we call PSSM (position specific scoring matrix), which represents the probability of each amino acid at each position. For the starting sequence, the amino acid at each position is selected that maximizes the probability. For the semi-greedy protocol, at each step, 10 independent mutations are evaluated and the mutation with best loss is fixed. The positions are sampled following 1-pLDDT74and the amino acid identity following PSSM. This is done for 32 steps. The sequence with best loss across the 32 steps is selected. The loss combines pLDDT over the binder positions plus contact score: 0.1 * (1-pLDDT) + 1.0 * contact score. The contact score minimizes the entropy of the distogram output (distance distribution between cP atoms) between the target and the peptide. More specifically, every peptide position must have at least one confident distance prediction to any target position, unless a hotspot is defined. The protocol is available via Google Colab at: colab.research.google.com / github / sokrypton / ColabDesign / blob / v1.1.1 / af / examples / peptide_binder_design.ipynb. Following the first round of design, the final peptide sequence was used as a seed to initialize the next round. This process was repeated a total of five times. The peptide sequence was then screened computationally by comparing the interface between the Navl.5cTD and the peptide in models of the complex generated with ColabFold40, ESMFold41, OmegaFold26and RoseTTAFold42. Contacts of structural units analysis was done with the COCOMAPS webserver75using cutoffs of 6.0 A and 4.5 A.Protein Expression and Purification

[0149] For protein expression Escherichia Coli BL21 (DE+) cells were co-transformed with plasmids for both the Nav1.5CTDand CaM and grown in 0.5 L cultures of CIRCLEGROW supplemented with carbenicillin (0.1 mg / mL) and kanamycin (0.05 mg / mL). The cells were grown at 37 °C until OD600of 0.7 was reached at which point expression was induced by the addition of 0.5 mL of 1 M IPTG and the temperature was set to 18°C. Cells were harvested the following morning by centrifugation (4,000 rpm, 4°C, 15 min) and stored at -20°C.

[0150] The Nav1.5CTDwas purified as described previously76. Cell pellets were thawed and resuspended in lysis buffer (50 mM HEPES, 500 mM KC1, 50 μM EGTA, 0.01% (w / v) NaNs, pH 7.4), disrupted via emulsification, and centrifuged (25,000 rpm, 4 °C, 50 min). The supernatant was then filtered (0.2 μM), mixed with 5 mL of Glutathione Sepharose 4B, and rocked at 22°C for 30 min. The resin was then rinsed with 100 mL of lysis buffer, and the Nav1.5CTD-GST+CaM complex was eluted in 15 mL of elution buffer (lysis buffer + 10 mM glutathione). The GST-tag and glutathione were removed by the addition of precision protease and overnight dialysis against lysis buffer, respectively. The sample was then mixed with 5 mLAttorney Docket 44010.196WO-PCT / / CU24196of glutathione resin and the Nav1.5CTD+CaM complex was collected and concentrated. Anion exchange chromatography (pH 8.5 to 7.4 and KC10 to 250 mM) was then used to separate the Navl.5cTD and CaM. Protein purity (>95%) was assessed by SDS-PAGE and UV-vis spectroscopy. Protein concentration was determined from the absorbance at 280 nm.Fluorescence Anisotropy

[0151] The ELIXIR peptide, with an N-terminal fluorescein and TAT sequence (YGRKKRRQRRRGSGMSPRREALYRGFRASYDVLRH, SEQ ID NO. 4, theoretical MW: 4826.48 g / mol), was synthesized by GenScript. Binding of the Nav1.5CTDto the ELIXIR peptide (100 nM) was monitored in 50 mM HEPES, 100 mM KC1, 1 mM MgCh, 100 mM EGTA, 0.01% (w / v) NaNs, 1 mM DTT, pH 7.4 at 22 °C. The fluorescence anisotropy (λem= 494 nm, λex= 518 nm, 5 nm excitation and emission bandpasses, 4 sec integration) was monitored throughout the titration with a fluorolog fluorometer (Horiba). Anisotropy (R) was calculated with,R = (IVV−GIVH) / IVV+2GIVHwhere Ivv and IVH are the intensities of the vertically excited and vertically emitted, and vertically excited and horizontally emitted light, respectively. G was determined prior to each titration as the ratio between the horizontally excited and vertically emitted light, and the horizontally excited and horizontally emitted light (IHV / IHH). Each data point was the average of the three measurements.

[0152] The normalized anisotropy was fit to Eq 2 with the Solver add-in in Excel,Y = Ka[NaV1.5CTD]free1+Ka[NaV1.5CTD]free

[0153] where [Nav1.5CTD]free was determined with Eq 3[NaV1.5CTD]free= (−b ± √(b²−4Ka(−[NaV1.5CTD]total)))2Ka

[0154] In Eq 3, b was (l+Ka[ELIXIR peptide]-Ka[Nav1.5CTD]totai). The reported KD and standard deviation were determined from 3 replicate titrations.Attorney Docket 44010.196WO-PCT / / CU24196Cell Culture and Transfection

[0155] HEK293 cells (ATCC CRL1573) were cultured in 60-mm dishes on glass coverslips and transfected with the Ca2+-phosphate method. For electrophysiology recordings, cells were co-transfected with 4-10 pg of cDNA encoding the desired Nav channel variant, 1 pg of YFP, and 0.5 pg of simian virus 40 T-antigen ± 3 pg of ELIXIR. For experiments investigating the phosphorylation-dependent late Na+current, 3 pg of cDNA encoding the catalytic domain of PKA or CaMKIIT286Dwas used. Four hours after transfection, the cells were rinsed, and the media replaced. Electrophysiological recordings were performed at room temperature 1-2 days after transfection.Whole-Cell Recordings

[0156] Whole-cell electrophysiology recordings were conducted at room temperature with an Axopatch 200B amplifier (Axon Instruments). Pipettes contained (in mM): 114 CsMeSO3, 5 CsCl, 1 MgCl2, 4 Mg-ATP, 10 HEPES (pH 7.4), and 10 BAPTA (l,2-bis(o-aminophenoxy)ethane-N, N, N', N'-tetraacetic acid), at adjusted to 290 mOsm with glucose. The bath contained (in mM) 20 NaCl, 1 CaCl2, 10 HEPES, pH 7.4 (adjusted with NaOH), at 300 mOsm (adjusted with glucose). Pipettes were pulled from borosilicate glass (World Precision Instruments, MTW 150-F4), with a horizontal micropipette P-97 puller (Sutter Instruments) and fire polished with a microforge (Narishige). Pipettes typically had a resistance of 2-3 MQ, which was >70% compensated. An ITC-18 (Instrutech), controlled by custom MATLAB software (Mathworks), was used during data acquisition. Currents were low-pass filtered at 2 kHz before being digitized at several times that frequency. P / 8 leak subtraction was used. Cells were kept at a holding potential of -120 mV.Multichannel Recordings

[0157] Multichannel recording from HEK293 cells, iPSC-CMs, or aMVMs were obtained in the on-cell configuration as in our previous work24’25. Pipettes contained a buffer comprised of (in mM): 140 NaCl, 10 HEPES, 1 CaCl2, pH 7.4 (adjusted with tetraethylammonium hydroxide), and at 300 mOsm (adjusted with tetraethylammonium methanesulfonate). To zero the membrane potential, the bath contained (in mM): 132 K-glutamate, 5 KC1; 5 NaCl, 3 MgCl2, 2 EGTA, 10 glucose, 20 HEPES, pH 7.4 (adjusted with NaOH), at 300 mOsm (adjusted with glucose). For experiments monitoring the dose-dependent effect of ELIXIR on INaL, HEK293 cells were treated with the indicated concentration of ELIXIR-cpp at 30°C for 4 hours after which multichannel recordings were collected. Recordings were conducted at roomAttorney Docket 44010.196WO-PCT / / CU24196temperature using the integrating mode of an Axopatch 200A amplifier (Axon Instruments, Molecular Devices). Patch pipettes were pulled from ultra-thick-walled borosilicate glass (BF200-116-10; Sutter Instruments) using a horizontal puller (P-97, Sutter Instruments), fire polished with a microforge (Narishige) and coated with Sylgard (Dow Coming). Pipettes typically had a 4 - 7 M resistance. Elementary currents were low-pass filtered at 2 kHz with a four-pole Bessel filter and digitized at 200 kHz with an ITC-18 (Instrutech) analog-to-digital converter, controlled by custom MATLAB software (Mathworks). For every pulse P / 8 leak pulses was obtained. Leak subtraction was done with an automated algorithm that fit the kinetics of the leak current and capacitive transient using convex optimization with LI regularization of the following objective function in equation 4: / Imine,s(½|IL− De − s|²2+ λ|s|1)e,s \2s.t. e > 0, s < 0, and s(t < t0) = 0 (4)

[0158] Here, ZL is the leak waveform that is to be fit as a sum of exponentials; D is a matrix composed of a library of exponential functions with various time constants; e is a sparse vector composed of the amplitude of each exponential; 5 is a vector representing an estimate of negative outliers from the baseline that correspond to channel openings; and 1 is a penalty term set to be 0.25. After leak subtraction, the unitary current for each patch was estimated from an amplitude histogram and each trace was subsequently idealized. The ensemble average from 50 - 120 stochastic traces was computed for each patch and normalized to the peak current. The average late current for each patch (persist) was computed as the average normalized Po following 50ms of depolarization. Concentration-response data were analyzed using a nonlinear least squares fit with a Hill equation (Graphpad Prism 10). The upper and lower plateau values of the curve were fixed to the Rpersit value of Nav1.5 ΔKPQ expressed alone (upper) and co-expressed with ELIXIR (lower).Flow Cytometric FRET

[0159] The flow cytometric two-hybrid binding assays were performed as previously described. HEK293 cells (ATCC CRL1573) cultured in 12-well plates were transfected with linear polyethylenimine 25 kDa polymer (PEI, Polysciences #2396602) using a 2:1 PEI to DNA mass ratio. For each experiment cells were transfected with 3 pg Venus-Nav1.5CTD, 1 pg Cerulean-ELIXIR, and 0.5 pg T antigen, after which cells were cultured for 2 days. To enhanceAttorney Docket 44010.196WO-PCT / / CU24196the level of mature fluorophores protein production was inhibited with Cycloheximide, 100 pM, 2 hours before fluorescence measurements were made.

[0160] Fluorescence was measured with an LSRII (BD Biosciences) flow cytometer, equipped with 405 nm, 488 nm and 633 nm lasers for excitation and 18 emission channels. Healthy single cells were selected based off the forward and side scatter signals. FRET efficiency was determined by measuring three different fluorescent signals: 1) Cerulean emission following direct excitation (Seer) through the BV421 channel (excitation 405 nm, emission 450 / 50), 2) Venus emission through direct excitation (Sven) with the fluorescein isocyanate channel (excitation 405 nm, dichroic 505 LP, emission 525 / 50) and 3) Venus emission due to FRET (SFRET) through the BV510 channel (excitation 405 nm, dichroic 505 LP, emission 525 / 50). These measurements were used to obtain Cerairect (Cerulean emission due to direct excitation), Venairect (Venus emission due to direct excitation) and FRET efficiency. Flow cytometric signals were collected at a medium rate (2,000 to 8,000 events / sec) and the data were exported as Flow Cytometry Standard 3.0 files for further processing and analysis with custom MATLAB software (Mathworks).

[0161] As previously described, several control experiments were conducted for experimental run of the flow cytometer. Firstly, the background fluorescence level of each channel (BGcer, BGven, and BGFRET) was determined using cells that were not exposed to any fluorophore containing plasmid. Secondly, cells expressing only Venus were used to determine the spectral crosstalk parameter RAI, which corresponds to the bleed-through of Venus fluorescence into the FRET channel. Thirdly, cells expressing Cerulean alone were used to measure the spectral crosstalk parameters RDI, and RD2, corresponding to the bleed-through of Cerulean fluorescence into the FRET and Venus channels, respectively. Fourth, the instrumentspecific calibration parameters fven / fcer and gven / gcer, corresponding to the ratios of the fluorescence excitation and emission of Venus and Cerulean, respectively, were measured on the day of the experiment using Cerulean-Venus dimers with known FRET efficiencies. Fifthly, cells co-expressing Cerulean and Venus were used to estimate concentrationdependent collisional FRET. For each cell, spectral crosstalk was then accounted for with Eq 5-7.Cerdirect= RD1× SCer(5)Vendirect= RA1× (SVen− RD2× SCer) (6)VenFRET= SFRET− RA1× (SVen− RD2× SCer) − RD1× SCer)Attorney Docket 44010.196WO-PCT / / CU24196

[0162] After unmixing the spectra, fA / fD and gA / gD were obtain by from the Cerulean-Venus dimer data by finding the slope and intercept of the linear relationship shown in Eq 8.VenFRET / Cerdirect= (gCer / gVen) × (Vendirect / Cerdirect) − fVen / fCerCer direct 9Ven ^erdirect f Cer

[0163] Donor-centric FRET efficiencies (ED) were then calculated with Eq 9.ED= VenFRET / (VenFRET+ (fVen / fCer) × Cerdirect)

[0164] The relative proportion of Cerulean (Neer) and Venus (Nven) fluorophores in each cell were then determined from Eq 10 and 11, respectively.NCer= Cerdirect / (1−ED) (10)NVen= Vendirect / (gVen / gCer) × (fVen / fCer)3Cer fcer

[0165] A 1:1 binding isotherm was then imposed as in previous studies, by iteratively fitting the data to Eq 12,ED= EMax× VenFree / (VenFree+ KD,EFF) (12)T / _„. j,VenFree+KD,EFF)

[0166] where VenFree is determined with Eq 13,VenFree= NVen− (NCer−CerFree) (13)

[0167] in which CerFree is computed from Eq 14.CerFree= 0.5(NCer− KD,EFF− NVen) + 0.5(√(NCer− KD,EFF− NVen)2+ 4NCerKD,EFF))(14)

[0168] For each FRET pair, KD,EFF, EMax, and 95% confidence intervals were obtained through iterative constrained least squares analysis. Data were obtained from two independent transfections.Attorney Docket 44010.196WO-PCT / / CU24196Phylogenetic tree construction

[0169] The sequences of the nine human Nav CTDs were obtained from UniProt. The residues used for each isoform were: NavLl: 1787-1954; Navi.2: 1777-1944; Navi.3: 1772-1939; Navl.4: 1599-1776; Navi.5: 1773-1940; NaV1.6: 1767-1934; Navi.7: 1761-1928; Navi.8: 1723-1890; Navi.9: 1605-1772. The sequences were aligned with the BlastP algorithm and the phylogenetic tree constructed using the BLAST server.Aden-associated virus Generation

[0170] Adeno-associated virus for the expression of ELIXIR, and control virus for the expression of GFP, were purchased from VectorBuilder. To monitor the expression of ELIXIR it was cloned with a C-terminal P2A site followed by mCherry (ELIXIR-P2A-mCherry).Quantification of ELIXIR-cpp Uptake by aMVMs

[0171] Uptake of the ELIXIR-cpp was monitored using the same peptide as used in the fluorescence anisotropy experiments. Freshly dissociated aMVM were plated in a 12 well plate and incubated with different concentrations (100 nM-10 pM) of ELIXIR-cpp for 2 hours at 37 °C in Tyrode’s solution (135 mM NaCl, 4mM KC1, 1 mM MgCh, 10 mM HEPES, 1 mM CaCh (pH 7.4 using NaOH)). Cellular uptake was monitored with an LSR II (BD Biosciences) flow cytometer equipped with 405 nm, 488 nm, and 633 nm lasers for excitation and 18 different emission channels as previously described25. Forward and side scatter signals were detected and used to gate for single cells that were living. Flow cytometric signals were collected at a medium flow rate (2k - 8k events / sec). Histograms corresponding to the singlecell population of the fluorescein channel were analyzed using custom MATLAB software (Math works).Mouse ModelsNavi.6 N1768D mice and brain slice electrophysiology

[0172] Congenic C57BL / 6J mice heterozygous for the Scn8a-N1768D allele were crossed with each other to generate experimental Scn8aN1768D / N1768Dmice and Scn8a+ / +littermate controls. Mice were kept in the vivarium on a 12-h light-dark cycle. Mice had access to regular chow and water, ad libitum. Mice were bred and procedures were conducted at the Columbia University Institute of Comparative Medicine, which is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care and were approved by the Columbia Institutional Animal Care and Use Committee (protocol #AC-AABH551).Attorney Docket 44010.196WO-PCT / / CU24196

[0173] Intracranial injection of adenovirus containing ELIXIR-mCherry was performed at P0 or P1. Pups were injected under hypothermic anesthesia. Bilateral intracerebroventricular injections of virus (1 pL) were performed using a Hamilton syringe to induce widespread expression in the cortex. Pups were quickly rewarmed and returned to their home cage following injection.

[0174] Slice electrophysiology was conducted at P18-P22, as homozygous expression of the Scn8a-N1768D allele causes early mortality starting at this age5. Animals were deeply anesthetized with isoflurane and decapitated, and the brain was quickly dissected into ice-cold high-sucrose slicing solution (26 mM NaHCO3, 2.5 mM KC1, 1.25 mM NaH2PO4, 10 mM MgCl2, and 0.5 mM CaCl2, 11 mM d-glucose, 234 mM sucrose, pH 7.3-7.4). 250 pm slices were cut using a Leica VT1500 Vibratome. Slices were transferred to 37°C aCSF (126 mM NaCl, 26 mM NaHCO3, 2.5 mM KC1, 1.25 mM NaH2PO4, 1.0 mM MgCl2, and 2.0 mM CaCl2, 10 mM d-glucose, pH 7.3-7.4) and incubated for 1 hour. All solutions were continuously bubbled with 95% 02 and 5% CO2. Individual slices were transferred to a recording chamber located on an upright microscope (BX51WI; Olympus) and were perfused with heated (32°C) oxygenated aCSF.

[0175] Whole-cell current-clamp recordings were obtained using Multiclamp 700B and Clampex 10.7 software (Molecular Devices). Layer V pyramidal cells were targeted for patching based on location, size, and shape. Cells expressing ELIXIRmCherry were identified using a fluorescent light source (Olympus). Both ELIXIRmCherry-expressing and nonexpressing cells were targeted for patch clamp to generate within-subject control data. Intracellular solution contained 127 mM K-gluconate, 10 mM HEPES, 8 mM NaCl, 0.6 mM EGTA, 4 mM ATP, and 0.3 mM GTP, pH 7.2. When patch electrodes were filled with intracellular solution, their resistance ranged from 3.5 to 6 MQ. Access resistance was monitored continuously for each cell and any cell that changed by more than 15% was excluded from analyses.

[0176] Holding current was delivered to adjust resting membrane potential to -75 mV. AP-generating properties of neurons were tested by injecting 1 sec square pulses of depolarizing current incrementing in 20 pA steps, up to 1 nA. AP threshold was defined as the membrane voltage at which the slope crossed 5 mV / ms. APs were automatically identified using this slope threshold and visually confirmed. APD90 was defined as the time during which the membrane voltage exceeded 10% of the peak amplitude. Half-width was defined as the time during which the membrane voltage exceeded 50% of the peak amplitude. Maximum rise rate was defined as the maximum of the first derivative of the rising phase of the AP. Maximum fall rate wasAttorney Docket 44010.196WO-PCT / / CU24196defined as the minimum of the first derivative of the falling phase of the AP. Depolarization block threshold was defined as the current step at which the number of APs produced fell to less than ’A of the maximum. Brain slice electrophysiology data was analyzed offline in MATLAB.Enzymatic isolation of ventricular CaMKIIδc,OE cardiomyocytes

[0177] Young adult (8 to 10-wk-old, male and female) CaMKIIδc,OE mice and WT littermate control mice in the C57BL6 / J background were used. Mice were kept at standard temperature, humidity, and lighting. Food (Teklad, 2018) and fresh, sterile drinking water were provided ad libitum. Before terminal surgery, mice were injected with heparin (400 U / kg body weight) and anesthetized with isoflurane (5% in an induction chamber, then 1.5 - 3% via nose cone). Hearts were excised and retrograde perfused on a constant flow Langendorff apparatus (4 min, 37 °C) with Ca2+-free normal Tyrode’s solution, gassed with 100% O2. The heart was then perfused for 12 - 17 min with 100 mg collagenase (type 2, Worthington Biochemical Corp) and 1.4 mg protease (type XIV, Sigma-Aldrich) in 50 mL of Tyrode’s solution (with 10 pmol / L Ca2+) to enzymatically isolate cardiomyocytes. Following digestion, the myocytes were gently triturated with a pipette, then filtered through a nylon mesh and allowed to sediment for ~ 10 min. The sedimentation was repeated three times with increasing [Ca2+] from 0.125 to 0.25 to 0.5 mmol / L. Finally, ventricular myocytes were kept in Tyrode’s solution (0.5 mmol / L Ca2+) at room temperature until use. All animal handling procedures were in strict compliance with an approved protocol (#23175) of the Institutional Animal Care and Use Committee at the University of California, Davis conforming to the NIH Guide for the Care and Use of Laboratory Animals (8th edition, 2011).CaMKIIδc,OE Cardiomyocyte

[0178] Isolated single murine ventricular cardiomyocytes were placed in a temperature-controlled perfusion chamber (Warner Instruments) and mounted on a Leica DMI3000 B inverted microscope (Leica Microsystems). Cells were bathed at 37°C, for 10 minutes, before starting, and continuously perfused (2 mL / min) during experiments with Tyrode’s solution containing (in mmol / L): NaCl 140, KC1 4, CaCh 1.8, MgCh 1, HEPES 5, Na-HEPES 5, glucose 5.5; pH=7.4. Electrodes were fabricated from borosilicate glass (World Precision Instruments) and had tip resistances of 2 to 3 MΩ when filled with internal solution which contained (in mmol / L): K-aspartate 100, KC1 30, NaCl 8, Mg-ATP 5, phosphocreatine dipotassium salt 10, HEPES 10, EGTA 0.01, cAMP 0.002, and calmodulin 0.0001; pH=7.2 (with KOH). Using this internal solution, the intracellular Ca2+transient and contraction of theAttorney Docket 44010.196WO-PCT / / CU24196cardiomyocyte are preserved. An Axopatch 200B amplifier (Axon Instruments Inc.) was used for recording and signals were digitized at 50 kHz by a Digidata 1322A A / D converter (Axon Instruments) under software control (pClamp10.4). Series resistance was typically 3 to 5 MΩ and was compensated by >90%. Experiments were discarded when the series resistance was high or increased by >20% during the recordings. All experiments were conducted at 37±0.1°C.

[0179] APs were recorded in whole-cell current-clamp conditions where cells were stimulated with supra-threshold depolarizing pulses (2 ms duration) delivered via the patch pipette at 1 Hz. AP duration at 90% repolarization (APD90) was used to characterize AP repolarization. Series of 50 consecutive APs were analyzed to estimate short-term variability of the APD90. Short-term variability was calculated with equation 5,STy _ — ■‘QP^nD / (nbeats−1) × √2where APDnand APDn+1indicate the durations of the nthand (n+l)thAP, and nbeats denotes the total number of consecutive beats analyzed.

[0180] For voltage-clamp late Na+current measurements, internal solution contained (in mmol / L): CsCl 110, tetraethylammonium chloride 20, Mg-ATP 5, HEPES 10, phosphocreatine di sodium salt 5, calmodulin 0.0001, EGTA 10, CaCl24.1 (free [Ca2+] = 100 nmol / L), pH=7.20. Bath solution contained (in mmol / L): NaCl 140, CsCl 4, CaCl21.8, MgCl21, HEPES 5, Na-HEPES 5, glucose 5.5, 4-aminopyridine 5, nifedipine 0.01, pH=7.4. The late Na+current magnitude was measured at the end of a 500 ms depolarizing pulse from a holding potential of -120 mV to -40 mV. The current amplitude was normalized to the capacitance (late Na+current density) of each cell. Chemicals and reagents were purchased from Sigma-Aldrich. A subset of cells was preincubated with ELIXIR-cpp (3 pmol / L, 4 hours).Navi.5 F1759A adeno-associated virus infection

[0181] The Institutional Animal Care and Use Committee at Columbia University approved all animal experiments (#s AC-AABH550). The Navi.5 F1759A mouse line was previously generated by breeding Navi.5 F1759A mice, in a B6CBA / F2 hybrid background, with cardiac rtTA mice in a FVB / N background to generate doxycycline-inducible transgenic mice (Marx lab paper Wan et al 2015). Mice were kept on a 12-hour light-dark cycle, and fresh food and water were provided ad libitum. In all experiments both male and female mice were used. At postnatal day 4, Navi.5 F1759A mouse pups were administered 1.03×1011GC / g of adeno-associated virus 9 encoding either GFP or ELIXIR through a dorsal subcutaneousAttorney Docket 44010.196WO-PCT / / CU24196injection. ECG and electrophysiology experiments were conducted eight weeks after the injection. Prior to experimentation, the expression of Navi.5 F1759A was induced with doxycycline chow overnight. Ventricular myocytes were isolated as described above.Confocal Imaging

[0182] Images of freshly dissociated myocytes were collected on a Nikon Ti Eclipse inverted microscope equipped with a Yokogawa CSU-X1 spinning disk confocal and a CMOS camera from Andor Zyla scientific using a 60x oil-immersion objective.ECG Analysis

[0183] Four-lead electrocardiograms were recorded from isoflurane-anesthetized mice using an Emka ECG and IOX software (Emka Technologies). The RR QRS and QT durations were measured manually with IOX software. The QT duration was corrected for heart rate using Bazett’s method with equation 16,where QT and RR are the measured QT and RR interval, respectively.Differentiation of Induced-Pluripotent Stem Cell Derived Cardiomyocytes

[0184] Induced pluripotent stem cells were obtained and differentiated into cardiomyocytes as described previously25. Prior to differentiation human iPSCs were maintained in mTeSR medium (Stem Cell Technologies) and passaged every 4 - 6 days onto Matrigel-coated plates (Coming). On the first day of differentiation (day 0), embryoid bodies (EBs) were generated by treating the iPSCs with 1 mg / mL Collagenase B (Roche, Cat #: 11088807001) for 1 h, or until cells detached from the plate. The cells were then collected and centrifuged at 300 ref for 3 min and resuspended as small clusters of 50-100 cells by gentle pipetting in differentiation medium (RPMI 1640 (Thermo Fisher Scientific, Cat #: 11875085) with 2 mM L-glutamine (Thermo Fisher Scientific, Cat #: 25030149), 0.4 mM monothioglycerol (Millipore Sigma, Cat #: M6145), and 50 pg / mL ascorbic acid (Millipore Sigma, Cat #: A4403)). Differentiation medium was supplemented with 2 ng / mL BMP4 (R& D Systems) and 10 pM Rock inhibitor (Y-27632 dihydrochloride, Tocris Fisher Cat #: 1254 / 50). EBs were cultured on ultra-low attachment 6-well plates (Corning Costar, Cat #: 3471) in a humidified incubator at 37°C, 5% CO2, and 5% O2. On day 1, the media was changed toAttorney Docket 44010.196WO-PCT / / CU24196differentiation media supplemented with 20 ng / mL BMP4 (R& D Systems), 20 ng / mL Activin A (R& D Systems), 5ng / mL bFGF (R& D Systems). On day 3, EBs were harvested and washed once with RPMI 1640. Medium was changed to differentiation media supplemented with 5 ng / mL VEGF (R& D Systems) and 5 pM XAV939 (Reprocell-Stemgent, Cat #: 04-0046). From this point on, every 2-3 days media was replaced with media supplemented with only 5 ng / mL VEGF (R& D Systems). Following differentiation, the cells were allowed to mature for 20 - 28 days before being used in experiments.Statistical Analysis

[0185] Pooled data are presented as the mean ± SEM. Statistical analysis was performed in Graphpad Prism 10.0.0. Normality was tested using a D’Agostino-Pearson normality test. For normally distributed data requiring multiple comparisons, a one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons was used. Multiple comparisons of nonnormally distributed data were done with a Kruskal -Wallis test followed by a Dunn’s post hoc test. Comparisons of two groups were conducted with a 2-tailed Student’ s t-test (normally distributed data), or a Mann-Whitney U test (nonnormally distributed data). Differences with a P-value < 0.05 were considered statistically significant. For recordings conducted on aMVMs, the normality of the data was assessed by Shapiro-Wilk test and the equality of group variance was tested using the Brown-Forsythe test. Statistical significance of differences was determined using Mann-Whitney U test, and ANOVA with an appropriate multiple comparisons test, when applicable. Interaction between genotype and treatment was determined using a two-way ANOVA. Animals were grouped with no blinding but randomized in cellular experiments. Fully blinded analysis was not performed in cellular studies because the same person carried out the experiments and analysis. Both male and female animals were used. Group sizes were determined by an a priori power analysis with an a of 0.05 and power of 0.8, to detect a 20% signal difference at the endpoint.Images of Protein Structure

[0186] All protein structure images and vacuum electrostatics were generated with PyMOL (Schrodinger LLC). Unless otherwise stated, the Nav1.5CTDis shown as gray cylinders and the ELIXIR peptide as a light blue (actinium) helix.Attorney Docket 44010.196WO-PCT / / CU24196Code Availability

[0187] The peptide design protocol described here is available through Google Colab at: colab.research.google.com / github / sokrypton / ColabDesign / blob / v1.1.1 / af / examples / peptide_binder_design.ipynb.

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Proc Natl Acad Sci U S A 120, e2217800120.10.1073 / pnas.2217800120.59. Morgenstern, T. J., Nirwan, N., Hernandez-Ochoa, E. O., Bibollet, H., Choudhury, P., Laloudakis, Y. D., Ben Johny, M., Bannister, R. A., Schneider, M. F., Minor, D. L., Jr., and Colecraft, H. M. (2022). Selective posttranslational inhibition of Ca(V)beta(l)-associated voltage-dependent calcium channels with a functionalized nanobody. Nat Commun 13, 7556.10.1038 / s41467-022-35025-7.60. AlQuraishi, Y. L.a. M. (2023). Generating Novel, Designable, and Diverse Protein Structures by Equivariantly Diffusing Oriented Residue Clouds. arXiv.61. Roy, A., Shi, L., Chang, A., Dong, X., Fernandez, A., Kraft, J. C., Li, J., Le, V. Q., Winegar, R. V., Cherf, G. M., et al. (2023). De novo design of highly selective miniprotein inhibitors of integrins avP6 and avP8. bioRxiv, 2023.2006.2012.544624.10.1101 / 2023.06.12.544624.Attorney Docket 44010.196WO-PCT / / CU2419662. Wilde, A. A. M., and Amin, A. S. (2018). 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A., Kilic, A., et al. (2015). SCN5A variant that blocks fibroblast growth factor homologous factor regulation causes human arrhythmia. Proc Natl Acad Sci U S A 112, 12528-12533. 10.1073 / pnas.1516430112.67. Pei, Z., Xiao, Y., Meng, J., Hudmon, A., and Cummins, T. R. (2016). Cardiac sodium channel palmitoylation regulates channel availability and myocyte excitability with implications for arrhythmia generation. Nat Commun 7, 12035. 10.1038 / ncomms12035. 68. Yu, P., Hu, L., Xie, J., Chen, S., Huang, L., Xu, Z., Liu, X., Zhou, Q., Yuan, P., Yan, X., et al. (2018). O-GlcNAcylation of cardiac Navi.5 contributes to the development of arrhythmias in diabetic hearts. Int J Cardiol 260, 74-81. 10.1016 / j.ijcard.2018.02.099.69. Lu, Z., Wu, C. Y., Jiang, Y. P., Ballou, L. M., Clausen, C., Cohen, I. S., and Lin, R. Z. (2012). Suppression of phosphoinositide 3 -kinase signaling and alteration of multiple ion currents in drug-induced long QT syndrome. 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[0189] Various modifications and variations of the described methods, pharmaceutical compositions, and kits of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it will be understood that it is capable of further modifications and that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the invention. This application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure come within known customary practice within the art to which the invention pertains and may be applied to the essential features herein before set forth.

Claims

Attorney Docket 44010.196WO-PCT / / CU24196CLAIMSWhat is claimed:

1. A synthetic peptide comprising a sequence of MSPRREALYRGFRAXYDVLRH (SEQ ID NO. 1) or a variant thereof with at least 90% sequence identity, wherein X is cysteine or serine.

2. The synthetic peptide of claim 1, wherein the peptide comprises a substitution in a residue other than L8, F12, and Y16.

3. The synthetic peptide of claim 1 or 2, wherein the dissociation constant (Kd) of the peptide for the CTD of Navi.5 is 0.89 ± 0.25 pM.

4. The synthetic peptide of any of one claims 1 to 3, wherein the peptide comprises a sequence of MSPRREALYRGFRACYDVLRH (SEQ ID NO. 2).

5. The synthetic peptide of any one of claims 1 to 3, wherein the peptide comprises a sequence of MSPRREALYRGFRASYDVLRH (SEQ ID NO. 3).

6. The synthetic peptide of any of claims 1 to 5, further comprising cell penetrating peptide (CPP) sequence, wherein SEQ ID NO. 1 is fused to the CPP sequence.

7. The synthetic peptide of claim 6, wherein the CPP is a viral tat sequence.

8. The synthetic peptide of claim 7, wherein the peptide comprises a sequence of YGRKKRRQRRRGSGMSPRREALYRGFRASYDVLRH (SEQ ID NO. 4).

9. The synthetic peptide of any one of claims 1 to 8, wherein the peptide is modified to increase stability, reduce immunogenicity, and / or optimize cellular uptake.

10. The synthetic peptide of claim 9, wherein peptide comprises an N-terminal acetyl group and / or a C-terminal amide.

11. The synthetic peptide of any one of claims 1 to 10, wherein the peptide is conjugated to a carrier.

12. The synthetic peptide of any one of claims 1 to 11, wherein the peptide is conjugated to a label.Attorney Docket 44010.196WO-PCT / / CU2419613. The synthetic peptide of claim 1, wherein the amino acid sequence of the synthetic peptide is set forth in SEQ ID NO. 2, SEQ ID NO. 3, or SEQ ID NO. 4.

14. A pharmaceutical composition comprising the peptide of any one of claims 1 to 13 and a pharmaceutically acceptable carrier.

15. A polynucleotide encoding the synthetic peptide of any one of claims 1 to 13.

16. A vector comprising the polynucleotide of claim 15, wherein the vector is selected from a viral vector, a non-viral plasmid, an mRNA delivery construct, a lipid nanoparticle formulation, an exosome-based construct, or a protein nanocarrier.

17. The vector of claim 16, wherein the vector is a viral vector selected from the group consisting of an adeno-associated virus (AAV) vector, a lentiviral vector, and an adenoviral vector.

18. The vector of claim 17, wherein the viral vector is an AAV vector.

19. A method of treating a disease associated with pathological late sodium current (iNaL) arising from impaired voltage-gated sodium (Nav) channel inactivation comprising administering to a subject in need thereof an effective amount of the synthetic peptide or vector of any one of claims 1 to 18, wherein the synthetic peptide selectively inhibits INOL.

20. The method of claim 19, wherein the subject exhibits increased late sodium current (INaL) relative to a wild-type baseline level.

21. The method of claim 19 or 20, wherein the subject expresses a disease-linked nonsense or truncation variant of a Nav channel carboxyl-terminal domain.

22. The method of any one of claims 19 to 21, wherein the disease is caused by a mutation that reduces the propensity of the inactivation gate to occupy the pore-proximal site of the sodium channel.

23. The method of claim 19, wherein the disease is caused by a mutation of the inactivation gate of a sodium channel.

24. The method of claim 19, wherein the disease is caused by hyperactivation of protein kinase A (PKA) or Ca2+ / calmodulin-dependent protein kinase II (CaMKII).Attorney Docket 44010.196WO-PCT / / CU2419625. The method of any one of claims 19 to 24, wherein the disease is selected from the group consisting of arrhythmogenic long-QT syndrome type 3, cardiac arrhythmia, atrial fibrillation, pathological cardiac remodeling related to heart failure or myocardial ischemia, congenital myotonia, myotonic syndromes, epilepsy, migraines, intellectual disability, painful neuropathies, and chronic pain conditions26. The method of any one of claims 19 to 25, wherein the synthetic peptide or vector is administered to skeletal muscle cells, neurons, or cardiomyocytes.

27. The method of any one of claims 19 to 26, wherein the peptide is administered systemically.

28. The method of any one of claims 19 to 27, wherein the peptide is delivered by viral vector, mRNA vector, lipid nanoparticle, protein nanocarrier, exosome, or a cell-penetrating peptide.

29. A method of identifying a modulator of binding to a voltage-gated sodium channel carboxyl-terminal domain (CTD), the method comprising:a) contacting a fluorescently labeled peptide that binds the CTD with a CTD polypeptide under conditions suitable for CTD-peptide complex formation;b) measuring fluorescence anisotropy of the mixture;c) contacting the mixture with a test agent;d) measuring fluorescence anisotropy following contact with the test agent; and e) identifying the test agent as a modulator of CTD-peptide binding when the fluorescence anisotropy differs from that measured in the absence of the test agent.

30. The method of claim 29, wherein the fluorescently labeled peptide comprises a sequence of SEQ ID NO. 2 or SEQ ID NO. 3.

31. The method of claim 29 or 30, wherein a decrease in fluorescence anisotropy indicates that the test agent inhibits binding of the peptide to the CTD.

32. The method of any one of claims 29 to 31, wherein an increase in fluorescence anisotropy indicates that the test agent stabilizes or enhances binding of the peptide to the CTD.

33. The method of any one of claims 29 to 32, wherein the CTD polypeptide comprises a CTD of Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, Nav1.8, or Nav1.9.Attorney Docket 44010.196WO-PCT / / CU2419634. The method of any one of claims 29 to 33, wherein the CTD polypeptide comprises a disease-associated mutant or truncation variant.

35. The method of any one of claims 29 to 34, wherein the test agent comprises a small molecule, peptide, peptidomimetic, antibody fragment, or protein.

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