Gene therapy for treating and preventing myocarditis

Abstract

Disclosed herein are methods and materials for treating myocarditis with gene therapy. In some cases, disclosed herein are methods and material for treating myocarditis in a subject in need thereof comprising administering to the subject an effective amount of a vector comprising a nucleic acid sequence encoding a connexin 43 polypeptide operably linked to a promoter active in cardiac muscle tissue. Also disclosed herein are methods of treating myocarditis by administering to a subject an agent that increases the level of a connexin 43 encoding nucleic acid molecule or connexin 43 polypeptide.

Description

[0001] Attorney Docket No. 15670-0429WO1 / SD2024-416

[0002] GENE THERAPY FOR TREATING AND PREVENTING MYOCARDITIS

[0003] CLAIM OF PRIORITY

[0004] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 725,055, filed on November 26, 2024. The entire contents of the foregoing are incorporated herein by reference.

[0005] SEQUENCE LISTING

[0006] This application contains a Sequence Listing that has been submitted electronically as an XML file named “T5670-0429W01_SL_ST26.xmU’ The XML file, created on November 21, 2025. is 308,801 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.

[0007] FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0008] This invention was made with government support under HL095780 and HL181001 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0009] TECHNICAL FIELD

[0010] The present disclosure relates to methods and materials of gene therapy for treating myocarditis.

[0011] BACKGROUND

[0012] Myocarditis is a health threatening heart disease and a major public health issue, being the predominant cause of sudden cardiac death, particularly among young people. Globally, it affects an estimated 10.2 to 105.6 individuals per 100,000 population, amounting to around 1.8 million cases annually. Myocarditis is a frequent contributor to sudden death, secondary dilated cardiomyopathy, and heart failure, particularly in the young population. Despite the high disease burden, there remains an unmet clinical need as no effective treatments exist. Current approaches involve guessing the underlying mechanism or cause to guide the use of steroid interventions and beta-blockers. Attorney Docket No. 15670-0429WO1 / SD-2024-416-2PCT

[0013] SUMMARY

[0014] Provided herein are methods for treating myocarditis in a subject in need thereof comprising administering to the subject an effective amount of a nucleic acid sequence encoding a connexin 43 polypeptide operably linked to a promoter active in cardiac muscle tissue, wherein connexin 43 polypeptide levels are increased in the cardiac muscle tissue of the subject after the administration relative to the levels prior to the administration, thereby treating the myocarditis.

[0015] In some embodiments, the subject further has a disease or disorder selected from arrhythmogenic right ventricular cardiomyopathy (ARVC); arrhythmogenic left ventricular cardiomyopathy (ALVC); right ventricular dysfunction: left ventricular dysfunction; fibro- fatty replacement of the myocardium; hypertrophic cardiomyopathy; cardiac electrical and physiological dysfunction in arrhythmogenic disease; or a combination thereof.

[0016] In some embodiments, the connexin 43 polypeptide comprises an amino acid sequence comprises of SEQ ID NO: 1 (P17302 (CXA_1 HUMAN; UniProtKB)), or a functional fragment thereof. In some embodiments, the nucleic acid sequence encoding the connexin 43 polypeptide is SEQ ID NO: 2.

[0017] In some embodiments, the nucleic acid sequence is in a vector, such as a viral vector, a plasmid or a nanoparticle delivery7vector. In some embodiments, the viral vector is an adenoviral vector, a lentiviral vector, or an adeno-associated viral vector (AAV). In some embodiments, the AAV vector is an AAV1. AAV2. AAV6. AAV8 or an AAV9 serotype.

[0018] In some embodiments, the promoter is a CMV immediate early enhancer / promoter. In some embodiments, the promoter is a cardiac-specific promoter. In some embodiments, the promoter is a troponin-T promoter. In some embodiments, the promoter is a cardiac myosin light chain promoter, cardiac myosin heavy chain promoter, or an a-cardiac actin enhancer attached to an elongation factor la promoter.

[0019] In some embodiments, the nucleic acid sequence or vector containing the nucleic acid sequence is administered locally or systemically.

[0020] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0021] In some embodiments, the effective amount is from about 2xlO l l to 2 / 10A14 viral genomes per kg of body weight of the subject.

[0022] In some embodiments, as a result of the administration, cardiac inflammation is reduced, adipose tissue in and / or around the heart is reduced, cardiac electrophysiological and physiological dysfunction is reduced, cardiac structural integrity is improved, or a Attorney Docket No. 15670-0429WO1 / SD-2024-416-2PCT combination thereof. In some embodiments, as a result of the administration, clinical symptoms of myocarditis are reduced. In some embodiments, as a result of the administration, the amount and / or duration of concomitant corticosteroid use is reduced.

[0023] In some embodiments, the methods described herein further comprise administering to the subject an anti-inflammatory molecule. In some embodiments, the anti-inflammatory molecule is selected from an NS AID, colchicine, a beta blocker or aspirin.

[0024] In some embodiments, administration of conn exin 43 to the subject increases the expression of one or more genes or level of one or more polypeptides in a cell of the subject, wherein the gene or polypeptide is selected from N-cadherin, desmoplakin (DSP), plakoglobin (JUP), plakophilin 2 (PKP2) and desmoglein 2 (DSG2).

[0025] In some embodiments, the cell is a cardiac muscle cell, a cardiac fibroblast, a cardiomyocyte, or a cardiac macrophage.

[0026] Also provided herein are methods for treating myocarditis in a subject in need thereof comprising administering to the subject an agent that increases the level of a connexin 43 encoding nucleic acid molecule or connexin 43 polypeptide in the subject, wherein connexin 43 levels are increased in heart of the subject relative to levels prior to administration, thereby treating the myocarditis. In some embodiments, the agent is a small molecule, a nucleic acid sequence, a peptide or a peptide mimetic.

[0027] In some embodiments, the myocarditis is a result of a gene mutation. In some embodiments, the gene is Desmoplakin. Titin. or a Cytoskeletal gene.

[0028] In some embodiments, the myocarditis is a result of a viral infection. In some embodiments, the viral infection is caused by SARS-CoV-2 or HIV. In some embodiments, the myocarditis is a result of a bacterial infection. In some embodiments, the bacterial infection is a Staphylococcus aureus infection, a Salmonella infection, a Rickettsia infection, or a Chlamydia infection. In some embodiments, the myocarditis is a result of a parasitic infection. In some embodiments, the parasitic infection is Chagas disease, Toxoplasmosis, or Trichinosis. In some embodiments, the myocarditis is a result of a fungal infection. In some embodiments, the fungal infection is Candida. Aspergillus, or Histoplasma.

[0029] In some embodiments, the myocarditis is a result of an autoimmune disease. In some embodiments, the autoimmune disorder is Lupus, Sarcoidosis, Rheumatoid Arthritis, or Eosinophilic Myocarditis.

[0030] In some embodiments, the myocarditis is a result of a toxic substance or cancer drug.

[0031] In some embodiments, the myocarditis is a result of an allergic reaction. In some embodiments, the allergic reaction is from an antibiotic or a vaccine. Attorney Docket No. 15670-0429WO1 / SD-2024-416-2PCT

[0032] In some embodiments, the myocarditis includes epicardial fat accumulation which decreases upon an increase in connexin 43 expression or polypeptide levels.

[0033] In some embodiments, the myocarditis is acute, chronic, or recurrent. In some embodiments, the myocarditis is Giant cell, lymphocytic, or toxic myocarditis. In some embodiments, the methods for treating myocarditis described herein comprise administering to the subject a JAK inhibitor prior to administering the nucleic acid sequence encoding connexin 43 or the agent that increases the level of a connexin 43 encoding nucleic acid molecule in the subject. In some embodiments, the JAK inhibitor is selected from ruxolitinib, tofacitinib, baricitinib, or orencia (abatacept).

[0034] In some embodiments, the methods for treating myocarditis described herein comprise administering to the subject a T-cell mediator prior to administering the nucleic acid sequence encoding connexin 43 or the agent that increases the level of a connexin 43 encoding nucleic acid molecule in the subject. In some embodiments, the T-cell mediator is a mediator of CD4+ cells. In some embodiments, the CD4+ cells are Tregs. In some embodiment, the T-cell mediator is an antibody, a small molecule, a nucleic acid sequence, a peptide, or a peptide mimetic. In some embodiments, the T-cell mediator is an inhibitor of IL- 12, gamma interferon, IL-4, IL-5, IL-6, IL-23, IL-10, TGF-P, or IL-35. In some embodiments, the JAK inhibitor or the T-cell mediator is administered for about 1-30 days prior to administering the nucleic acid sequence encoding connexin 43 or the agent that increases the level of a connexin 43 encoding nucleic acid molecule in the subject.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0036] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. Attorney Docket No. 15670-0429WO1 / SD-2024-416-2PCT

[0037] BRIEF DESCRIPTION OF DRAWINGS

[0038] FIGs. 1A-1G show that cardiac-specific desmoplakin heterozygous knockout mice (DSP Het mice) are predisposed to myocarditis-driven cardiomyopathy following acute inflammatory' stress. FIG. 1A is a schematic diagram illustrating the application of inflammatory' stressors on Day 0 (alpha-myosin heavy' chain peptide (a-MyHC pep) + pertussis toxin (PT)) and Day 7 (a-MyHC pep) in 6-8-week-old mice to induce myocarditis. This model was used to assess both acute (21 days) and chronic (4 months) outcomes post- inflammatory' stress. FIG. IB shows hematoxylin and eosin (H&E) stained cardiac sections, demonstrating intense lymphocytic infiltrates in the hearts of wild type (WT) and DSP Het mice at acute phase (21 days post-acute inflammatory stress). While inflammation infiltrates resolved in WT mouse hearts, they persisted in DSP Het mouse hearts at chronic phase (4 months post-acute inflammatory stress). FIG. 1C shows H&E stained cardiac sections from the chronic phase in mice, including WT mice treated with alpha-MyHC peptide + pertussis toxin (WT + a-MyHC pep + PT), DSP Het mice, and DSP Het mice treated with alpha- MyHC peptide + pertussis toxin (DSP Het + a-MyHC pep + PT). DSP Het mice exhibited dilated right ventricular (RV) chambers at chronic phase (arrow) following inflammatory' stress. FIGs. 1D-1E are representative pictures of cardiac gross morphology (FIG. ID) and H&E stained cardiac sections (FIG. IE), highlighting fat deposition and fibrosis in DSP Het mouse hearts at chronic phase post-inflammatory stress, but not in WT mouse hearts. PKP2 Het mouse hearts do not exhibit significant remodeling at chronic phase post-inflammatory stress. FIG. IF shows surface electrocardiogram (ECG) analysis in mice. Premature ventricular contractions (PVC, arrows) were observed only in DSP Het + a-MyHC pep + PT mice at chronic phase. FIG. 1G shows representative MRI images of hearts from WT + a- MyHC pep + PT mice. DSP Het mice, and DSP Het + a-MyHC pep + PT mice. MRI analysis measured the left ventricle (LV) and right ventricle (RV) for cardiac dimensions, including end-diastolic volume (EDV) and end-systolic volume (ESV), as well as heart function, expressed as ejection fraction (EF). RV dilatation and dysfunction were observed in DS Het mice at chronic phase post-inflammatory’ stress.

[0039] FIGs. 2A-2C show that connexin 43 protein levels were upregulated in response to inflammatory' stress and this response was suppressed in DSP Het mice. FIG. 2A is a schematic diagram illustrating connexin 43 protein expression before and after inflammatory' stress in 6-8-week-old WT (black and dashed line) and DSP Het mice (black line) at baseline (before inflammatory stress) and 4 months post-inflammatory stress (chronic). Connexin 43 and DSP protein levels were assessed by protein blot (FIG. 2B) and quantitative analysis Attorney Docket No. 15670-0429WO1 / SD-2024-416-2PCT

[0040] (FIG. 2C). A reduction / loss of connexin 43 protein at baseline and after acute inflammatory stress was observed in DSP Het mouse hearts. Beta-actin was used as a loading control.

[0041] FIG. 3 is a schematic diagram illustrating the treatment protocol and its anticipated effect of AAV9-connexin 43 (AAV9-Cx43) gene therapy that was tested in treating myocarditis-related inflammation in WT and DSP Het mice. The lines represent the DSP Het mouse trajectory before and after inflammatory stress, WT mouse trajectory before and after inflammatory stress, and the anticipated effect of connexin 43 restoration by AAV9-Cx43 gene therapy in DSP Het mice before and after inflammatory stress.

[0042] FIGs. 4A-4D show the effect of AAV9-Cx43 gene therapy on the late onset of morphological abnormalities, immune cell infiltration, and fibrosis associated with myocarditis-related inflammation in DSP Het mouse hearts. Cardiac gross morphology (FIG. 4A), H&E stained cardiac sections (FIG. 4B), immune cell filtration (FIG. 4C), and fibrosis (FIG. 4D) in hearts were shown in DSP Het mice (left), DSP Het + a-MyHC pep + PT mice (middle), and DSP Het + a-MyHC pep + PT + AVV9-Cx43 mice (right). Smaller hearts, decreased immune cell infiltration, and reduced fibrosis were observed in DSP Het + a- MyHC pep + PT + AVV9-Cx43 (right) when compared to DSP Het + a-MyHC pep + PT (middle). DSP Het mice were used as a negative control. Cardiac fibrosis was assessed by Masson’s tri chrome staining on cardiac sections.

[0043] FIGs. 5A-5C show that AAV9-Cx43 gene therapy prevented electrophysiological deficits in Z S’P Het mice post-inflammatory stress. FIG. 5A shows representative surface ECG tracings (averaged over 100 beats), along with average heart rate, PR intervals, and QRS intervals in mice. FIG. 5B shows the quantification of QRS intervals from surface ECG tracings. QRS intervals in DSP Het + a-MyHC pep + PT + AVV9-Cx43 mice were significantly reduced compared to DSP Het + a-MyHC pep + PT mice. FIG. 5C shows representative surface ECG tracings demonstrating the presence of PVCs only in DSP Het + a-MyHC pep + PT mice.

[0044] FIGs. 6A-6B show that AAV9-Cx43 gene therapy prevented cardiac functional deficits (especially in the right ventricle) in DSP Het mice post-inflammatory stress. FIG. 6A shows representative MRI images from DSP Het mice without and with acute inflammatory stress, as well as DSP Het mice with acute inflammatory stress that were treated with AAV9- Cx43 gene therapy. FIG. 6B shows the quantitative analysis of MRI measurements of right ventricle (RV) for cardiac dimensions, including end-diastolic volume (EDV) and end- systolic volume (ESV), as well as heart function, expressed as ejection fraction (EF). Attorney Docket No. 15670-0429WO1 / SD-2024-416-2PCT

[0045] FIG. 7 shows that AAV9-Cx43 alleviated long term deficits of connexin and DSP proteins at time points coincident with alleviation of myocarditis-related inflammation in DSP Het mouse hearts pose-inflammatory stress. Representative images of protein blots show connexin 43 and DSP protein levels in DSP Het mouse hearts at baseline (5 months of age) and following inflammatory stress (a-MyHC peptide), with or without AAV9-Cx43 gene therapy. WT mice were used as a control. Beta- actin and ponceau staining were utilized as loading controls for protein blots.

[0046] FIG. 8A-C shows that neutralizing antibodies targeting CD4 positive T cells can alleviate cardiac inflammation and subsequent cardiac enlargement in cardiac-specific DSP homozy gous knockout (DSP Homo-cKO) mice. Fig. 8A is a schematic diagram illustrating the treatment protocol to interrogate the role of CD4 positive T cells in cardiac inflammation and enlargement in DSP Homo-cKO mice. DSP-cKO mice were treated with 300 pg of neutralizing antibodies targeting CD4 T cells at postnatal day 4, designated as Day 0 of the treatment, and subsequently treatments were administered with 100 pg of neutralizing antibodies to CD4 T cells at Day 7 (Week 1), Day 14 (Week 2) and Day 21 (Week 3). Hearts from these mice were assessed at 4 weeks post-treatment. Fig. 8B shows representative cardiac gross morphology and quantitative effects of a significant reduction in heart weight- to-body weight ratios in DSP Homo-cKO mice treated with CD4-neutralizing antibodies at 4 weeks post-treatment, when compared to untreated DSP Homo-cKO mice. These treated DSP Homo-cKO mice exhibit heart weight-to-body weight ratios comparable to wild type mouse hearts. Fig. 8C shows that this reduction in cardiac enlargement at 4 weeks post-treatment in DSP Homo-cKO mice treated with CD4 neutralizing antibodies was accompanied by a histopathological reduction in cardiac inflammatory cell infiltrates and fibrosis, when compared to untreated DSP Homo-cKO mice. These treated DSP Homo-cKO mice exhibit cardiac inflammatory cell infiltrates and fibrosis comparable to wild type mouse hearts. H&E and Trichrome staining were utilized to visualize cardiac inflammatory cell infiltrates and fibrosis.

[0047] DETAILED DESCRIPTION

[0048] Provided herein are methods for treating myocarditis in a subject in need thereof comprising administering to the subject an effective amount of a vector comprising a nucleic acid sequence encoding a connexin 43 polypeptide operably linked to a promoter active in cardiac muscle tissue, wherein connexin 43 polypeptide levels are increased in the cardiac Attorney Docket No. 15670-0429WO1 / SD-2024-416-2PCT muscle tissue of the subject after the administration relative to the levels prior to the administration, thereby treating the myocarditis.

[0049] Also provided herein methods for treating myocarditis in a subject in need thereof including administering to the subject an agent that increases the level of a connexin 43 encoding nucleic acid molecule or connexin 43 polypeptide in the subject, wherein connexin 43 levels are increased in the heart of the subject relative to levels prior to administration, thereby treating the myocarditis.

[0050] As used in the specification and the appended claims, the singular forms ‘'a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0051] As used herein, the term “about,” when used herein in reference to a value, refers to a value that is ± 10% of the referenced value.

[0052] Connexin 43 and Desmoplakin (DSP)

[0053] Connexin 43 and DSP are two key proteins involved in cellular communication and structural integrity, particularly in the heart. Connexin 43 is a protein that forms gap junctions and specialized structures that allow direct communication between neighboring cells by facilitating the exchange of ions, metabolites, and other small molecules. It is encoded by the GJA1 gene and is highly expressed in the heart, where it plays helps maintain synchronous contraction of cardiac muscle cells. Connexin 43 ensures the electrical coupling of cardiomyocytes, which facilitates the propagation of electrical impulses during the cardiac cycle. Mutations or alterations in connexin 43 expression can lead to arrhythmias and other heart disorders, some of which can include inflammation. During the acute phase following inflammatory stress in the cardiac tissue of a subject, connexin 43 gene expression and / or protein production may be upregulated to protect the cardiac tissue from inflammatory stress. In some cases, connexin 43 gene expression and / or protein production can recover to prestress levels and resolve so that it is not susceptible to progressing into the chronic inflammatory phase. However, in myocarditis predisposing cases (e.g., myocarditis associated with genetics (e.g., DSP mutations) and viral infections), connexin 43 gene expression and / or protein production can be reduced; and this chronic reduction of connexin 43 establishes a susceptible state facilitating the cardiac tissue to enter the chronic phase of inflammation. Thus, provided herein are methods and materials of restoring connexin 43 expression to resolve inflammation in myocarditis.

[0054] In some embodiments, the connexin 43 polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 1 (Pl 7302 (CXA_1 HUMAN; UniProtKB)), or a functional Attorney Docket No. 15670-0429WO1 / SD-2024-416-2PCT fragment thereof. The nucleic acid sequence encoding connexin 43 polypeptide (P17302) is set forth in SEQ ID NO: 2. There are at least four smaller truncated isoforms of connexin 43 in human heart, which include the 32kDa (100-382AA, SEQ ID NO: 3), 29kDa (125-382AA, SEQ ID NO: 4), 26kDa (147-382AA, SEQ ID NO: 5) and 20 kDa (213-382AA, SEQ ID NO: 6), that may act as functional fragments thereof (Smith and Shaw, Autoregulation of connexin 43 gap junction formation by internally translated isoforms. Cell Rep. 5(3): 611-8 (2013), which is incorporated by reference herein in its entirety).

[0055] Amino acid (AA) sequence of connexin 43 polypeptide, 382 AA (SEQ ID NO: 1)

[0056] MGDWSALGKLLDKVQAYSTAGGKVWLSVLFIFRILLLGTAVESAWGDEQSA

[0057] FRCNTQQPGCENVCYDKSFPISHVRFWVLQIIFVSVPTLLYLAHVFYVMRKEE

[0058] KLNKKEEELKVAQTDGVNVDMHLKQIEIKKFKYGIEEHGKVKMRGGLLRTY

[0059] IISILFKSIFEVAFLLIQWYIYGFSLSAVYTCKRDPCPHQVDCFLSRPTEKTIFIIF

[0060] MLVVSLVSLALNIIELFYVFFKGVKDRVKGKSDPYHATSGALSPAKDCGSQK

[0061] YAYFNGCSSPTAPLSPMSPPGYKLVTGDRNNSSCRNYNKQASEQNWANYSA

[0062] EQNRMGQAGSTISNSHAQPFDFPDDNQNSKKLAAGHELQPLAIVDQRPSSRA

[0063] SSRASSRPRPDDLEI

[0064] Nucleic acid sequence encoding connexin 43 polypeptide. 382 AA (SEQ ID

[0065] NO: 2) NCB1 Reference Sequence: NM_000165.5

[0066] AAAAGCTTTTACGAGGTATCAGCACTTTTCTTTCATTAGGGGGAAGGCGT

[0067] GAGGAAAGTACCAAACAGCAGCGGAGTTTTAAACTTTAAATAGACAGGT

[0068] CTGAGTGCCTGAACTTGCCTTTTCATTTTACTTCATCCTCCAAGGAGTTCA

[0069] ATCACTTGGCGTGACTTCACTACTTTTAAGCAAAAGAGTGGTGCCCAGGC

[0070] AACATGGGTGACTGGAGCGCCTTAGGCAAACTCCTTGACAAGGTTCAAGC

[0071] CTACTCAACTGCTGGAGGGAAGGTGTGGCTGTCAGTACTTTTCATTTTCCG

[0072] AATCCTGCTGCTGGGGACAGCGGTTGAGTCAGCCTGGGGAGATGAGCAGT

[0073] CTGCCTTTCGTTGTAACACTCAGCAACCTGGTTGTGAAAATGTCTGCTATG

[0074] ACAAGTCTTTCCCAATCTCTCATGTGCGCTTCTGGGTCCTGCAGATCATAT

[0075] TTGTGTCTGTACCCACACTCTTGTACCTGGCTCATGTGTTCTATGTGATGC

[0076] GAAAGGAAGAGAAACTGAACAAGAAAGAGGAAGAACTCAAGGTTGCCC

[0077] AAACTGATGGTGTCAATGTGGACATGCACTTGAAGCAGATTGAGATAAAG

[0078] AAGTTCAAGTACGGTATTGAAGAGCATGGTAAGGTGAAAATGCGAGGGG

[0079] GGTTGCTGCGAACCTACATCATCAGTATCCTCTTCAAGTCTATCTTTGAGG

[0080] TGGCCTTCTTGCTGATCCAGTGGTACATCTATGGATTCAGCTTGAGTGCTG

[0081] TTTACACTTGCAAAAGAGATCCCTGCCCACATCAGGTGGACTGTTTCCTCT

[0082] CTCGCCCCACGGAGAAAACCATCTTCATCATCTTCATGCTGGTGGTGTCCT

[0083] TGGTGTCCCTGGCCTTGAATATCATTGAACTCTTCTATGTTTTCTTCAAGG

[0084] GCGTTAAGGATCGGGTTAAGGGAAAGAGCGACCCTTACCATGCGACCAG

[0085] TGGTGCGCTGAGCCCTGCCAAAGACTGTGGGTCTCAAAAATATGCTTATT

[0086] TCAATGGCTGCTCCTCACCAACCGCTCCCCTCTCGCCTATGTCTCCTCCTG

[0087] GGTACAAGCTGGTTACTGGCGACAGAAACAATTCTTCTTGCCGCAATTAC

[0088] AACAAGCAAGCAAGTGAGCAAAACTGGGCTAATTACAGTGCAGAACAAA

[0089] ATCGAATGGGGCAGGCGGGAAGCACCATCTCTAACTCCCATGCACAGCCT

[0090] TTTGATTTCCCCGATGATAACCAGAATTCTAAAAAACTAGCTGCTGGACA

[0091] TGAATTACAGCCACTAGCCATTGTGGACCAGCGACCTTCAAGCAGAGCCA

[0092] GCAGTCGTGCCAGCAGCAGACCTCGGCCTGATGACCTGGAGATCTAGATA Attorney Docket No. 15670-0429WO1 / SD-2024-416-2PCT

[0093] CAGGCTTGAAAGCATCAAGATTCCACTCAATTGTGGAGAAGAAAAAAGG

[0094] TGCTGTAGAAAGTGCACCAGGTGTTAATTTTGATCCGGTGGAGGTGGTAC

[0095] TCAACAGCCTTATTCATGAGGCTTAGAAAACACAAAGACATTAGAATACC

[0096] TAGGTTCACTGGGGGTGTATGGGGTAGATGGGTGGAGAGGGAGGGGATA

[0097] AGAGAGGTGCATGTTGGTATTTAAAGTAGTGGATTCAAAGAACTTAGATT

[0098] ATAAATAAGAGTTCCATTAGGTGATACATAGATAAGGGCTTTTTCTCCCC

[0099] GCAAACACCCCTAAGAATGGTTCTGTGTATGTGAATGAGCGGGTGGTAAT

[0100] TGTGGCTAAATATTTTTGTTTTACCAAGAAACTGAAATAATTCTGGCCAGG

[0101] AATAAATACTTCCTGAACATCTTAGGTCTTTTCAACAAGAAAAAGACAGA

[0102] GGATTGTCCTTAAGTCCCTGCTAAAACATTCCATTGTTAAAATTTGCACTT

[0103] TGAAGGTAAGCTTTCTAGGCCTGACCCTCCAGGTGTCAATGGACTTGTGC

[0104] TACTATATTTTTTTATTCTTGGTATCAGTTTAAAATTCAGACAAGGCCCAC

[0105] AGAATAAGATTTTCCATGCATTTGCAAATACGTATATTCTTTTTCCATCCA

[0106] CTTGCACAATATCATTACCATCACTTTTTCATCATTCCTCAGCTACTACTC

[0107] ACATTCATTTAATGGTTTCTGTAAACATTTTTAAGACAGTTGGGATGTCAC

[0108] TTAACATTTTTTTTTTGAGCTAAAGTCAGGGAATCAAGCCATGCTTAATAT

[0109] TTAACAATCACTTATATGTGTGTCGAAGAGTTTGTTTTGTTTGTCATGTAT

[0110] TGGTACAAGCAGATACAGTATAAACTCACAAACACAGATTTGAAAATAAT

[0111] GCACATATGGTGTTCAAATTTGAACCTTTCTCATGGATTTTTGTGGTGTGG

[0112] GCCAATATGGTGTTTACATTATATAATTCCTGCTGTGGCAAGTAAAGCAC

[0113] ACTTTTTTTTTCTCCTAAAATGTTTTTCCCTGTGTATCCTATTATGGATACT

[0114] GGTTTTGTTAATTATGATTCTTTATTTTCTCTCCTTTTTTTAGGATATAGCA

[0115] GTAATGCTATTACTGAAATGAATTTCCTTTTTCTGAAATGTAATCATTGAT

[0116] GCTTGAATGATAGAATTTTAGTACTGTAAACAGGCTTTAGTCATTAATGTG

[0117] AGAGACTTAGAAAAAATGCTTAGAGTGGACTATTAAATGTGCCTAAATGA

[0118] ATTTTGCAGTAACTGGTATTCTTGGGTTTTCCTACTTAATACACAGTAATT

[0119] CAGAACTTGTATTCTATTATGAGTTTAGCAGTCTTTTGGAGTGACCAGCAA

[0120] CTTTGATGTTTGCACTAAGATTTTATTTGGAATGCAAGAGAGGTTGAAAG

[0121] AGGATTCAGTAGTACACATACAACTAATTTATTTGAACTATATGTTGAAG

[0122] ACATCTACCAGTTTCTCCAAATGCCTTTTTTAAAACTCATCACAGAAGATT

[0123] GGTGAAAATGCTGAGTATGACACTTTTCTTCTTGCATGCATGTCAGCTACA

[0124] TAAACAGTTTTGTACAATGAAAATTACTAATTTGTTTGACATTCCATGTTA

[0125] AACTACGGTCATGTTCAGCTTCATTGCATGTAATGTAGACCTAGTCCATCA

[0126] GATCATGTGTTCTGGAGAGTGTTCTTTATTCAATAAAGTTTTAATTTAGTA

[0127] TAAACATA

[0128] Amino acid sequence of connexin 43 truncated isoform 1, 100-382 AA (SEQ

[0129] ID NO: 3)

[0130] MRKEEKLNKKEEELKVAQTDGVNVDMHLKQIEIKKFKYGIEEHGKVKMRG GLLRTYIISILFKSIFEVAFLLIQWYIYGFSLSAVYTCKRDPCPHQVDCFLSRPT EKTIFIIFMLVVSLVSLALNIIELFYVFFKGVKDRVKGKSDPYHATSGALSPAK DCGSQKYAYFNGCSSPTAPLSPMSPPGYKLVTGDRNNSSCRNYNKQASEQN WANYSAEQNRMGQAGSTISNSHAQPFDFPDDNQNSKKLAAGHELQPLAIVD QRPSSRASSRASSRPRPDDLEI

[0131] Amino acid sequence of connexin 43 truncated isoform 2, 125-382 AA (SEQ

[0132] ID NO: 4)

[0133] MHLKQIEIKKFKYGIEEHGKVKMRGGLLRTYIISILFKSIFEVAFLLIQWYIYGF

[0134] SLSAVYTCKRDPCPHQVDCFLSRPTEKTIFIIFMLVVSLVSLALNIIELFYVFFK

[0135] GVKDRVKGKSDPYHATSGALSPAKDCGSQKYAYFNGCSSPTAPLSPMSPPGY Attorney Docket No. 15670-0429WO1 / SD-2024-416-2PCT

[0136] KLVTGDRNNSSCRNYNKQASEQNWANYSAEQNRMGQAGSTISNSHAQPFDF PDDNQNSKKLAAGHELQPLAIVDQRPSSRASSRASSRPRPDDLEI

[0137] Amino acid sequence of connexin 43 truncated isoform 3. 147-382 AA (SEQ

[0138] ID NO: 5)

[0139] MRGGLLRTYIISILFKSIFEVAFLLIQWYIYGFSLSAVYTCKRDPCPHQVDCFLS RPTEKTIFIIFMLVVSLVSLALNIIELFYVFFKGVKDRVKGKSDPYHATSGALSP AKDCGSQKYAYFNGCSSPTAPLSPMSPPGYKLVTGDRNNSSCRNYNKQASE QNWANYSAEQNRMGQAGSTISNSHAQPFDFPDDNQNSKKLAAGHELQPLAI

[0140] VDQRPSSRASSRASSRPRPDDLEI

[0141] Amino acid sequence of connexin 43 truncated isoform 4, 213-382 AA (SEQ

[0142] ID NO: 6)

[0143] MLVVSLVSLALNIIELFYVFFKGVKDRVKGKSDPYHATSGALSPAKDCGSQK

[0144] YAYFNGCSSPTAPLSPMSPPGYKLVTGDRNNSSCRNYNKQASEQNWANYSA EQNRMGQAGSTISNSHAQPFDFPDDNQNSKKLAAGHELQPLAIVDQRPSSRA SSRASSRPRPDDLEI

[0145] Nucleic acid sequence encoding connexin 43 truncated isoform 1. 100-382

[0146] AA (SEQ ID NO: 7)

[0147] ATGCGAAAGGAAGAGAAACTGAACAAGAAAGAGGAAGAACTCAAGGTT

[0148] GCCCAAACTGATGGTGTCAATGTGGACATGCACTTGAAGCAGATTGAGAT

[0149] AAAGAAGTTCAAGTACGGTATTGAAGAGCATGGTAAGGTGAAAATGGGA

[0150] GGGGGGTTGCTGCGAACCTACATCATCAGTATCCTCTTCAAGTCTATCTTT

[0151] GAGGTGGCCTTCTTGCTGATCCAGTGGTACATCTATGGATTCAGCTTGAGT

[0152] GCTGTTTACACTTGCAAAAGAGATCCCTGCCCACATCAGGTGGACTGTTT

[0153] CCTCTCTCGCCCCACGGAGAAAACCATCTTCATCATCTTCATGCTGGTGGT

[0154] GTCCTTGGTGTCCCTGGCCTTGAATATCATTGAACTCTTCTATGTTTTCTTC

[0155] AAGGGCGTTAAGGATCGGGTTAAGGGAAAGAGCGACCCTTACCATGCGA CCAGTGGTGCGCTGAGCCCTGCCAAAGACTGTGGGTCTCAAAAATATGCT TATTTCAATGGCTGCTCCTCACCAACCGCTCCCCTCTCGCCTATGTCTCCT CCTGGGTACAAGCTGGTTACTGGCGACAGAAACAATTCTTCTTGCCGCAA

[0156] TTACAACAAGCAAGCAAGTGAGCAAAACTGGGCTAATTACAGTGCAGAA

[0157] CAAAATCGAATGGGGCAGGCGGGAAGCACCATCTCTAACTCCCATGCAC AGCCTTTTGATTTCCCCGATGATAACCAGAATTCTAAAAAACTAGCTGCT

[0158] GGACATGAATTACAGCCACTAGCCATTGTGGACCAGCGACCTTCAAGCAG

[0159] AGCCAGCAGTCGTGCCAGCAGCAGACCTCGGCCTGATGACCTGGAGATC

[0160] Nucleic acid sequence encoding connexin 43 truncated isoform 2. 125-382

[0161] AA (SEQ ID NO: 8)

[0162] ATGCACTTGAAGCAGATTGAGATAAAGAAGTTCAAGTACGGTATTGA

[0163] AGAGCATGGTAAGGTGAAAATGCGAGGGGGGTTGCTGCGAACCTACA

[0164] TCATCAGTATCCTCTTCAAGTCTATCTTTGAGGTGGCCTTCTTGCTGAT

[0165] CCAGTGGTACATCTATGGATTCAGCTTGAGTGCTGTTTACACTTGCAA

[0166] AAGAGATCCCTGCCCACATCAGGTGGACTGTTTCCTCTCTCGCCCCAC

[0167] GGAGAAAACCATCTTCATCATCTTCATGCTGGTGGTGTCCTTGGTGTCC

[0168] CTGGCCTTGAATATCATTGAACTCTTCTATGTTTTCTTCAAGGGCGTTA

[0169] AGGATCGGGTTAAGGGAAAGAGCGACCCTTACCATGCGACCAGTGGT Attorney Docket No. 15670-0429WO1 / SD-2024-416-2PCT

[0170] GCGCTGAGCCCTGCCAAAGACTGTGGGTCTCAAAAATATGCTTATTTC

[0171] AATGGCTGCTCCTCACCAACCGCTCCCCTCTCGCCTATGTCTCCTCCTG

[0172] GGTACAAGCTGGTTACTGGCGACAGAAACAATTCTTCTTGCCGCAATT

[0173] ACAACAAGCAAGCAAGTGAGCAAAACTGGGCTAATTACAGTGCAGAA

[0174] CAAAATCGAATGGGGCAGGCGGGAAGCACCATCTCTAACTCCCATGC

[0175] ACAGCCTTTTGATTTCCCCGATGATAACCAGAATTCTAAAAAACTAGC

[0176] TGCTGGACATGAATTACAGCCACTAGCCATTGTGGACCAGCGACCTTC

[0177] AAGCAGAGCCAGCAGTCGTGCCAGCAGCAGACCTCGGCCTGATGACC

[0178] TGGAGATC

[0179] Nucleic acid sequence encoding connexin 43 truncated isoform 3. 147-382

[0180] AA (SEQ ID NO: 9)

[0181] ATGCGAGGGGGGTTGCTGCGAACCTACATCATCAGTATCCTCTTCAAGTC

[0182] TATCTTTGAGGTGGCCTTCTTGCTGATCCAGTGGTACATCTATGGATTCAG

[0183] CTTGAGTGCTGTTTACACTTGCAAAAGAGATCCCTGCCCACATCAGGTGG

[0184] ACTGTTTCCTCTCTCGCCCCACGGAGAAAACCATCTTCATCATCTTCATGC

[0185] TGGTGGTGTCCTTGGTGTCCCTGGCCTTGAATATCATTGAACTCTTCTATG

[0186] TTTTC / IGCAAGGGCGTTAAGGATCGGGTTAAGGGAAAGAGCGACCCTTAC

[0187] CATGCGACCAGTGGTGCGCTGAGCCCTGCCAAAGACTGTGGGTCTCAAAA

[0188] ATATGCTTATTTCAATGGCTGCTCCTCACCAACCGCTCCCCTCTCGCCTAT

[0189] GTCTCCTCCTGGGTACAAGCTGGTTACTGGCGACAGAAACAATTCTTCTTG

[0190] CCGCAATTACAACAAGCAAGCAAGTGAGCAAAACTGGGCTAATTACAGT

[0191] GCAGAACAAAATCGAATGGGGCAGGCGGGAAGCACCATCTCTAACTCCC

[0192] ATGCACAGCCTTTTGATTTCCCCGATGATAACCAGAATTCTAAAAAACTA

[0193] GCTGCTGGACATGAATTACAGCCACTAGCCATTGTGGACCAGCGACCTTC

[0194] AAGCAGAGCCAGCAGTCGTGCCAGCAGCAGACCTCGGCCTGATGACCTG

[0195] GAGATC

[0196] Nucleic acid sequence encoding connexin 43 truncated isoform 4. 213-382

[0197] AA (SEQ ID NO: 10)

[0198] ATGCTGGTGGTGTCCTTGGTGTCCCTGGCCTTGAATATCATTGAACTCTTC

[0199] TATGTTTTCTTCAAGGGCGTTAAGGATCGGGTTAAGGGAAAGAGCGACCC

[0200] TTACCATGCGACCAGTGGTGCGCTGAGCCCTGCCAAAGACTGTGGGTCTC

[0201] AAAAATATGCTTATTTCAATGGCTGCTCCTCACCAACCGCTCCCCTCTCGC

[0202] CTATGTCTCCTCCTGGGTACAAGCTGGTTACTGGCGACAGAAACAATTCTT

[0203] CTTGCCGCAATTACAACAAGGAAGCAAGTGAGCAAAACTGGGCTAATTAC

[0204] AGTGCAGAACAAAATCGAATGGGGCAGGCGGGAAGCACCATCTCTAACT

[0205] CCCATGCACAGCCTTTTGATTTCCCCGATGATAACCAGAATTCTAAAAAA

[0206] CTAGCTGCTGGACATGAATTACAGCCACTAGCCATTGTGGACCAGCGACC

[0207] TTCAAGCAGAGCCAGCAGTCGTGCCAGCAGCAGACCTCGGCCTGATGACC

[0208] TGGAGATC

[0209] DSP is a member of the plakin family of cytolinkers, serving as an important linker between junctional components, including junction plakoglobin (JUP) and plakophilin-2 (PKP2), and the intermediate filament cytoskeleton (desmin). Patients carrying DSP mutations frequently present with recurrent myocarditis and worsened clinical outcomes. Attorney Docket No. 15670-0429WO1 / SD-2024-416-2PCT

[0210] In some embodiments, the gene expression and / or protein production of connexin 43 can be increased or decreased in response to inflammatory stress. In some embodiments. DSP gene expression and / or protein production is reduced. In some embodiments, connexin 43 gene expression and / or protein production can be decreased when DSP gene expression and / or protein production is reduced. In some embodiments, connexin 43 gene expression and / or protein production can be reduced / lost from the acute phase throughout the chronic phase following inflammatory stress in a subject having cardiac-specific DS heterozygous deficiency.

[0211] Myocarditis

[0212] As an inflammatory disease of cardiomyopathy, myocarditis is characterized byinfiltration of inflammatory cells into the myocardium, nonischemic myocyte necrosis, and either regional or diffuse inflammation. Patients with myocarditis may have a variety of clinical manifestations, including chest pain, fatigue, dyspnea or shortness of breath, syncope, palpitations, swelling, flu-like symptoms, and prodromal respiratory and gastrointestinal infections.

[0213] The etiology of myocarditis is mainly divided into infectious and noninfectious factors. Infectious factors include, but are not limited to, a variety- of pathogen infections such as viral infections (e.g., SARS-CoV-2 or HIV), bacterial infections (e.g., Staphylococcus aureus. Salmonella, Chlamydia, or Rickettsia), fungal infections (e.g., Candida, Aspergillus. or Histoplasma), and parasite infections (e.g., Chagas disease. Toxoplasmosis, or Trichinosis). In some embodiments, viral infections associated with myocarditis include, but are not limited to, Picomaviridae such as Enteroviruses (e.g., Coxsackievirus A / B or Echoviruses); Adenoviridae (e.g., Adenovirus serotypes 2 or 5); Parvoviridae (e.g., Parvovirus B19); Herpesviridae (e.g., Human herpesvirus 6 HHV-6, Cytomegalovirus, Epstein-Barr virus, Herpes simplex virus, or Varicella-zoster virus); Orthomyxoviridae (e.g., Influenza A and B); Coronaviridae (e.g., SARS-CoV-2); Retroviridae (e.g., HIV); Hepadnaviridae (e.g., Hepatitis B virus (rare)); and Flaviviridae (e.g.. Hepatitis C virus (rare)).

[0214] Noninfectious factors include, but are not limited to, gene mutations (e.g., mutations in DSP, Titin, or cytoskeletal genes), systemic autoimmune diseases (e.g., systemic Lupus erythematosus, Sarcoidosis, rheumatoid arthritis, eosinophilic myocarditis), drugs (e.g., aminophylline, amphetamines, anthracyclines, catecholamines, and immune checkpoint inhibitors), hypersensitivity reactions (e.g., antibiotics or vaccines), and tumors. Attorney Docket No. 15670-0429WO1 / SD-2024-416-2PCT

[0215] The pathogenesis of myocarditis can be summarized as a classic 3-phase model, including the acute infection phase, the subacute immune response phase, and the chronic myopathy phase. The acute infection phase is mainly caused by the first invasion of the pathogen or reactivation of the pathogen in a dormant state. The subacute immune response phase is the time when an appropriate immune response contributes to pathogen clearance, whereas an excessive immune response can cause tissue damage. The chronic myopathy phase is when there is persistent overreaction of the immune response, myocardial tissue undergoes matrix remodeling and fibrosis, which can lead to heart failure and secondary dilated cardiomyopathy. In some embodiments, the acute phase of myocarditis in a subject can occur about 14 to 28 days (e.g., about 16 to 26 days, about 18 to 24 days, about 20 to 22 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, or about 28 days) after the first stimulation of inflammatory stress. In some embodiments, the acute phase can occur about 21 days after the first stimulation of inflammatory stress (e.g., a-MyHC peptide and PT). In some embodiments, the chronic phase of myocarditis in a subject refers to about two or more months (e.g., about three or more months, about four or more months, about five or more months, about six or more months, about two months, about three months, about four months, about five months, or about six months) after the first stimulation of inflammatory stress. In some embodiments, the chronic phase of myocarditis in a subject can occur about four months after the first stimulation of inflammatory stress. In some embodiments, the myocarditis can be acute, chronic, recurrent or fulminant.

[0216] Acute myocarditis is the most common form of myocarditis. In some embodiments, the acute myocarditis presents with features including sudden onset of symptoms (e.g.. within days to weeks), often triggered by a viral infection described herein, and / or mimics acute coronary syndrome. In some embodiments, a subject with acute myocarditis may present symptoms including, but not limited to, chest pain, dyspnea, arrhythmias, and / or fatigue. In some embodiments, the acute myocarditis may progress to fulminant myocarditis (e.g., severe or rapid heart failure). In some embodiments, a subject with acute myocarditis may make complete recovery. In some embodiments, the acute myocarditis can transition to chronic inflammatory cardiomyopathy.

[0217] Chronic myocarditis can include inflammation that persists beyond three months. In some embodiments, chronic myocarditis persists with ongoing viral infection or autoimmune mechanisms. In some embodiments, chronic myocarditis presents features including slow or Attorney Docket No. 15670-0429WO1 / SD-2024-416-2PCT smoldering course, chronic heart failure symptoms, and / or dilated cardiomyopathy (DCM) risk. In some embodiments, chronic myocarditis may occur due to inadequate resolution of the acute myocarditis.

[0218] Recurrent myocarditis can be a new episode of myocarditis that occurs after clinical recovery from a previous episode of myocarditis. In some embodiments, recurrent myocarditis presents features including repeated chest pain, troponin elevation, and inflammation. In some embodiments, recurrent myocarditis present symptoms that recur months or years after initial episode of myocarditis. In some embodiments, recurrent myocarditis is associated with viral infections described herein (e.g., HHV-6), autoimmune myocarditis, immune-related conditions, and / or complications after mRNA-vaccine myocarditis.

[0219] Fulminant myocarditis can include a severe and rapidly progressing subset of acute myocarditis. In some embodiments, fulminant myocarditis presents features including cardiogenic shock over hours to days and / or high inflammation burden on biopsy. In some embodiments, a subject with fulminant myocarditis can require intensive care in an intensive care unit (ICU) and mechanical circulatory support (e.g., extracorporeal membrane oxygenation (ECMO) or Impella). In some embodiments, a subject with fulminant myocarditis can paradoxically have better long-term prognosis if survived.

[0220] Myocarditis is characterized by an inflammatory cell infiltration utilizing any appropriate methods known in art (e.g., magnetic resonance imaging (MRI), PET, or endomyocardial biopsy with histopathology). Cardiac inflammation can be either focal or diffuse, with or without myocardial cell damage in the myocardium. The infiltrating immune cells contributing to the development of myocarditis can be neutrophils, eosinophils, mast cells, innate lymphoid cells (ILCs), monocytes, macrophages, dendritic cells (DCs), T lymphocytes, B lymphocytes, or any combination thereof. See, e.g., Liu and Han, Role of immune cells in the pathogenesis of myocarditis, J. Leukoc. Biol, 115 (2): 253-275 (2024); Aretz et al., Myocarditis. A histopathologic definition and classification, Am J Cardiovasc Pathol, 1 : 3- 14 (1987); Ammirati et al., Management of acute myocarditis and chronic inflammatory cardiomyopathy: an expert consensus document. Giro Pieart Fail, 663-687 (2020). These articles are incorporated herein by reference in their entirety.

[0221] In some embodiments, the myocarditis is Giant cell, lymphocytic, or toxic myocarditis. In some embodiments, the methods described herein can be used for treating myocarditis induced by inflammatory stress (e.g., alpha myosin heavy chain (a-MyHC) peptide and / or pertussis toxin (PT). In some embodiments, the cardiac inflammation Attorney Docket No. 15670-0429WO1 / SD-2024-416-2PCT following acute inflammatory stress (e.g., a-MyHC peptide and / or PT) persists from acute phase throughout the chronic phase. In some embodiments, the cardiac inflammation following acute inflammatory stress (e.g., a-MyHC peptide and / or PT) occurs during the acute phase but resolves at chronic phase. In some embodiments, the cardiac inflammation following acute inflammatory stress persists from acute phase throughout the chronic phase in a subject having cardiac-specific DSP heterozygous deficiency.

[0222] In some embodiments, the methods described herein can be used for treating myocarditis in a subject having cardiac-specific DSP homozygous or heterozygous deficiency. In some embodiments, the methods described herein can be used for treating myocarditis induced by inflammatory stress (e.g., a-MyHC peptide and / or PT) in a subject having cardiac-specific DSP heterozygous deficiency. In some embodiments, a subject may sustain multiple occurrences of inflammatory stress (e.g., a-MyHC peptide and / or PT) (e.g., two or more times, three or more times, four or more times, one time, two times, three times, four times, or five times). In other embodiments, a subject may encounter different sources of inflammatory stress (e.g., two different sources of inflammatory stress, three different sources of inflammatory stress, etc.). In some embodiments, the inflammatory stress can be cumulative.

[0223] Myocarditis generally reduces cardiac ability7to pump and can cause rapid or abnormal heart rhythms (or arrhythmias, e.g., premature ventricular contractions (PVC)). In some embodiments, the methods provided herein can be used for treating myocarditis-related disorders including, but not limited to, cardiac inflammation (e.g., immune cell infiltration), adipose tissue accumulation in and / or around the heart, cardiac electrophysiological dysfunction (e.g., PVC), cardiac physiological dysfunction (e.g., reduced ejection fraction (EF)), cardiac fibrosis, or any combination thereof. In some embodiments, the subject with myocarditis has reduced expression of connexin 43 gene and / or protein.

[0224] Patients often present with chest pain, heart failure, abnormal ECGs (e.g., PVC), and elevated cardiac biomarkers. Diagnosis is typically confirmed through MRI or PET imaging, showing evidence of cardiac inflammation. In some embodiments, MRI can be used to measure cardiac dimensions (e.g., end-diastolic volume (EDV) and end-systolic volume (ESV)) and cardiac function assessed by ejection fraction (EF). The typical treatments for myocarditis aim to manage symptoms and reduce inflammation. Common approaches include supportive care (e.g., rest and activity limitation, and fluid and electrolyte management), medications (e.g.. anti-inflammatory drugs, heart failure medications, antiviral therapy, or antibiotics), arrhythmia management (e.g., antiarrhythmic drugs, pacemaker, Attorney Docket No. 15670-0429WO1 / SD-2024-416-2PCT implantable cardioverter-defibrillator), advanced therapies (e.g., ventricular assist devices, extracorporeal membrane oxygenation, or heart transplantation), or any combination thereof.

[0225] Methods of Treatment

[0226] Provided herein are methods for treating myocarditis in a subject in need thereof including administering to the subject an effective amount of a vector including a nucleic acid sequence encoding a connexin 43 polypeptide operably linked to a promoter active in cardiac muscle tissue, wherein connexin 43 levels are increased in the cardiac muscle tissue of the subject after the administration relative to the levels prior to the administration, thereby treating the myocarditis. In some embodiments, administering an effective amount of a vector including a nucleic acid sequence encoding a connexin 43 polypeptide operably linked to a promoter active in cardiac muscle tissue chronically upregulates the expression of connexin 43 in the cardiac muscle tissue. As used herein, the terms “subject” and “patient” are used interchangeably throughout the specification and describe an animal, human or non-human. Veterinary and non-veterinary applications are contemplated by the present invention. Human patients can be adult humans or juvenile humans (e.g., humans below the age of 18 years old). In addition to humans, patients include, but are not limited to, mice, rats, hamsters, guinea-pigs, rabbits, ferrets, cats, dogs, and primates. Included are, for example, rodents (e.g., mice, rats, gerbils, hamsters, ferrets, rabbits), non-human primates (e g., monkey, chimpanzee, gorilla, and the like), lagomorphs, swine (e.g., pig. miniature pig), equine, canine, feline, bovine, as well as other domestic, farm, and zoo animals. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the human has a gene mutation in a Desmoplakin, Titin, or a Cytoskeletal gene. In some embodiments, the subject is a mouse. In some embodiments, the subject is a mouse that is predisposed to developing myocarditis when exposed to inflammatory stress. In some embodiments, the subject is a cardiac-specific DS heterozy gous deficient (DSP Het) mouse.

[0227] Provided herein are methods for treating myocarditis in a subject in need thereof including administering to a subject an effective amount of a vector including a nucleic acid sequence encoding a connexin 43 polypeptide for the purpose of treating myocarditis.

[0228] The methods described in this disclosure can include identifying a subject as having, being at risk of developing, or suspected of having a disorder associated with myocarditis. Myocarditis can co-occur in some subjects with heart conditions. In some embodiments, the subject further has a disease or disorder selected from arrhythmogenic right ventricular Attorney Docket No. 15670-0429WO1 / SD-2024-416-2PCT cardiomyopathy (ARVC), arrhythmogenic left ventricular cardiomyopathy (ALVC), right ventricular dysfunction, left ventricular dysfunction, fibro-fatty replacement of the myocardium, hypertrophic cardiomyopathy, cardiac electrical and physiological dysfunction in arrhythmogenic disease, or a combination thereof. In some embodiments, the heart condition develops or is uncovered as a result of the myocarditis. In some embodiments, the subject does not have a co-occurring heart condition other than the myocarditis.

[0229] The terms “effective amount,” “effective dose”, and “effective to treat,” as used herein, refer to an amount or a concentration of an agent, or composition including the agent, utilized for a period of time (including acute or chronic administration and periodic or continuous administration) that is effective within the context of its administration for causing an intended effect or physiological outcome. In some embodiments, the methods described herein include using the effective amount of vectors from about 2x 10A11 to 2x lO 14 viral genomes per kg of body weight of the subject. For example, the effective amount of vectors can be, but is not limited to, about 4xlOAl l to l x lO 14, about 8x lOAl l to 5x l0A13, about 2xlOA12 to 2x lOA13, about 4x lOA12 to lx lOA13, about 2x lOAl l, about 4x lOAl l, about 8 x 10Al 1 , about 2xlOA12, about 4xlOA12, about 8x lO 12, about 2xlOA13, about 4x lOA13, about 8x l0 13, or about 2x lOA14 viral genomes per kg of body weight of the subject. Further, as used herein, the effective amount results in improvement of the pathology of myocarditis in the subject, e.g., improvement in symptoms of heart failure and / or reduction of inflammation in the heart muscle, adipose tissue in and / or around the heart is reduced, cardiac electrophysiological and physiological dysfunction is reduced, cardiac structural integrity is improved, or a combination thereof.

[0230] Administration of vectors is typically by injection or infusion. In some embodiments, intravenous administration is used. In other embodiments, the vectors are administered into a tissue, organ, or body cavity that is, or is in communication with, the site where treatment is to take effect, such as the heart itself, or the pericardial space. Suitable doses of the vector can be administered to a subject in need thereof. Non-limiting examples of methods of administration include subcutaneous administration, intravenous administration, intramuscular administration, intraperitoneal administration, infusion, intrathecal administration, intranasal administration and intra-arterial. In some cases, administration can involve injection of a formulation of the vector composition. Continuous and discontinuous administration schedules include dosing schedules in which the dose of vector is modulated throughout the administration, such that, for example, at the beginning of the connexin 43 administration period; the dose is low and increased until the end of the connexin 43 Attorney Docket No. 15670-0429WO1 / SD-2024-416-2PCT administration period; the dose is initially high and decreased during the administration period; the dose is initially low, increased to a peak level, then reduced towards the end of the administration period; and any combination thereof. Also, the dosing schedules may be performed using any method of standard in the art, such as a catheter system.

[0231] The methods described herein further include administering to the subject medications for the treatment of myocarditis. The medications can include an anti-inflammatory molecule, a heart failure medication, an antiviral medication, and / or an antibiotic. In some embodiments, the methods described herein further include administering to the subject antiinflammatory molecules (e.g., neutralizing antibodies, JAK inhibitors, T cell mediators, corticosteroids, nonsteroidal anti-inflammatory drugs (NSAIDs), colchicine, immunosuppressive drugs, intravenous immunoglobulin, or biological agents for targeted immunotherapy) and any combination thereof. In some embodiments, the methods described herein further include administering neutralizing antibodies (e.g., CD4 neutralizing antibodies). In some embodiments, the methods described herein further include administering to the subject JAK inhibitors prior to administering the nucleic acid sequence encoding connexin 43 or an agent that increases the level of a connexin 43 encoding nucleic acid molecule in a subject. In some embodiments, the JAK inhibitors can be selected from ruxolitinib, tofacitinib, baricitinib, or orencia (abatacept). In some embodiments, the methods described herein further include administering to the subject T-cell mediators prior to administering the nucleic acid sequence encoding connexin 43 or the agent that increases the level of a connexin 43 encoding nucleic acid molecule in the subject. In some embodiments, the T-cell mediators can be mediators of CD4+ cells. In some embodiments, the CD4+ cells are Tregs. In some embodiments, the T-cell mediators are antibodies, small molecules, nucleic acid sequences, peptides, or peptide mimetics. In some embodiments, the T-cell mediators are inhibitors of IL-12, gamma interferon, IL-4, IL-5, IL-6, IL-23, IL-10, TGF- , or IL-35. In some embodiments, the JAK inhibitors or the T-cell mediators are administered for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days prior to administering an nucleic acid sequence encoding connexin 43 or an agent that increases the level of a connexin 43 encoding nucleic acid molecule in a subject.

[0232] In some embodiments, the methods described herein further include administering corticosteroids (e.g., prednisone or methylprednisolone). In some embodiments, the methods described herein further include administering NSAIDs (e.g., aspirin, ibuprofen, or naproxen). In some embodiments, the methods described herein further include administering Attorney Docket No. 15670-0429WO1 / SD-2024-416-2PCT colchicine. In some embodiments, the methods described herein further include administering immunosuppressive drugs (e.g., Azathioprine. mycophenolate mofetil). In some embodiments, the methods described herein further include administering intravenous immunoglobulin (IVIG). In some embodiments, the methods described herein further include administering biological agents for targeted immunotherapy (e.g., anti-TNF agent or IL-1 receptor antagonist). In some embodiments, the methods described herein further include administering any combination of the anti-inflammatory molecules described herein. In some embodiments, the methods described herein further include administering to the subject the heart failure medications (e.g., ACE inhibitors, beta-blockers, or diuretics). In some embodiments, the methods described herein further include administering any combination of the heart failure medications described herein. In some embodiments, the methods described herein further include administering to the subject antiviral medications. In some embodiments, the methods described herein further include administering to the subject antibiotics.

[0233] The medications described herein (e g., CD4 neutralizing antibodies, JAK inhibitors, or T cell mediators) can be administered before, simultaneously, or after the administration of the nucleic acid sequence encoding connexin 43 or the agent that increases the level of a connexin 43 encoding nucleic acid molecule in the subject. In some embodiments, the medications described herein (e.g., CD4 neutralizing antibodies, JAK inhibitors, or T cell mediators) can be administered about 1 day. 2 days. 3 days. 4 days. 5 days. 6 days. 7 days. 8 days, 9 days, 10 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, and 8 weeks before the administration of the nucleic acid sequence encoding connexin 43 or the agent that increases the level of a connexin 43 encoding nucleic acid molecule in the subject. In some embodiments, the medications described herein (e.g., CD4 neutralizing antibodies, JAK inhibitors, or T cell mediators) can be administered simultaneously with the nucleic acid sequence encoding connexin 43 or the agent that increases the level of a connexin 43 encoding nucleic acid molecule in the subject. In some embodiments, the medications described herein (e.g., CD4 neutralizing antibodies, JAK inhibitors, or T cell mediators) can be administered the same day as the nucleic acid sequence encoding connexin 43 or the agent that increases the level of a connexin 43 encoding nucleic acid molecule in the subject. In some embodiments, the medications described herein (e.g., CD4 neutralizing antibodies, JAK inhibitors, or T cell mediators) can be administered about 1 day, 2 days, 3 days, 4 days, 5 days. 6 days. 7 days. 8 days, 9 days, 10 days, 14 days. 3 weeks. 4 weeks, 5 weeks, 6 weeks, 7 weeks, and 8 weeks after the administration of the nucleic acid sequence encoding connexin Attorney Docket No. 15670-0429WO1 / SD-2024-416-2PCT

[0234] 43 or the agent that increases the level of a connexin 43 encoding nucleic acid molecule in the subject.

[0235] The medications described herein (e.g., CD4 neutralizing antibodies, JAK inhibitors, or T cell mediators) can be administered for one time or multiple times. In some embodiments, the medications described herein (CD4 neutralizing antibodies, JAK inhibitors, or T cell mediators) can be administered one time before, simultaneously, or after the administration of the nucleic acid sequence encoding connexin 43 or the agent that increases the level of a connexin 43 encoding nucleic acid molecule in the subject. In some embodiments, the medications described herein (e.g., CD4 neutralizing antibodies, JAK inhibitors, or T cell mediators) can be administered multiple times before, simultaneously, or after the administration of the nucleic acid sequence encoding connexin 43 or the agent that increases the level of a connexin 43 encoding nucleic acid molecule in the subject. In some embodiments, the medications described herein (e.g., CD4 neutralizing antibodies, JAK inhibitors, or T cell mediators) can be administered the same or different doses at different time points during multiple administration.

[0236] Also provided herein are methods of treating myocarditis by administering to a subject an agent that increases the level of a connexin 43 encoding nucleic acid molecule or connexin 43 polypeptide, with or without additional anti-inflammatory molecules (e.g., neutralizing antibodies, JAK inhibitors, T cell mediators, corticosteroids, nonsteroidal antiinflammatory drugs (NSAIDs), colchicine, immunosuppressive drugs, intravenous immunoglobulin, or biological agents for targeted immunotherapy). As used in this context, to “treat” means to ameliorate at least one symptom of the myocarditis, such as cardiac inflammation, or myocarditis-related cardiomyopathy. In some embodiments, as a result of the administration in the methods described herein, connexin 43 polypeptide levels in the cardiac muscle tissue can be increased relative to the levels prior to the administration. In some embodiments, as a result of the administration in the methods described herein, cardiac inflammation (e.g., inflammatory cell infiltration) can be reduced, adipose tissue in and / or around the heart can be reduced, cardiac electrophysiological (e.g., PVC incidence), physiological dysfunction (e.g., increased EDV of RV, increased ESV of RV, decreased EF) can be improved, cardiac structural integrity (e g., enlarged heart or dilated RV) can be improved, heart weight-to-body weight ratio can be reduced, cardiac fibrosis can be improved, or a combination thereof. In some embodiments, as a result of the administration in the methods described herein, clinical symptoms of myocarditis can be reduced, wherein the clinical symptoms of myocarditis include, but are not limited to, chest pain, fatigue. Attorney Docket No. 15670-0429WO1 / SD-2024-416-2PCT dyspnea or shortness of breath, syncope, palpitations, swelling, flu-like symptoms, and prodromal respiratory and gastrointestinal infections. In some embodiments, as a result of the administration in the methods described herein, the amount and / or duration of concomitant medication (e.g., neutralizing antibodies, JAK inhibitors, T cell mediators, corticosteroids, NSAIDs, or other anti-inflammatory medications) use can be reduced.

[0237] In some embodiments, as a result of the administration in the methods described herein, the expression of one or more genes or level of one or more polypeptides in a cell (e.g., cardiac muscle cell, a cardiac fibroblast, a cardiomyocyte, or a cardiac macrophage) of the subject can be increased, wherein the one or more genes or the one or more polypeptides can be selected fromN-cadherin, desmoplakin (DSP), plakoglobin (JUP), plakophillin 2 (PKP2), and desmoglein 2 (DSG2).

[0238] Also provided herein are methods for treating myocarditis in a subject in need thereof including administering to the subject an agent that increases the level of a connexin 43 encoding nucleic acid molecule or connexin 43 polypeptide in the subject, wherein connexin 43 levels are increased in heart of the subject relative to levels prior to administration, thereby treating the myocarditis. Any appropriate agent known in art to increase the level of a connexin 43 encoding nucleic acid molecule or connexin 43 polypeptide can be used in the methods described herein. In some embodiments, the agent used in methods described herein include, but is not limited to. a small molecule, a nucleic acid sequence, a peptide, or a peptide mimetic.

[0239] Vectors and Adeno-associated virus (AA V)

[0240] In some embodiments, the methods described herein include introducing an exogenous nucleic acid molecule into cells (e.g., cardiac muscle cell, a cardiac fibroblast, a cardiomyocyte, or a cardiac macrophage) by a vector. For example, a vector can be an expression vector where the expression vector includes a promoter sequence operably linked to the sequence encoding the molecule (e.g., a nucleic acid molecule). Non-limiting examples of vectors include viral vectors, plasmids, transposons (e.g., DNA transposons, RNA transposons or retrotransposons, and class III transposons), cosmids, nanoparticle delivery vector, and any Gateway® vectors. Non-limiting examples of viral vectors include adeno- associated virus (AAV) vectors, any adenoviral derived (AV) vectors, cytomegaloviral derived (CMV) vectors, simian viral derived (SV40) vectors, lentivirus vectors, and retroviral vectors. A vector can, for example, include sufficient cis-acting elements for expression where other elements for expression can be supplied by the host mammalian cell or in an in Attorney Docket No. 15670-0429WO1 / SD-2024-416-2PCT vitro expression system. Skilled practitioners will be capable of selecting suitable vectors and mammalian cells for introducing any of spatial profiling reagents described herein.

[0241] AAV is a tiny non-enveloped virus having a 25 nm capsid. No disease is known or has been shown to be associated with the wildty pe virus. AAV has a single-stranded DNA (ssDNA) genome. AAV has been shown to exhibit long-term episomal transgene expression, and AAV has demonstrated excellent transgene expression in the brain, particularly in neurons. Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate. Space for exogenous DNA is limited to about 4.7 kb. An AAV vector such as that described in Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985) can be used to introduce DNA into cells. A variety of nucleic acids have been introduced into different cell ty pes using AAV vectors (see for example Hermonat et al., Proc. Natl. Acad. Sci. USA 81:6466- 6470 (1984); Tratschin et al.. Mol. Cell. Biol. 4:2072-2081 (1985); Wondisford et al.. Mol. Endocrinol. 2:32-39 (1988); Tratschin et al., J. Virol. 51:611-619 (1984); and Flotte et al., J. Biol. Chem. 268:3781-3790 (1993). There are numerous alternative AAV variants (over 100 have been cloned), and AAV variants have been identified based on desirable characteristics. In some embodiments, the AAV is AAV1, AAV2, AAV4, AAV5, AAV6, AV6.2. AAV7. AAV8, AAV9, rh. 10, rh.39, rh.43 or CSp3. In some embodiments, the AAV is chosen based on its natural tropism (see, e.g., Table 1).

[0242] Table 1. Characterization of AAV natural serotypes. Attorney Docket No. 15670-0429WO1 / SD-2024-416-2PCT

[0243] (Table 1 adapted from Table 1 of Komeyenkov and Zamyatnin. Jr.. Pharmaceutics 2021. 13(5), 750).

[0244] In some embodiments, the vector used in the methods provided herein can be a viral vector, a plasmid, or a nanoparticle delivery7vector. In some embodiment, the vector can be an adenoviral vector, a lentiviral vector, or an AAV vector. A preferred viral vector system used for delivery of nucleic acids in the present methods is the adeno-associated virus (AAV). In some embodiment, the AAV vector is an AAV1, AAV2, AAV6, AAV8, or an AAV9 seroty pe. In some embodiments, the methods provided herein include adeno- associated virus (AAV) vector (e.g., AAV9) for introducing a nucleic acid sequence encoding a connexin 43 polypeptide operably linked to a promoter active in cardiac muscle tissue. AAV9 has been shown to be highly efficient in delivering therapeutic genes (e.g., connexin 43) into cardiac muscle tissue, owing to its strong tropism for cardiac muscle cells.

[0245] In some embodiments, the vector can be a recombinant AAV (rAAV) delivered directly to muscle by injection with a needle, catheter or related device, using techniques known in the art. For in vivo delivery, the rAAV virions can be formulated into pharmaceutical compositions and one or more dosages may be administered.

[0246] In some embodiments, the methods provided herein include any appropriate promoter active in cardiac muscle tissue that can be operably linked to a nucleic acid sequence encoding a connexin 43 polypeptide. In some embodiments, the promoter used in the methods provided herein can be a Cytomegalovirus (CMV) immediate early enhancer / promoter. In some embodiments, the promoter used in the methods provided herein can be a cardiac-specific promoter. In some embodiments, the promoter is a troponin-T promoter. In some embodiments, the promoter is a cardiac myosin light chain promoter, cardiac myosin heavy chain promoter, or an a-cardiac actin enhancer attached to an elongation factor la promoter. Attorney Docket No. 15670-0429WO1 / SD-2024-416-2PCT

[0247] The methods provided herein can use any appropriate methods of administration to deliver the vector into cardiac tissue. In some embodiments, the methods provided herein including administering the vector locally or systemically. Non-limiting exemplary methods of administration include intracoronary injection, intramyocardial injection, epicardial delivery, pericardial injection, intravenous injection, intramuscular injection, or subcutaneous injection.

[0248] Pharmaceutical Compositions

[0249] Also provided herein are pharmaceutical compositions including a vector comprising a nucleic acid sequence encoding a connexin 43 polypeptide operably linked to a promoter active in cardiac muscle tissue and methods of using the pharmaceutical compositions described herein for treating myocarditis.

[0250] Pharmaceutical compositions typically include a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.

[0251] Pharmaceutical compositions are typically formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.

[0252] Pharmaceutical compositions are typically formulated to be compatible with its intended route of administration into subjects. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.

[0253] Methods of formulating suitable pharmaceutical compositions are known in the art, see, e.g., Remington: The Science and Practice of Pharmacy, 21st ed., 2005; and the books in the series Drugs and the Pharmaceutical Sciences: A Series of Textbooks and Monographs (Dekker, NY). For example, solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benz l 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. pH can be adjusted with Attorney Docket No. 15670-0429WO1 / SD-2024-416-2PCT acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0254] EXAMPLES

[0255] Example 1. Cardiac-specific desmoplakin heterozygous knockout mice (DSP Het mice) were predisposed to myocarditis-driven cardiomyopathy following acute inflammatory stress

[0256] A DSP-related myocarditis mouse model was established to assess myocarditis-driven cardiomyopathy induced by acute inflammation stress using 6-8-week-old DSP Het mice (FIG. 1A). WT mice were used as control. Inflammatory stressors, alpha-myosin heavy chain peptide (a-MyHC pep) and pertussis toxin (PT) were administered to mice on Day 0 and only a-MyHC pep was administered on Day 7 to induce myocarditis. Cardiac inflammation was assessed by H&E staining on cardiac sections at acute phase (21 days post-inflammatory stress) and at chronic phase (4 months post-inflammatory stress). Surface ECG and MRI were utilized to evaluate cardiac rhythm, remodeling, and function.

[0257] As shown in FIG. IB, intense lymphocytic infiltrates in the hearts of both WT and DSP Het mice at acute phase (21 days) post-in fl ammatory stress. Inflammatory infiltrates persisted in DSP Het mouse hearts throughout chronic phase (4 months) post-inflammatory stress, while they resolved in WT mouse hearts. FIG. 1C showed that DSP Het mice, after acute inflammatory stress, exhibited dilated right ventricular chambers at chronic phase (indicated by the arrow), in contrast to WT mice after acute inflammatory stress and DSP Het mice without acute inflammatory' stress.

[0258] Fat deposition and fibrosis in DSP Het mouse hearts at chronic phase post- inflammatory stress were observed (FIGs. 1D-1E), but not in WT mouse hearts. Surface ECG (FIG. IF) showed premature ventricular contractions (PVC, arrows) only in DSP Het + a-MyHC pep + PT mice at chronic phase. MRI analysis (FIG. 1G) indicated right ventricular (RV) dilatation and heart dysfunction in D P Het mice at chronic phase.

[0259] Example 2. DSP deficiency suppressed the responsive elevation of connexin 43 following inflammatory stress Attorney Docket No. 15670-0429WO1 / SD-2024-416-2PCT

[0260] To determine connexin 43 expression in DSP-related myocarditis following acute inflammatory’ stress, connexin 43 protein were assessed utilizing Western Blot at baseline and after acute inflammatory stress.

[0261] As shown in FIG. 2A, connexin 43 protein levels were upregulated in response to inflammatory’ stress and this response was suppressed in DSP Het mice. FIGs. 2B-2C demonstrated a reduction / loss of connexin 43 protein at baseline and 4 months after acute inflammatory’ stress in DSP Het mouse hearts.

[0262] Example 3. Restoration of connexin 43 alleviated myocarditis-related cardiomyopathy in DSP Het mice

[0263] Connexin 43 restoration was achieved through AAV9-Cx43 gene therapy in DSP Het mice. The treatment protocol and its anticipated effect of AAV9-Cx43 gene therapy are shown in FIG. 3.

[0264] As shown in FIG. 7, AAV9-Cx43 gene therapy restored the expression of proteins connexin 43 and DSP in DSP Het mouse hearts, where a reduction in both connexin 43 and DSP proteins was observed following inflammatory stress. Concurrently, reduced heart size (FIGs.4A-4B), decreased immune cell infiltration (FIG. 4C), and reduced fibrosis at the chronic phase were observed in DSP Het + a-MyHC pep + PT + AVV9-Cx43 mice. Surface ECG analysis indicated that AAV9-Cx43 gene therapy prevented electrophysiological deficits in DSP Het mice post-inflammatory stress (FIGs. 5A-5C). Specifically, QRS intervals in DSP Het + a-MyHC pep + PT + AVV9-Cx43 mice was significantly reduced compared to DSP Het + a-MyHC pep + PT mice (FIGs. 5A-5B). PVCs were only observed in DSP Het + a-MyHC pep + PT mice (FIG. 5C).

[0265] FIGs. 6A-6B showed that AAV9-Cx43 gene therapy prevented cardiac functional deficits, particularly in the right ventricle, in DSP Het mice post-inflammatory stress. MRI measurements of the RV indicated significant improvements in end-diastolic volume (EDV), end-systolic volume (ESV), and ejection fraction (EF).

[0266] Example 4. Neutralizing antibodies to CD4 were sufficient to alleviate cardiac enlargement, inflammation, and fibrosis in cardiac-specific desmoplakin homozygous knockout mice (DSP Homo-cKO mice).

[0267] To test whether anti-inflammatory’ treatment will revert the cardiac enlargement and fibrosis, DSP Homo-cKO mice were treated with 300 pg of CD4-neutralizing antibodies to Attorney Docket No. 15670-0429WO1 / SD-2024-416-2PCT target CD4 T cells at postnatal day 4, designated as Day 0 of the treatment. Subsequent treatments were administered with 100 pg of CD4-neutralizing antibodies at Day 7 (Week 1), Day 14 (Week 2) and Day 21 (Week 3). Hearts from these mice were assessed at 4 weeks post-treatment. Heart weight-to-body weight ratio was measured. Inflammatory cell infiltration and fibrosis were assessed histopathologically using H&E staining and Masson’s trichrome staining on cardiac tissue sections.

[0268] As shown in FIGs. 8A-C, DSP Homo-cKO mice treated with CD4-neutralizing antibodies demonstrated a significant reduction in heart weight-to-body weight ratio, cardiac inflammatory cell infiltration, and fibrosis compared to untreated DSP Homo-cKO mice. The cardiac inflammatory cell infiltration and fibrosis in the treated DSP Homo-cKO mice was shown to be comparable to wild type mouse hearts. These results indicated that pre-treatment with CD4-neutralizing antibodies can be used as a potential adjuvant treatment to prevent / alleviate cardiac immune response prior to cardiac connexin 43 gene therapy.

Claims

Attorney Docket No. 15670-0429WO1 / SD-2024-416-2PCTWHAT IS CLAIMED IS:1 . A method for treating myocarditis in a subject in need thereof comprising: administering to the subject an effective amount of a nucleic acid sequence encoding a connexin 43 polypeptide operably linked to a promoter active in cardiac muscle tissue, wherein connexin 43 levels are increased in the cardiac muscle tissue of the subject after the administration relative to the levels prior to the administration, thereby treating the myocarditis.

2. The method of claim 1, wherein the subject further has a disease or disorder selected from arrhythmogenic right ventricular cardiomyopathy (ARVC); arrhythmogenic left ventricular cardiomyopathy (ALVC); right ventricular dysfunction; left ventricular dysfunction; fi bro- Patty replacement of the myocardium; hypertrophic cardiomyopathy; cardiac electrical and physiological dysfunction in arrhythmogenic disease; or a combination thereof.

3. The method of claim 1, wherein the connexin 43 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1 (Pl 7302 (CXA_1 HUMAN; UniProtKB)), or a functional fragment thereof.

4. The method of claim 3, wherein the functional fragment comprises the amino acid sequence set forth in any one of SEQ ID NOs: 3-6.

5. The method of claim 1, wherein the nucleic acid sequence encoding the connexin 43 polypeptide comprises SEQ ID NO: 2.

6. The method of claim 3, wherein the functional fragment is encoded by the nucleic acid sequence set forth in any one of SEQ ID NOs: 7-10.

7. The method of claim 1 , wherein the nucleic acid sequence is in a vector.

8. The method of claim 7, wherein the vector is a viral vector, a plasmid, a liposome, or a nanoparticle delivery vector.Attorney Docket No. 15670-0429WO1 / SD-2024-416-2PCT9. The method of claim 8, wherein the viral vector is an adenoviral vector, a lentiviral vector, or an adeno-associated viral vector (AAV).

10. The method of claim 9, wherein the AAV vector is an AAV1, AAV2, AAV6, AAV8, or an AAV9 serotype.

11. The method according to claim 1 , wherein the promoter is a CMV immediate early enhancer / promoter.

12. The method of claim 1, wherein the promoter is a cardiac-specific promoter.

13. The method of claim 1, wherein the promoter is a troponin-T promoter.

14. The method of claim 1, wherein the promoter is a cardiac myosin light chain promoter, cardiac myosin heavy chain promoter, or an a-cardiac actin enhancer attached to an elongation factor la promoter.

15. The method of claim 1, wherein the nucleic acid sequence is administered locally or systemically.

16. The method of claim 1 , wherein the subject is a mammal.

17. The method of claim 16, wherein the subject is a human.

18. The method of claim 1, wherein the effective amount is from about 2x 10Al 1 to 2 / I OAI4 viral genomes per kg of body weight of the subject.

19. The method of claim 1, further comprising administering to the subject an antiinflammatory molecule.

20. The method of claim 19, wherein the anti-inflammat ory molecule comprises an NS AID, colchicine, a beta blocker, or aspirin.Attorney Docket No. 15670-0429WO1 / SD-2024-416-2PCT21. The method of claim 1, wherein the administration of the nucleic acid sequence encoding the connexin 43 polypeptide to the subject increases the expression of one or more genes or level of one or more polypeptides in a cell of the subject, wherein the gene or polypeptide is selected from the group consisting of N-cadherin, desmoplakin (DSP), plakoglobin (JUP), plakophillin 2 (PKP2), and desmoglein 2 (DSG2).

22. The method of claim 21, wherein the cell is a cardiac muscle cell, a cardiac fibroblast, a cardiomyocyte, or a cardiac macrophage.

23. A method for treating myocarditis in a subject in need thereof comprising: administering to the subject an agent that increases the level of a connexin 43 encoding nucleic acid molecule or connexin 43 polypeptide in the subject, wherein connexin 43 levels are increased in the heart of the subject relative to levels prior to administration, thereby treating the myocarditis.

24. The method of claim 23, wherein the agent is a small molecule, a nucleic acid sequence, a peptide or a peptide mimetic.

25. The method of claims 1 or 23, wherein the myocarditis is a result of a gene mutation.

26. The method of claim 25, wherein the gene comprises Desmoplakin, Titin, or aCytoskeletal gene.

27. The method of claims 1 or 23, wherein the myocarditis is a result of a viral infection.

28. The method of claim 27, wherein the viral infection is SARS-CoV-2 infection, HIV infection, Picomaviridae infection, Adenoviridae infection, Parvoviridae infection, Herpesviridae infection, Orthomyxoviridae infection, Coronaviridae infection, Retroviridae infection, Hepadnaviridae infection, or Flaviviridae infection.

29. The method of claims 1 or 23, wherein the myocarditis is a result of a bacterial infection.Attorney Docket No. 15670-0429WO1 / SD-2024-416-2PCT30. The method of claim 29, wherein the bacterial infection is a Staphylococcus aureus infection, a Salmonella infection, a Rickettsia infection, or a Chlamydia infection.

31. The method of claims 1 or 23, wherein the myocarditis is a result of a parasitic infection.

32. The method of claim 31, wherein the parasitic infection is Chagas disease, Toxoplasmosis, or Trichinosis.

33. The method of claims 1 or 23, wherein the myocarditis is a result of a fungal infection.

34. The method of claim 33, wherein the fungal infection is a Candida infection, an Aspergillus infection, or a Histoplasma infection.

35. The method of claims 1 or 23, wherein the myocarditis is a result of an autoimmune disease.

36. The method of claim 35, wherein the autoimmune disorder is Lupus, Sarcoidosis, Rheumatoid Arthritis, or Eosinophilic Myocarditis.

37. The method of claims 1 or 23, wherein the myocarditis is a result of a toxic substance or cancer drug.

38. The method of claims 1 or 23, wherein the myocarditis is a result of an allergic reaction.

39. The method of claim 38, wherein the allergic reaction is from an antibiotic or a vaccine.

40. The method of claims 1 or 23, wherein the administration of the nucleic acid sequence encoding the connexin 43 polypeptide or the agent that increases the level of a connexin 43 encoding nucleic acid molecule or connexin 43 polypeptide reduces epicardia fat accumulation in the subject.Attorney Docket No. 15670-0429WO1 / SD-2024-416-2PCT41. The method of claims 1 or 23, wherein the myocarditis is acute, chronic, or recurrent.

42. The method of claims 1 or 23, wherein the myocarditis is Giant cell, lymphocytic, or toxic myocarditis.

43. The method of claim 1 or 23, further comprising administering to the subject a JAK inhibitor prior to administering the nucleic acid sequence encoding connexin 43 polypeptide or the agent that increases the level of a connexin 43 encoding nucleic acid molecule or connexin 43 polypeptide.

44. The method of claim 43, wherein the JAK inhibitor is selected from the group consisting of ruxolitinib, tofacitinib, baricitinib, and orencia (abatacept).

45. The method of claim 1 or 23, further comprising administering to the subject a T-cell mediator prior to administering the nucleic acid sequence encoding connexin 43 polypeptide or the agent that increases the level of a connexin 43 encoding nucleic acid molecule or connexin 43 polypeptide.

46. The method of claim 45. wherein the T-cell mediator is a mediator of CD4+ cell.

47. The method of claim 46, wherein the CD4+ cell is regulatory T cell (Treg).

48. The method of claim 45, wherein the T-cell mediator is an antibody, a small molecule, a nucleic acid sequence, a peptide, or a peptide mimetic.

49. The method of claim 48, wherein the T-cell mediator is an inhibitor of IL-12, gamma interferon, IL-4, IL-5, IL-6, IL-23. IL-10, TGF-0, or IL-35.

50. The method of claim 43 or 45, wherein the JAK inhibitor or the T-cell mediator is administered for about 1-30 days prior to administering the nucleic acid sequence encoding connexin 43 polypeptide or the agent that increases the level of a connexin 43 encoding nucleic acid molecule in the subject.

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