Compositions and methods for treating heart disease

Abstract

The invention of the present disclosure features Ca2 +-calmodulin-dependent kinase II (CaMKII) inhibiting multimeric polypeptides, polynucleotides encoding such polypeptides, and methods of treating heart diseases (e.g., catecholamine-sensitive polymorphic ventricular tachycardia (CPVT) or atrial fibrillation (AF)) using the same.

Description

Technical Field

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is a PCT international patent application, claiming priority and benefits to U.S. Provisional Application No. 63 / 342,311 filed on May 16, 2022, the contents of which are incorporated herein by reference in their entirety.

[0003] STATEMENT OF RIGHTS TO INVENTS FROM FEDERALLY SPONSORED RESEARCH

[0004] This invention was made with government support under grant number W81XWH1910473 from the U.S. Department of Defense. The government has certain rights in this invention. Background Art

[0005] Catecholamine-sensitive polymorphic ventricular tachycardia (CPVT) is a disease characterized by abnormal heart rhythm that affects one in every 10,000 people. Symptoms of CPVT include dizziness or fainting associated with exercise or emotional stress. Ventricular tachycardia episodes may cause the heart to stop beating effectively (cardiac arrest), leading to sudden death in children and young people with no known cardiac abnormalities. Treatment of CPVT includes exercise restriction, use of beta blockers, and automatic implantable cardioverter-defibrillators. Other treatments include surgical sympathectomy and flecainide therapy. Unfortunately, these treatments are not effective for all subjects and are limited by subject compliance, drug side effects, or the risk of adverse events (such as fatal electrical storms caused by implantable defibrillators). Therefore, there is currently an unmet need for improved compositions and methods for treating CPVT and other heart diseases characterized by abnormal heart rhythms.

[0006] Atrial fibrillation (AF) currently affects more than 2 million adults in the United States alone, and is the most common type of sustained cardiac arrhythmia seen in clinical practice. While methods exist to treat AF, there is currently no cure, and AF is associated with a shortened life expectancy. Furthermore, approximately 158,000 people die from AF each year in the United States alone. Therefore, there is a need for improved compositions and methods for treating AF. Summary of the invention

[0007] As described below, the invention disclosed herein is characterized by Ca 2+- Calmodulin-dependent kinase II (CaMKII) inhibitory multimeric polypeptides (e.g., comprising autocamtide-2-related inhibitory peptide (AIP) multimers), polynucleotides encoding CaMKII inhibitory multimeric polypeptides, and methods of using such polypeptides and polynucleotides to treat cardiac diseases, conditions or disorders characterized by arrhythmias (e.g., atrial fibrillation (AF), catecholamine-sensitive polymorphic ventricular tachycardia (CPVT), ischemic heart disease, arrhythmias, heart failure (e.g., heart failure associated with aortic tethering), hypertensive heart disease and pulmonary hypertensive heart disease, myocardial infarction, valvular disease, congenital heart disease, cardiac hypertrophy, ventricular arrhythmias or Timothy syndrome).

[0008] In one aspect, the disclosed invention features a multimeric polypeptide that inhibits CaMKII.

[0009] In one aspect, the disclosed invention features an expression vector comprising a polynucleotide encoding a multimeric polypeptide of the above aspects or embodiments thereof.

[0010] In one aspect, the disclosed invention features a pharmaceutical composition comprising an effective amount of a multimeric polypeptide of any of the above aspects or embodiments thereof.

[0011] In one aspect, the disclosed invention features a pharmaceutical composition comprising an effective amount of an expression vector of any of the above aspects or embodiments thereof.

[0012] In one aspect, the disclosed invention features a cell comprising an expression vector of any of the above aspects or embodiments thereof.

[0013] In one aspect, the disclosed invention features a method of regulating arrhythmia in a subject. The method comprises contacting a cell comprising a cardiac ryanodine channel (RYR2) in the subject with a multimeric polypeptide of any of the above aspects or embodiments thereof or a polynucleotide encoding the multimeric polypeptide.

[0014] In one aspect, the invention of the present disclosure features a method of inhibiting phosphorylation of a RYR2 polypeptide in a cell. The method comprises contacting a cell containing a cardiac RYR2 polypeptide with a multimeric polypeptide of any of the above aspects or embodiments thereof or a polynucleotide encoding the multimeric polypeptide.

[0015] In one aspect, the disclosed invention features a method of treating a subject comprising a mutation associated with arrhythmia. The method comprises administering to the subject a multimeric polypeptide of any of the above aspects or embodiments thereof or a polynucleotide encoding the multimeric polypeptide.

[0016] In one aspect, the disclosed invention features a method of treating a subject having a cardiac disease, condition, or disorder characterized by cardiac arrhythmia, comprising administering to the subject a multimeric AIP polypeptide or a polynucleotide encoding the polypeptide.

[0017] In one aspect, the disclosed invention features a method of treating a subject having a cardiac disease, condition, or disorder characterized by cardiac arrhythmia, comprising administering to the subject an adeno-associated viral vector comprising a polynucleotide encoding a multimeric AIP polypeptide.

[0018] In one aspect, the disclosed invention features a method of reducing cardiac variability in a subject with atrial fibrillation, comprising administering to the subject a multimeric AIP polypeptide or a polynucleotide encoding the multimeric AIP polypeptide.

[0019] In any of the above aspects or embodiments thereof, the multimeric polypeptide comprises two or more AIP peptides. In any of the above aspects or embodiments thereof, the multimeric polypeptide comprises about 3 to about 20 AIP peptide repeats. In any of the above aspects or embodiments thereof, the multimeric polypeptide comprises 3, 4, 5 or 6 AIP peptide repeats. In any of the above aspects or embodiments thereof, the multimeric polypeptide comprises 3 AIP peptides. In any of the above aspects or embodiments thereof, the multimeric polypeptide comprises 5 AIP peptides. In any of the above aspects or embodiments thereof, the AIP repeats are continuous and / or separated by a linker. In any of the above aspects or embodiments thereof, the multimeric polypeptide comprises a sequence having at least 85% amino acid sequence identity with the following:

[0020] YKKALHRQEAVDALYKKALHRQEAVDALYKKALHRQEAVDAL(AIPx3); or

[0021] YKKALHRQEAVDALYKKALHRQEAVDALYKKALHRQEAVDALYKKALHRQEAVDALYKKALHRQEAVDAL(AIPx5).

[0022] In any of the above aspects or embodiments thereof, the multimeric polypeptide is fused to a 12.6 kDa FK506 binding protein (FKBP12.6) polypeptide.

[0023] In any of the above aspects or embodiments thereof, the multimeric polypeptide inhibits the EC of CaMKII 50 Less than the EC of AIP to inhibit CaMKII 50 In any of the above aspects or embodiments thereof, the multimeric polypeptide inhibits the EC of CaMKII 50 Less than the EC of AIP to inhibit CaMKII50 5%.

[0024] In any of the above aspects or embodiments thereof, the multimeric polypeptide is operably linked to a promoter suitable for driving expression of the multimeric polypeptide in mammalian cardiac cells. In any of the above aspects or embodiments thereof, the promoter is selected from one or more of the group consisting of a cardiac troponin T promoter, an α-myosin heavy chain (α-MHC) promoter, a myosin light chain-2v (MLC-2v) promoter, and a cardiac NCX1 promoter.

[0025] In any of the above aspects or embodiments thereof, the vector is a retrovirus, an adenovirus or an adeno-associated virus vector (AAV). In embodiments, the AAV is selected from one or more of AAV9, AAV6, AAV2i8, AAVrh10, AAVrh74, MyoAAV, Anc80 and Anc82.

[0026] In any of the above aspects or embodiments thereof, the cell is in vivo or in vitro. In any of the above aspects or embodiments thereof, the cell is an in vivo human cell.

[0027] In any of the above aspects or embodiments thereof, the mutation is in the cardiac ryanodine channel (RYR2). In any of the above aspects or embodiments thereof, the mutation is R4651I .

[0028] In any of the above aspects or embodiments thereof, the method inhibits arrhythmia. In any of the above aspects or embodiments thereof, the arrhythmia is catecholamine-sensitive polymorphic ventricular tachycardia. In any of the above aspects or embodiments thereof, the arrhythmia is atrial fibrillation.

[0029] In any of the above aspects or embodiments thereof, the subject is a mammal and / or the cell is from a mammal. In any of the above aspects or embodiments thereof, the mammal is a human.

[0030] In any of the above aspects or embodiments thereof, the polynucleotide is DNA, RNA or a combination thereof. In any of the above aspects or embodiments thereof, the polynucleotide comprises one or more modified nucleobases. In any of the above aspects or embodiments thereof, the polynucleotide is present in a vector.

[0031] In any of the above aspects or embodiments thereof, the adeno-associated viral vector is an effective amount of adeno-associated viral vector. In any of the above aspects or embodiments thereof, the effective amount is between about 1x10 10 Viral genomes / kg is about 1x10 14In any of the above aspects or embodiments thereof, the effective amount is effective to transfect between about 20% and about 40% of the muscle cells in the subject.

[0032] In any of the above aspects or embodiments thereof, the administering is effective to reduce heart rate variability in the subject. In any of the above aspects or embodiments thereof, the administering is effective to reduce atrial scar in the subject.

[0033] In any of the above aspects or embodiments thereof, the cardiac disease, condition or disorder is atrial fibrillation.

[0034] In any of the above aspects or embodiments thereof, the multimeric AIP comprises between about 3 and 10 AIP repeats.

[0035] The compositions and articles defined herein are isolated or otherwise made with respect to the examples provided hereinafter. Other features and advantages of the invention will be apparent from the detailed description and claims.

[0036] definition

[0037] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art. The following references provide general definitions of many of the terms used in the present invention by technicians: Singleton et al., Dictionary of Microbiology and Molecular Biology (Second Edition 1994); The Cambridge Dictionary of Science and Technology (Walker Edition, 1988); The Glossary of Genetics, 5th Edition, R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). Unless otherwise indicated, the following terms used herein have the meanings assigned thereto.

[0038] "Multimer" or "multimeric polypeptide" means a polypeptide sequence containing two or more repeats of an amino acid sequence. In one embodiment, the multimeric polypeptide inhibits CaMKII. Such multimeric polypeptides are referred to as "CaMKII-inhibiting multimeric polypeptides."

[0039] "Autocamtide-2-related inhibitory peptide (AIP)" means a peptide or fragment thereof having at least about 85% amino acid sequence identity with YKKALRRQEAVDAL (SEQ ID NO: 1); comprising or consisting of at least about 9 to 14 consecutive amino acids of SEQ ID NO: 1; and having cardiac modulatory activity and / or CaMKII inhibitory activity. In one embodiment, AIP has at least about 85% amino acid sequence identity with KKALRRQEAVDAL. In some cases, AIP has at least about 85% amino acid sequence identity with kkKlrrqeaFdal (AIPo), wherein capital letters represent changes to the amino acid sequence KKALRRQEAVDAL. In some cases, AIP contains one or more changes described in Ishida et al., "Critical amino acid residues of AIP, a highly specific inhibitory peptide ofcalmodulin-dependent protein kinase II", FEBS Letters, 427: 115-118 (1998), the contents of which are incorporated herein by reference in their entirety for all purposes, and / or AIP contains changes in one or more key amino acid residues described therein (e.g., at positions corresponding to the capitalized amino acid residues provided in the amino acid sequence kkKlrrqeaFdal). In some cases, AIP has at least 70%, 75%, 80%, 85%, or 90% amino acid sequence identity to KKALRRQEAVDAL and contains one or more of the following amino acid substitutions or combinations thereof: K1A, K1Y, K2A, K2Y, A3K, A3Y, L4A, L4F, L4Nle (norleucine), L4G, L4I, LrNva (norvaline), L4M, L4V, L4Y, R5A, R5K, R5H, R5Y, R6A, R6Orn, R6K, R6dmR (N G ,N G, R6Cit (citrulline), R6H, R6Y, Q7A, Q7E, Q7D, Q7N, Q7Orn, Q7Y, E8A, E8Y, A9G, A9C, A9V, A9I, A9L, A9Y, V10A, V10F, V10I, V10L, V10Nva (norvaline), V10Abu (2-aminobutyric acid), V10G, V10Y, D11A, D11Y, A12Y, L13A, L13Y, and A3K / V10F. In an embodiment, AIPo has improved selectivity for CaMKII inhibition compared to PKC inhibition. In one embodiment, the AIP peptide comprises one or more changes in the peptide sequence. In one embodiment, the AIP peptide consists essentially of SEQ.ID.NO:1. In another embodiment, the AIP peptide consists of SEQ.ID.NO: 1 or consists of about 9 to 13 consecutive amino acids of SEQ.ID.NO: 1. In one embodiment, the AIP peptide consists essentially of SEQ.ID.NO: 1 or consists essentially of about 9 to 13 consecutive amino acids of SEQ.ID.NO: 1. In another embodiment, the AIP peptide comprises one or more modified amino acids. In another embodiment, the AIP peptide comprises 1, 2, 3, 4, 5 or more changes in SEQ.ID.NO: 1.

[0040] "Autocamtide-2-related inhibitory (AIP) multimeric polypeptide" means a polypeptide comprising two or more AIP peptide repeats. In one embodiment, the AIP multimer comprises 2 to 20 repeats (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20). In one embodiment, the AIP multimeric polypeptide comprises the sequence YKKALHRQEAVDALYKKALHRQEAVDALYKKALHRQEAVDAL or the sequence YKKALHRQEAVDALYKKALHRQEAVDALYKKALHRQEAVDALYKKALHRQEAVDALYKKALHRQEAVDAL. In another embodiment, the repeats are consecutive.

[0041] "AIP multimeric polynucleotide" means a polynucleotide encoding an AIP multimeric polypeptide.

[0042] "Calcium / calmodulin-dependent protein kinase II (CaMKII) polypeptide" means a polypeptide or a fragment thereof having at least 85% sequence identity to the amino acid sequence provided in GenBank Accession No. AAH32784.1 below, or a fragment thereof having kinase activity.

[0043] >AAH32784.1 Calcium / calmodulin-dependent protein kinase IIδ [Homo sapiens]

[0044] MASTTTCTRFTDEYQLFEELGKGAFSVVRRCMKIPTGQEYAAKIINTKKLSARDHQKLEREARICRLLKHPNIVRLHDSISEEGFHYLVFDLVTGGELFEDIVAREYYSEADASHCIQQ ILESVNHCHLNGIVHRDLKPENLLLASKSKGAAVKLADFGLAIEVQGDQQAWFGFAGTPGYLSPEVLRKDPYGKPVDMWACGVILYILLVGYPPFWDEDQHRLYQQIKAGAYDFPSPEWD TVTPEAKDLINKMLTINPAKRITASEALKHPWICQRSTVASMMHRQETVDCLKKFNARRKLKGAILTTMLATRNFSAAKSLLKKPDGVKESTESSNTTIEDEDVKARKQEIIKVTEQLIE AINNGDFEAYTKICDPGLTAFEPEALGNLVEGMDFHRFYFENALSKSNKPIHTIILNPHVHLVGDDAACIAYIRLTQYMDGSGMPKTMQSEETRVWHRRDGKWQNVHFHRSGSPTVPIK.

[0045] "Calcium / calmodulin-dependent protein kinase II (CaMKII) polynucleotide" means a polynucleotide encoding a CaMKII polypeptide. A representative CaMKII polynucleotide sequence is provided below (GenBank Accession No. BC032784.1).

[0046] >BC032784.1:63-1499 Homo sapiens calcium / calmodulin-dependent protein kinase IIδ, mRNA (cDNA clone MGC:44911 IMAGE:5178265), complete coding sequence (cds)

[0047]

[0048] "CN19o peptide" means a peptide or fragment thereof having at least about 85% amino acid sequence identity to KRAPKLGQIGRQKAVDIED (SEQ ID NO: 2); comprising or consisting of at least about 9 to 19 consecutive amino acids of SEQ ID NO: 2; and having cardiomodulatory activity and / or CaMKII inhibitory activity. In one embodiment, the CN19o peptide comprises one or more changes in the peptide sequence.

[0049] "CN19o multimeric polypeptide" means a polypeptide comprising two or more CN19o peptide repeats or a fragment thereof. In one embodiment, the CN19o multimer comprises the sequence

[0050] KRAPKLGQIGRQKAVDIEDKRAPKLGQIGRQKAVDIEDKRAPKLGQIGRQKAVDIED or sequence

[0051] KRAPKLGQIGRQKAVDIEDKRAPKLGQIGRQKAVDIEDKRAPKLGQIGRQKAVDIEDKRAPKLGQIGRQKAVDIEDKRAPKLGQIGRQKAVDIEDKRAPKLGQIGRQKAVDIED. In another embodiment, the repetitions are consecutive.

[0052] "CN19o multimeric polynucleotide" means a polynucleotide encoding a CN19o multimeric polypeptide.

[0053] "12.6-kDa FK506-binding protein (FKBP12.6) polypeptide" means a polypeptide or a fragment thereof having at least 85% sequence identity to the amino acid sequence provided in the following NCBI Reference Sequence Accession No. NP_004107.1, or a fragment thereof that can bind to a RYR2 polypeptide.

[0054] >NP_004107.1 Peptidylprolyl cis-trans isomerase FKBP1B isoform a [Homo sapiens]

[0055] MGVEIETISPGDGRTFPKKGQTCVVHYTGMLQNGKKFDSSRDRNKPF KFRIGKQEVIKGFEEGAAQMSLGQRAKLTCTPDVAYGATGHPGVIPPNAT LIFDVELLNLE.

[0056] "12.6-kDa FK506-binding protein (FKBP12.6) polynucleotide" means a polynucleotide encoding a FKBP12.6 polypeptide. A representative FKBP12.6 polynucleotide sequence is provided below (NCBI Reference Sequence Accession No. NM_004116.5).

[0057] >NM_004116.5:110-436 Homo sapiens FKBP prolyl isomerase 1B (FKBP1B), transcript variant 1, mRNA

[0058] ATGGGCGTGGAGATCGAGACCATCTCCCCCGGAGACGGAAGGACATTCCCCAAGAAGGGCCAAACGTGTGTGGTGCACTACACAGGAATGCTCCAAAATGGGAAGAAGTTTGATTCATCCAGAGACAGAAACAAACCTTTCAAGTTCAGAATTGGCAAACAGGA AGTCATCAAAGGTTTTGAAGAGGGTGCAGCCCAGATGAGCTTGGGGCAGAGGGCGAAGCTGACCTGCACCCTGATGTGGCATATGGAGCCACGGGCCACCCCGGTGTCATCCCTCCCAATGCCACCCTCATCTTTGACGTGGAGCTGCTCAACTTAGAGTGA.

[0059] "Ryanodine receptor 2 (RYR2) polypeptide" means a polypeptide or a fragment thereof having at least 85% sequence identity to the amino acid sequence provided in the following GenBank Accession No. CAA62975.1, or a fragment thereof that can form a homotetramer that functions as a calcium channel.

[0060] >CAA62975.1 Ranodine receptor, partial [Homo sapiens]

[0061] LYELLAALIRGNRKNCAQFSGSLDWLISRLERLEASSGILEVLHCVLVESPEALNIIKEGHIKSIISLLDKHGRNHKVLDVLCSLCVCHGVAVRSNQHLICDNLLPGRDLLLQTRLVNHVSSMRPNIFLGVSEGSAQYKKWYYELMVDH.

[0062] "Ryanodine receptor 2 (RYR2) polynucleotide" means a polynucleotide encoding a RYR2 polypeptide. A representative RYR2 polynucleotide sequence is provided below (GenBank Accession No. X91869.1).

[0063] >X91869.1 Homo sapiens ryanodine receptor mRNA

[0064] CTGTATGAGTTGCTGGCGGCTCTAATTAGAGGAAATCGTAAAAACTGTGCTCAATTTTCTGGCTCCCTCGACTGGTTGATCAGCAGATTGGAAAGACTGGAAGCTTCTTCAGGCATTCTGGAAGTTTTACACTGTGTTTTAGTAGAAAGTCCAGAAGCTCTAAATATTATTAAAGAAGGACATATTAAATCTATTATCTCACTTTTAGACAAACATGGAAGAAA TCACAAGGTTCTGGATGTCTTGTGCTCACTCTGTGTTTGCCACGGGGTTGCAGTCCGTTCTAACCAGCATCTCATCTGTGACAATCTCCTACCAGGAAGAGACTTGTTATTGCAGACACGTCTTGTGAACCATGTCAGCAGCATGAGACCCAATATTTTTCTGGGCGTCAGTGAAGGTTCTGCTCAGTATAAGAAATGGTACTATGAATTGATGGTGGACCAC.

[0065] "CaMKII inhibitor" means a peptide or small molecule that inhibits CaMKII activity. Exemplary inhibitors are known in the art (e.g., AIP, CN19, CN27, CN19o, CN21), and are described, for example, by Coultrap et al., PLOS One e25245, Vol. 6, No. 10, 2011 and Pellicena et al., Frontiers in Pharmacology 21: 1-20, 2014. Other inhibitors include:

[0066]

[0067]

[0068] "Agent" means a peptide, polypeptide, nucleic acid molecule or small chemical compound.

[0069] "Ameliorate" means to reduce, inhibit, attenuate, alleviate, arrest or stabilize the development or progression of a disease. In some embodiments, the disease is a cardiac disease or disorder.

[0070] With respect to an amino acid sequence, an "alteration" refers to a change in the identity of one or more amino acids in the amino acid sequence.

[0071] "Analog" means a molecule that is not identical but has similar functional or structural features. For example, a polypeptide analog retains the biological activity of the corresponding naturally occurring polypeptide while having certain biochemical modifications that enhance the function of the analog relative to the naturally occurring polypeptide. Such biochemical modifications may increase the protease resistance, membrane permeability, or half-life of the analog without changing, for example, ligand binding. Analogs may include non-natural amino acids. Polynucleotide analogs retain the biological activity of the corresponding naturally occurring polypeptide while having certain biochemical modifications that enhance the function of the analog relative to the naturally occurring polynucleotide. Such biochemical modifications may increase the nuclease resistance, membrane permeability, or half-life of the analog without changing, for example, its functional activity (such as its protein encoding function). Analogs may include modified nucleic acid molecules.

[0072] The term "cardiomyocyte" as used herein refers broadly to a muscle cell of the heart. In one embodiment, the mammalian heart cell is a cardiomyocyte. In another embodiment, the cardiomyocyte differentiated from induced pluripotent stem cells is a cardiomyocyte.

[0073] As used herein, the phrase "cardiac condition, disease, or disorder" is intended to include all disorders characterized by insufficient, poor or abnormal cardiac function. Exemplary cardiac conditions, diseases or disorders include, but are not limited to, atrial fibrillation (AF), catecholamine-sensitive polymorphic ventricular tachycardia (CPVT), ischemic heart disease, arrhythmias, heart failure (e.g., heart failure associated with aortic tethering), hypertensive heart disease and pulmonary hypertensive heart disease, myocardial infarction, valvular disease, congenital heart disease, myocardial hypertrophy, ventricular arrhythmias or Timothy syndrome) and any condition that causes congestive heart failure in a subject (particularly a human subject). Insufficient or abnormal cardiac function may be the result of disease, injury, genetic mutation and / or aging. Background is that the response to myocardial injury follows a clear path, in which some cells die and others enter a dormant state, which is not yet dead but dysfunctional. Subsequently, inflammatory cells infiltrate and collagen deposition becomes part of the scar, all of which occur simultaneously with the inward growth of new blood vessels and a certain degree of continuous cell death.

[0074] "Effective amount" or "therapeutically effective amount" means the amount of drug required to improve the symptoms of a disease relative to an untreated patient. The effective amount of the active compound used to practice the present invention to treat a disease varies depending on the mode of administration, the age, weight, and general health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate amount and dosage regimen. This amount is referred to as an "effective" amount. Therefore, the term "therapeutically effective amount" refers to an amount of a composition disclosed herein that, when administered to a typical subject with a cardiovascular condition, disease, or disorder, is sufficient, for example, to significantly reduce symptoms or clinical indicators associated with cardiac dysfunction or disorder in treatment or prevention.

[0075] In terms of treatment (e.g., a subject's cardiovascular condition or disease), the term "therapeutically effective amount" refers to an amount that is safe and sufficient to prevent or postpone the development of a cardiovascular disease or disorder (e.g., arrhythmia). Thus, the amount can cure or inhibit arrhythmia, or relieve a cardiovascular disease or disorder, slow down the progression of a cardiovascular disease, slow down or inhibit the symptoms of a cardiovascular disease or disorder, slow down or inhibit the formation of secondary symptoms of a cardiovascular disease or disorder, or inhibit the development of secondary symptoms of a cardiovascular disease or disorder. The effective amount for treating a cardiovascular disease or disorder depends on the type of cardiovascular disease being treated, the severity of the symptoms, the subject being treated, the age and overall condition of the subject, the mode of administration, etc. Therefore, it is impossible to specify an exact "effective amount". However, for any given situation, those skilled in the art can determine a suitable "effective amount" using only routine experiments. The efficacy of treatment can be judged by a general practitioner, for example, efficacy can be assessed in an animal model of a cardiovascular disease or disorder discussed herein, such as treating a rodent with acute myocardial infarction or ischemia-reperfusion injury, and any treatment or administration of a composition or formulation that results in a reduction in at least one symptom of a cardiovascular disease or disorder as disclosed herein, such as an increase in cardiac ejection fraction, a decrease in the rate of heart failure, a decrease in infarct size, a decrease in related morbidity (pulmonary edema, renal failure, arrhythmia), an improvement in exercise tolerance or other quality of life indicators, and a decrease in mortality, all indicate that the treatment is effective. In embodiments where the composition is used to treat a cardiovascular disease or disorder, experimental animal models of cardiovascular disease can be used to judge the efficacy of the composition, such as an animal model of ischemia-reperfusion injury (Headrick JP, Am JPhysiol Heartcirc Physiol 285; H1797; 2003) and an animal model of acute myocardial infarction. (Yang Z, Am J Physiol Heart Circ. Physiol 282:H949:2002; Guo Y, J Mol Cell Cardiol 33;825-830, 2001). When using an experimental animal model, the treatment is effective when the symptoms of the cardiovascular disease or disorder are reduced, such as when one or more of the symptoms of dyspnea, chest pain, palpitations, dizziness, syncope, edema, cyanosis, pallor, fatigue and hypertension are reduced in the treated animals earlier than in the untreated animals.

[0076] Subjects suitable for treatment by the methods disclosed herein can be identified by any method for diagnosing cardiac arrhythmias. Methods for diagnosing these conditions are well known to those skilled in the art. As a non-limiting example, cardiac arrhythmias can be diagnosed by an electrocardiogram (ECG or EKG), which is a graphical record of heart activity on paper or a computer monitor.

[0077] The terms "coronary artery disease" and "acute coronary syndrome" are used interchangeably herein and refer to myocardial infarction associated with a cardiovascular condition, disease or disorder, including all disorders characterized by insufficient, poor or abnormal cardiac function, such as ischemic heart disease, hypertensive heart disease and pulmonary hypertensive heart disease, valvular disease, congenital heart disease, and any condition that leads to congestive heart failure in a subject, particularly a human subject. Insufficient or abnormal cardiac function may be the result of disease, injury and / or aging. Background is that the response to myocardial injury follows a clear path, in which some cells die and others enter a dormant state, which is not yet dead but is dysfunctional. Subsequently, inflammatory cells infiltrate and collagen is deposited as part of the scar, all of which occur simultaneously with the ingrowth of new blood vessels and a certain degree of continued cell death.

[0078] In the present disclosure, "comprises", "comprising", "containing", and "having" and the like may have the meanings ascribed thereto in U.S. patent law, and may refer to "includes", "including", and the like; "consisting essentially of" or "consists essentially of" also have the meanings ascribed thereto in U.S. patent law, and the terms are open ended, allowing for more content than that recited, but excluding prior art embodiments, as long as the basic or novel features of the recited content are not altered by the presence of content other than that recited. In some embodiments, any embodiment designated as "comprising" a particular component or element is also deemed to be "consisting of" or "consisting essentially of" the particular component or element.

[0079] By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. This portion preferably contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the full length of a reference nucleic acid molecule or polypeptide. A fragment may contain 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 or 1000 nucleotides or amino acids.

[0080] "Heart rate variability" or "beat-to-beat variability" means the variability of the RR interval (the interval between two consecutive R waves measured by an electrocardiogram), particularly as measured by the standard deviation of the RR interval. In some embodiments, an increase in a subject's heart rate variability or beat-to-beat variability relative to a reference indicates an arrhythmia or is a symptom of a cardiovascular disease, condition or disorder. In some embodiments, administration of an agent disclosed herein to a subject having an increased heart rate variability or beat-to-beat variability relative to a reference is effective to reduce the heart rate variability or beat-to-beat variability, preferably, such administration is effective to reduce the heart rate variability or beat-to-beat variability to a reference level.

[0081] "Hybridization" refers to hydrogen bonding between complementary nucleobases, which may be Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonding. For example, adenine and thymine are complementary nucleobases that pair by forming hydrogen bonds.

[0082] The terms "isolated", "purified" or "biologically pure" refer to materials that are free to varying degrees from components that normally accompany them in their natural state. "Isolate" means a certain degree of separation from the original source or surrounding environment. "Purify" means a degree of separation greater than isolation. A "purified" or "biologically pure" protein is sufficiently free of other materials so that any impurities do not substantially affect the biological properties of the protein or cause other adverse consequences. That is, if the nucleic acid or peptide of the present invention is substantially free of cellular material, viral material or culture medium when produced by recombinant DNA technology, or substantially free of chemical precursors or other chemicals when chemically synthesized, the nucleic acid or peptide is purified. Purity and homogeneity are generally determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" may mean that a nucleic acid or protein produces essentially one band in an electrophoretic gel. For proteins that can be modified (e.g., phosphorylated or glycosylated), different modifications may produce different isolated proteins that can be purified separately.

[0083] "Isolated polynucleotide" means a nucleic acid (e.g., DNA) that is free of flanking genes located in the naturally occurring genome of the organism from which the nucleic acid molecule of the invention is derived. Thus, the term includes, for example, recombinant DNA that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryotic or eukaryotic organism, or that exists as a separate molecule (e.g., cDNA or genomic or cDNA fragments generated by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes RNA molecules transcribed from a DNA molecule, and recombinant DNA that is part of a hybrid gene encoding an additional polypeptide sequence.

[0084] "Isolated polypeptide" means a polypeptide of the present invention that has been separated from naturally associated components. Generally, a polypeptide is isolated when it is at least 60% by weight free of the proteins and naturally occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99% by weight of the polypeptide of the present invention. An isolated polypeptide of the present invention can be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide, or by chemically synthesizing the protein. Purity can be measured by any suitable method, such as column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

[0085] As used herein, "obtaining" in "obtaining an agent" includes synthesizing, purchasing or otherwise obtaining the agent.

[0086] "Polypeptide" or "amino acid sequence" means any amino acid chain, regardless of length or post-translational modification. In various embodiments, the post-translational modification is glycosylation or phosphorylation. In various embodiments, conservative amino acid substitutions may be made to the polypeptide to provide functionally equivalent variants or homologs of the polypeptide. In some aspects, the present invention encompasses sequence changes that result in conservative amino acid substitutions. In some embodiments, "conservative amino acid substitutions" refer to amino acid substitutions in proteins to which the conservative amino acid substitutions are directed that do not change relative charge or size characteristics. Variants may be prepared according to methods known to those skilled in the art for altering polypeptide sequences, as can be seen in references for compiling such methods, such as Molecular Cloning: A Laboratory Manual, J. Sambrook et al., ed., 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989 or Current Protocols in Molecular Biology, FM Ausubel et al., ed., John Wiley & Sons, Inc., New York. Non-limiting examples of conservative amino acid substitutions include substitutions between amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. In various embodiments, conservative amino acid substitutions can be made to the amino acid sequences of the proteins and polypeptides disclosed herein.

[0087] "Reduces" means a negative change relative to a reference. In the context of arrhythmias, the term reduces refers to any significant reduction in symptoms. For example, the incidence of arrhythmias or symptoms or the severity of symptoms is reduced by at least 1%, at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, or at least 100%, including integer percentages from 1% to 100%.

[0088] "Reference" means a corresponding control condition. In one embodiment, the reference is an untreated control. In another embodiment, the reference is a wild-type healthy control. In another embodiment, a subject with a cardiac disease or disorder is treated with a multimeric polypeptide as described herein, and the effect of such treatment is evaluated relative to a subject before treatment or an untreated subject also suffering from a cardiac disease or disorder.

[0089] A "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence; for example, a fragment of a full-length cDNA or gene sequence, or a complete cDNA or gene sequence. For polypeptides, the length of a reference polypeptide sequence is typically at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of a reference nucleic acid sequence is typically at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 nucleotides, or any integer near or in between.

[0090] Nucleic acid molecules useful in the methods of the present invention include any nucleic acid molecules encoding polypeptides of the present invention or fragments thereof. Such nucleic acid molecules need not be 100% identical to endogenous nucleic acid sequences, but will generally exhibit substantial identity. Polynucleotides having "substantial identity" to endogenous sequences are generally capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the present invention include any nucleic acid molecules encoding polypeptides of the present invention or fragments thereof. Such nucleic acid molecules need not be 100% identical to endogenous nucleic acid sequences, but will generally exhibit substantial identity. Polynucleotides having "substantial identity" to endogenous sequences are generally capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule. "Hybridize" refers to pairing between complementary polynucleotide sequences (e.g., genes described herein) or portions thereof to form double-stranded molecules under various stringent conditions. (See, e.g., Wahl, GM and SL Berger (1987) Methods Enzymol. 152: 399; Kimmel, AR (1987) Methods Enzymol. 152: 507).

[0091] For example, stringent salt concentrations are typically less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of an organic solvent (e.g., formamide), while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions typically include a temperature of at least about 30°C, more preferably at least about 37°C, and most preferably at least about 42°C. Additional parameter variations, such as hybridization time, detergent (e.g., sodium dodecyl sulfate (SDS)) concentration, and inclusion or exclusion of carrier DNA are well known to those skilled in the art. Various levels of stringency can be achieved by combining these different conditions as desired. In a preferred embodiment, hybridization will be carried out at 30°C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will be conducted in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg / ml denatured salmon sperm DNA (ssDNA) at 37° C. In a most preferred embodiment, hybridization will be conducted in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA at 42° C. Useful variations of these conditions will be apparent to those skilled in the art.

[0092] For most applications, the stringency of the washing step after hybridization will also vary. Washing stringency conditions can be defined by salt concentration and temperature. As described above, washing stringency can be increased by reducing salt concentration or increasing temperature. For example, the stringent salt concentration of the washing step is preferably less than about 30mM NaCl and 3mM trisodium citrate, and most preferably less than about 15mM NaCl and 1.5mM trisodium citrate. The stringent temperature conditions of the washing step generally include a temperature of at least about 25°C, more preferably at least about 42°C, and even more preferably at least about 68°C. In a preferred embodiment, the washing step will be carried out at 25°C in 30mM NaCl, 3mM trisodium citrate and 0.1% SDS. In a more preferred embodiment, the washing step will be carried out at 42°C in 15mM NaCl, 1.5mM trisodium citrate and 0.1% SDS. In a more preferred embodiment, the wash step will be performed at 68°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations of these conditions will be apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., U.S. 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

[0093] "Substantially identical" means a polypeptide or nucleic acid molecule that exhibits at least 85% identity to a reference amino acid sequence or nucleic acid sequence. In embodiments, such sequences are at least 90%, 95% or even 99% identical to a reference sequence at the amino acid level or nucleic acid.

[0094] Sequence identity is typically measured using sequence analysis software (e.g., the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wisconsin 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by specifying the degree of homology for various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary method for determining the degree of identity, the BLAST program can be used, with a probability score in the range of e -3 and e -100 Between indicates closely related sequences.

[0095] As used herein, the term "modulate" means to adjust or regulate to a certain extent.

[0096] As used herein, the terms "pharmaceutically acceptable", "physiologically tolerable" and grammatical variations thereof, when referring to compositions, carriers, diluents and agents, are used interchangeably and represent that the material can be administered to or to a mammal without causing adverse physiological effects, such as nausea, dizziness, stomach discomfort, and the like. Unless so desired, a pharmaceutically acceptable carrier will not promote an increased immune response to the agent mixed therewith. The preparation of pharmaceutical compositions containing active ingredients dissolved or dispersed therein is well known in the art and need not be limited based on the formulation. Typically, such compositions are prepared as injectable liquid solutions or suspensions, but may also be prepared in a solid form suitable for dissolution or suspension in a liquid prior to use. The preparation may also be emulsified or presented as a liposomal composition. The active ingredient may be mixed with a pharmaceutically acceptable and compatible excipient in an amount suitable for use in the treatment methods described herein. Suitable excipients include, for example, water, saline, glucose, glycerol, ethanol, and the like and combinations thereof. In addition, if necessary, the composition may contain a small amount of auxiliary substances, such as wetting agents or emulsifiers, pH buffers, etc., which enhance the effectiveness of the active ingredient. The therapeutic composition of the present invention may include a pharmaceutically acceptable salt of the component. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the polypeptide) formed with inorganic acids (e.g., hydrochloric acid or phosphoric acid) or organic acids (e.g., acetic acid, tartaric acid, mandelic acid, etc.). Salts formed with free carboxyl groups may also be derived from inorganic bases (e.g., sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide) and organic bases (e.g., isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, etc.). Physiologically tolerable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions that do not contain any substance except the active ingredient and water, or contain buffers (e.g., sodium phosphate, saline, or both at physiological pH), such as phosphate-buffered saline. Further, the aqueous carrier may contain more than one buffer salt, as well as salts such as sodium chloride and potassium chloride, glucose, polyethylene glycol and other solutes. The liquid composition may contain a liquid phase in addition to water, and may contain a liquid phase other than water. Examples of such additional liquid phases are glycerol, vegetable oils (such as cottonseed oil) and water-oil emulsions. The amount of active agent used in the methods described herein to effectively treat a particular disorder or condition will depend on the nature of the disorder or condition and can be determined by standard clinical techniques. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, A. Osol (a standard reference in this area). For example, 1.5% by weight of the active ingredient is dissolved in a 0.9% sodium chloride solution to prepare a parenteral composition suitable for injection.

[0097] In one embodiment, a "pharmaceutically acceptable" carrier does not include an in vitro cell culture medium.

[0098] In one embodiment, the term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms that are approved by the U.S. federal or state regulatory agencies or listed in the U.S. Pharmacopeia or other generally recognized pharmacopoeias for use in animals (especially for humans) within the scope of reasonable medical judgment and are suitable for contact with human and animal tissues without excessive toxicity, irritation, allergic reaction or other problems or complications, and commensurate with a reasonable benefit / risk ratio.

[0099] The term "carrier" refers to a diluent, adjuvant, excipient or vehicle that is administered with the therapeutic agent. Such pharmaceutical carriers may be sterile liquids, such as water and oils, including oils of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, water is a preferred carrier. Saline solutions and aqueous glucose and glycerol solutions may also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc. If desired, the composition may also contain a small amount of a wetting agent or emulsifier, or a pH buffer. These compositions may be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. The composition may be formulated into suppositories with conventional adhesives and carriers (such as triglycerides). Oral formulations can include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, 18th edition, edited by Gennaro (Mack Publishing Co., 1990). The formulation should suit the mode of administration.

[0100] "Subject" means a mammal, including but not limited to a human or non-human mammal, such as a bovine, equine, canine, ovine, rodent, or feline. In one embodiment, the subject suffers from or has a predisposition to suffer from a monogenic disease, disorder, or condition that can be treated with the gene therapy vectors, cell-based therapies, and methods disclosed elsewhere herein.

[0101] 43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,

[0102] The term "tissue" refers to a group or layer of similar specialized cells that work together to perform some specific function. The term "tissue-specific" refers to the origin or defining characteristics of cells from a particular tissue.

[0103] As used herein, the terms "treat," "treating," "treatment," and the like refer to the reduction or amelioration of a disorder and / or symptoms associated therewith. It should be appreciated that, although not excluded, treatment of a disorder or condition does not require complete elimination of the disorder, condition, or symptoms associated therewith.

[0104] Unless specifically stated or obvious from the context, as used herein, the term "or" should be understood as inclusive. Unless specifically stated or obvious from the context, as used herein, the terms "a", "an" and "the" should be understood as singular or plural.

[0105] Unless specifically stated or obvious from the context, as used herein, the term "about" should be understood as being within the normal tolerance range in the art.

[0106] The recitation of any defined variable in a list of chemical groups herein includes the definition of that variable as any single group or combination of the listed groups. The recitation of an embodiment of a variable or aspect herein includes the definition of that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0107] Any composition or method provided herein can be combined with one or more of any other compositions and methods provided herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0108] Figures 1A to 1C Schematic diagrams, graphs and bar charts are provided showing how the peptide inhibitor autocamphin 2-related inhibitory peptide (AIP) can be used as part of gene therapy for catecholamine-sensitive polymorphic ventricular tachycardia (CPVT). Figure 1AA schematic diagram is provided showing how to use a clinical vector (e.g., adeno-associated virus (AAV)) to deliver AIP to cells (e.g., mouse cells) to inhibit or prevent CaMKII phosphorylation of CPVT mutant RYR2 (R4650I in the mouse polypeptide is shown as an example). In addition to the inhibitory peptide cargo, the clinical vector contains a promoter and a capsid, either of which can be optimized to improve the efficacy of the vector in therapy. Figure 1A In the present invention, RYR2 stands for "cardiac ryanodine receptor 2". Figure 1B Electrocardiograms of mice treated with AAV expressing a negative control (green fluorescent protein (GFP)) or AIP are provided. Mice treated with AIP showed reduced ventricular tachycardia (VT) after pacing compared to mice treated with GFP. Figure 1C Provided is a bar graph showing the percentage of mice that exhibited induced ventricular tachycardia (VT) in wild-type mice or RYR2-R176Q+ / 1 mice (CPVT mice with RyR2 R176Q mutation) treated with AIP or GFP. The scores in the bars represent the number of mice that exhibited induced VT as a proportion of the total number of mice studied. Administration of AIP resulted in a reduction in induced VT in RYR2-R176Q+ / - mice.

[0109] Figure 2A-2G Provides diagrams, protein structures, schematics, bar graphs and images showing how to optimize therapeutic vehicles containing CaMKII inhibitors for the treatment of catecholamine-sensitive polymorphic ventricular tachycardia (CPVT) by altering the potency, binding and / or multimerization of the CaMKII inhibitors. Figure 2AA graph showing the IC50 concentrations of apocampal inhibitory peptide 2-related (AIP) and CN19o is provided (Coultrap et al., "Improving a Natural CaMKII Inhibitor by Random and Rational Design," PLoS One, Oct. 3, 2011, doi.org / 10.1371 / journal.pone.0025245; and Ishida et al., "Critical amino acid residues of AIP, a highly specific inhibitory peptide of calmodulin-dependent protein kinase II," FEBS Letters, 427:115-118 (1998), the disclosures of which are incorporated herein by reference in their entirety for all purposes). CN19o has a lower IC50 than AIP and is therefore a more effective inhibitor of CaMKII activity in the in vitro systems used for these assays. Figure 2B Provides EM images of RYR2 at 6 angstrom resolution. Figure 2B In the present invention, FKBP12.6 represents "FK506 binding protein" that stabilizes RyR2 and prevents abnormal activation of the channel during the resting phase of the cardiac cycle. FKBP12.6 stabilizes RyR2 in the closed state. FKBP12.6 acts as a stabilizer to enhance the rigidity of the HD2 (HD2 and HD2') and P2 (P2 and P2') domains (Chi et al., "Molecular basis for allosteric regulation of the type 2ryanodine receptor channel gating by key modulators", PNAS, 116: 25575-25582 (2019), the disclosure of which is incorporated herein by reference in its entirety for all purposes). Figure 2C A schematic diagram showing AIPx5 multimers is provided. Figure 2D A schematic diagram showing the experimental design for evaluating the effectiveness of different candidate therapeutic vehicles in reducing arrhythmias in a CPVT mouse model (RYR2-R46450I) is provided. Figure 2D The left portion of provides a schematic diagram of different polypeptides that may be contained within a therapeutic vehicle to be delivered to a subject. Figure 2D, P2A represents a self-cleaving peptide, AIP represents an example of autocamphinone 2-related inhibitory peptide (AIP), mCherry represents fluorescent protein mCherry, CN19o represents the CaMKII inhibitor CN19o, FKBP12.6 represents the FKBP12.6 polypeptide bound to RYR2, and the polygon represents the viral capsid. Figure 2D ( Figure 2D The rightmost part of (continued) shows WT mice and RyR2 R4650 / WT Electrocardiogram of mouse RYR2 R4650 / WT The mice's heartbeats appeared irregular. Figure 2E Provide display application Figure 2D The polypeptide described in Ryr2 R4650I / WT Graph of the percentage of ectopic beats in mice. Administration of AIPx5 minimized the instances of ectopic beats observed in mice. Figure 2F Provide display application Figure 2D The polypeptide described in Ryr2 R4650I / WT Bar graph showing the percentage of mice that experienced inducible ventricular tachycardia (VT). Administration of AIPx5 was associated with a reduction in instances of inducible VT in mice. Figure 2G Provides images of cardiac tissue showing mCherry expression in the tissue, confirming Figure 2D The constructs described in were delivered to Ryr2 R4650I / WT Mouse heart tissue and expression.

[0110] Figure 3 Provide display Figure 2D Graphs, tubulin immunoblot images and figures of the effects of the indicated constructs on in vivo CaMKII activity. To confirm inhibition of CaMKII activation in vivo, phosphorylation of phospholamban (PLB) at threonine-17, a known target of CaMKII, was analyzed from whole heart lysates. Figure 3 The middle panel provides images of tubulin immunoblots showing the expression of glyceraldehyde-3-phosphate dehydrogenase polypeptide (GAPDH) and the levels of phosphorylated PLB (pPLB) and non-phosphorylated PLB (PLB) in cells exposed (+) or not exposed (-) to isoproterenol (ISO). Figure 3 The right panel of the Figure 2D Graph of the ratio of pPLB to PLB in hearts treated with mCherry ("ISO" sample), AIP, AIPx5, AIP-FKBP12.6, CN19o, CN19oX3, or CN19o-FKBP12.6 as described.

[0111] Figure 4 Provides a schematic illustration of the design of an in vitro experiment to evaluate Figure 2D Described and shown in Figure 4 The purified peptides on the left side have an effect on the activity of CaMKIIδ (an isoform of CaMKII). Table 1 provides the peptide sequences used in the in vitro experiments. Bioluminescence and homogeneous ADP kinase monitoring assays (ADP-GLO TM ) measures the activity of CaMKIIδ, which is described in Zegzouti et al., "ADP-Glo: A Bioluminescent and Homogeneous ADP Monitoring Assay for Kinases," ASSAY and Drug Development Technologies, December 2009, 560-572, doi.org / 10.1089 / adt.2009.0222, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0112] Figure 5 Provided evidence showing that AIP multimerization increases the potency of CaMKII inhibition (i.e., EC 50 Figure 3 shows in vitro bioluminescence and homogenous ADP monitoring assays of CaMKII inhibition in vitro. CN19o multimerization resulted in a decrease in CaMKII inhibition (i.e., EC 50 Table 1 provides the sequences of the peptides.

[0113] Figure 6 Provide display to RYR2 R176Q / WT and RYR2 R4560I / WT Schematic diagram of AAV vector administration in mice. R176Q / WT and RYR2 R4560I / WT Mouse. Figure 6 The upper portion of FIG. 1 provides a schematic diagram showing the polynucleotide components of an AAV vector. Figure 6 In the expression, ITR stands for “inverted terminal repeat”, CASQ2 stands for calcineurin 2 enhancer, cTnT stands for “cardiac troponin” promoter, INT stands for hemoglobin enhancer, AIPx5 stands for a polymer containing five autocamphin-2-related inhibitory peptide (AIP) units, mScarlet stands for the fluorescent polypeptide mScarlet, nls stands for nuclear localization signal, “stop” stands for stop codon, and WPRE stands for woodchuck hepatitis virus (WHV) posttranscriptional regulatory element.

[0114] Fig. 7A and 7BProvides a graph showing the effect of AIPx5 administration on atrial fibrillation in a mouse model. LKB Flox / Flo mice were dually injected with AAV-NPPA-Cre and AAV-AIPx5-mcherry (see Figure 6 ). Fig. 7A Results are presented for experiments in which mice expressing LKB1 positivity and flanked by LoxP sites (LKB1flox / flox) were injected with AAV9-NPPA-RFP, AAV9-NPPA-Cre, or dual injections of AAV9-NPPA-Cre+AAV9-cTnT-AIPx5 at postnatal day 3 (P3). Electrocardiographic recordings were performed under anesthesia for 3 minutes at two-week intervals. The time difference between two QRS complexes (RR interval) was plotted as a function of the subsequent interval (RR+1). Figure 7B Graphs showing the standard deviation of the RR intervals recorded for each animal for 1 min at the indicated time points are provided.

[0115] Figure 8 A schematic diagram of the treatment and testing regimen of Example 5 disclosed herein is provided.

[0116] Fig. 9 Graphs and icons are provided illustrating the beat-to-beat variability of control mice compared to LKB1 knockout mice. (A) shows that the beat-to-beat variability of control mice (conditional knockout (floxed) LKB1 mice without Cre) is very small. (B) shows that the beat-to-beat variability of LKB1 knockout mice (conditional knockout LKB1 mice in the presence of Cre) is significantly increased compared to control mice.

[0117] Fig.10 Provided are graphs showing that AIPx5 is associated with reduced beat-to-beat variability in LKB1 knockout mice.

[0118] Fig.11 Provided are graphs showing that AIPx5 is associated with reduced beat-to-beat variability in Tbx5 knockout mice. DETAILED DESCRIPTION

[0119] The invention disclosed herein is characterized in that Ca 2+ - Multimeric polypeptides of calmodulin-dependent kinase II (CaMKII) (e.g., AIP multimeric polypeptides), polynucleotides encoding CaMKII inhibitory multimeric polypeptides, and methods of using the same to treat cardiac diseases or disorders (e.g., catecholamine-sensitive polymorphic ventricular tachycardia (CPVT) or atrial fibrillation (AF)).

[0120] As described in detail below, the invention of the present disclosure is based at least in part on the discovery that AIP multimeric polypeptides show increased CaMKII inhibition and increased arrhythmia inhibition relative to non-multimeric AIP. Inhibition of CaMKII activation and subsequent downstream signaling can significantly reduce potential arrhythmias stimulated by catecholamines associated with mutations in the calcium reynolds channel RYR2. Therefore, the invention of the present disclosure provides compositions containing AIP multimers or polynucleotides encoding them and methods for treating cardiac diseases and disorders (e.g., CPVT or AF). In an embodiment, an AIP multimeric polypeptide is delivered to a subject using an expression vector (e.g., an AAV vector).

[0121] Catecholaminergic polymorphic ventricular tachycardia (CPVT)

[0122] Catecholamine-sensitive polymorphic ventricular tachycardia (CPVT) is an inherited cardiac arrhythmia caused primarily by autosomal dominant mutations in the gene encoding the cardiac ryanodine receptor (RYR2), the major intracellular calcium release channel in cardiomyocytes. Typically, CPVT patients are asymptomatic at rest but develop potentially lethal ventricular tachycardia during exercise or emotional distress. In wild-type cardiomyocytes, when the cardiac action potential opens the voltage-sensitive L-type Ca2+ channel located in the plasma membrane, the ventricular tachycardia is activated. 2+ The Ca generated 2+ Local influx triggers sarcoplasmic reticulum Ca release via RyR2 2+ The cytoplasmic Ca generated 2+ When the cell enters diastole, RyR2 is closed and the cytoplasmic Ca 2+ Sarcoplasmic reticulum Ca 2+ -ATPase pumps back into the sarcoplasmic reticulum. In cells carrying mutations associated with CPVT, RYR2 releases more Ca 2+ to the cytoplasm, leading to diastolic Ca 2+ The increase in RYR2 drives the exchange of sodium and calcium across the plasma membrane via the sodium-calcium exchanger (NCX1), resulting in afterdepolarizations that may trigger additional action potentials. The molecular mechanisms by which catecholamine stimulation reveals the arrhythmic nature of CPVT mutations are unknown. The mechanism by which RYR2 mutations produce the clinical phenotype of ventricular tachycardia is also uncertain.

[0123] CPVT mutations impair diastolic Ca release from the sarcoplasmic reticulum into the cytoplasm via RYR2 2+ In a single myocardial cell, diastolic Ca 2+ Elevated Ca induces reverse sodium-calcium exchange via NCX1 on the plasma membrane, leading to afterdepolarizations that may trigger additional action potentials. The molecular mechanisms by which catechol stimulation reveals the arrhythmic nature of CPVT mutations are still unclear, although catechol-induced Ca 2+- Activation of calmodulin-dependent protein kinase II (CaMKII) has been implicated. The mechanism by which RYR2 mutations produce the clinical phenotype of ventricular tachycardia is also uncertain, although one theory is that cardiomyocyte-triggered activity produces ventricular tachycardia.

[0124] CPVT is associated with recurrent atrial or ventricular arrhythmias during exercise or emotional distress. The average age of onset is about 10 years, with 30% of patients presenting with cardiac arrest. About 60% to about 70% of patients with CPVT have a RYR2 mutation. If atrial or ventricular arrhythmias recur, CPVT is usually treated with medications, left cardiac sympathectomy (LCSD), or an implantable cardioverter-defibrillator (ICD).

[0125] The advent of induced pluripotent stem cell (iPSC) technology and efficient methods for differentiating iPSCs into cardiomyocytes (iPSC-CMs) have created exciting opportunities for studying inherited cardiac arrhythmias. iPSC-CMs have been generated from patients with CPVT and other inherited arrhythmias and have been shown to capture key features of these diseases, including abnormal action potential duration and drug response.

[0126] Atrial Fibrillation (AF)

[0127] Atrial fibrillation (AF or A-fib) is an abnormal heart rhythm (arrhythmia) characterized by rapid, irregular beatings of the heart's atria. It usually starts with brief abnormal beats that become longer or more persistent over time. It may also start with other forms of arrhythmia (such as atrial flutter) and then turn into AF. Episodes may be asymptomatic. Symptomatic episodes may include palpitations, fainting, dizziness, shortness of breath, or chest pain. Atrial fibrillation is associated with an increased risk of heart failure, dementia, and stroke. It is a type of supraventricular tachycardia.

[0128] Atrial fibrillation is the most common serious heart rhythm disorder and affects more than 33 million people worldwide as of 2020. As of 2014, it affects approximately 2% to 3% of the population in Europe and North America. This has increased from the 0.4% to 1% of the population that was affected around 2005. In developing countries, approximately 0.6% of men and 0.4% of women are affected. The proportion of people with AF increases with age, with 0.1% of people under 50 years old, 4% between 60 and 70 years old, and 14% over 80 years old. Atrial fibrillation and atrial flutter caused 193,300 deaths in 2015, up from 29,000 in 1990.

[0129] Atrial fibrillation can be diagnosed using an electrocardiogram, blood tests, a Holter monitor, an event recorder, an echocardiogram, a stress test, a chest X-ray, a combination thereof, etc. Typically, treatment of AF includes medications (e.g., beta blockers, calcium channel blockers, digoxin, antiarrhythmic drugs, and / or blood thinners), cardioversion therapy (e.g., electrical or pharmacological cardioversion), or surgery or catheter procedures (e.g., atrioventricular (AV) node ablation or a maze procedure).

[0130] Multimeric Peptides

[0131] CaMKII inhibitory multimeric polypeptides (eg, multimeric polypeptides comprising AIP repeat sequences) are those multimeric polypeptides that significantly reduce CaMKII activity, such as CaMKII kinase activity (eg, phosphorylation of RYR2 by CaMKII).

[0132] In embodiments, the multimeric polypeptides of the invention contain about or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 repeats of a sequence containing, for example, about or at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids of the sequence YKKALHRQEAVDAL (AIP). In embodiments, the repeats are continuous and adjacent to each other. In some cases, one or more repeats are separated by a linker, wherein the linker can be a stretch of amino acid residues of about, at least about and / or no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues in length.

[0133] In embodiments, the CaMKII inhibition multimeric polypeptide (e.g., a multimeric polypeptide comprising AIP repeats) reduces CaMKII activity by at least about 10%, 25%, 50%, 75%, or 100%. In some embodiments, the CaMKII inhibition multimeric polypeptide (e.g., a multimeric polypeptide comprising AIP repeats) makes CaMKII activity undetectable.

[0134] In embodiments, a CaMKII-inhibiting multimeric polypeptide (e.g., a multimeric polypeptide comprising AIP repeats) has an EC for inhibiting CaMKII that is lower than that of a non-multimeric CaMKII-inhibiting polypeptide. 50 In embodiments, the CaMKII-inhibiting multimeric polypeptide (e.g., a multimeric polypeptide comprising AIP repeats) has an EC for inhibiting CaMKII that is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold lower than a reference peptide. 50In some cases, a CaMKII inhibitory multimeric polypeptide (e.g., a multimeric polypeptide comprising AIP repeats) has an EC for inhibiting CaMKII that is less than 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 1%, or 0.1% of a non-multimeric CaMKII inhibitory peptide (e.g., AIP). 50 In some cases, bioluminescence and homogeneous ADP kinase monitoring assays (ADP-GLO TM )Measurement of EC 50 , which is described in Zegzouti et al., "ADP-Glo: A Bioluminescent and Homogeneous ADP Monitoring Assay for Kinases," ASSAY and Drug Development Technologies, December 2009, 560-572, doi.org / 10.1089 / adt.2009.0222, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0135] In an embodiment, a CaMKII inhibitory multimeric polypeptide (e.g., a multimeric polypeptide comprising AIP) is modified in a manner that enhances or does not inhibit its ability to regulate cardiac rhythm. In one embodiment, the present invention provides a method for optimizing an amino acid sequence or a nucleic acid sequence by generating changes. Such changes may include certain mutations, deletions, insertions, post-translational modifications, and tandem replication. In a preferred embodiment, the amino acid sequence of a CaMKII inhibitory multimeric polypeptide (e.g., a multimeric polypeptide comprising AIP repeats) is modified to enhance protease resistance, particularly metalloprotease resistance. Therefore, the present invention also includes analogs of any naturally occurring polypeptide of the present invention. Analogs may differ from naturally occurring polypeptides of the present invention due to amino acid sequence differences, post-translational modifications, or both. The analogs of the present invention will generally exhibit at least 85%, more preferably 90%, and most preferably 95% or even 99% identity to all or part of the naturally occurring amino acid sequence of the present invention. The length of the sequence comparison is at least 10, 13, or 15 amino acid residues. Again, in an exemplary method for determining the degree of identity, the BLAST program can be used, with a probability score in the range of e -3 and e -100between denotes closely related sequences. Modifications include in vivo and in vitro chemical derivatization of polypeptides, such as acetylation, carboxylation, phosphorylation or glycosylation; such modifications may occur during polypeptide synthesis or processing or after treatment with a separate modifying enzyme. Analogs may also differ from naturally occurring polypeptides of the invention by changes in primary sequence. These include natural and induced genetic variation (e.g., random mutagenesis by radiation or exposure to ethane methylsulfate or by site-specific mutagenesis, as described in Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual (2nd ed.), CSH Press, 1989 or as described in Ausubel et al. above). Also included are cyclized peptides, molecules and analogs containing residues other than L-amino acids, such as D-amino acids or non-naturally occurring or synthetic amino acids (e.g., β or γ amino acids).

[0136] In addition to full-length polypeptides, the present invention also includes fragments of any polypeptide of the present invention. As used herein, "fragment" refers to a length of at least 5, 6, 7, 8, 9, 10, 11, 12 or 13 amino acids. Fragments of the present invention may be generated by methods known to those skilled in the art or may be caused by normal protein processing (e.g., removing amino acids not required for biological activity from nascent polypeptides or removing amino acids by alternative mRNA splicing or alternative protein processing events).

[0137] The physiological activity of analogs of CaMKII inhibitory multimeric polypeptides (e.g., multimeric polypeptides comprising AIP) may exceed that of natural CaMKII inhibitory polypeptides (e.g., non-multimeric AIP). Analog design methods are well known in the art, and the synthesis of analogs can be performed according to such methods by modifying the chemical structure so that the resulting analogs exhibit the activity of unmodified CaMKII inhibitory multimeric polypeptides. These chemical modifications include, but are not limited to, replacing the R groups that replace and changing the saturation of specific carbon atoms of the CaMKII inhibitory multimeric polypeptide. Preferably, the CaMKII inhibitory multimeric polypeptide analogs are relatively resistant to in vivo degradation and produce a more lasting therapeutic effect after administration. Assays for measuring functional activity include, but are not limited to, those described in the following examples.

[0138] Polynucleotide therapy

[0139] Polynucleotide therapy characterized by polynucleotides encoding CaMKII inhibitory multimeric polypeptides (e.g., multimers of AIP), analogs, variants, or fragments thereof is another therapeutic method for treating arrhythmias (e.g., CPVT or AF). The expression of such proteins in cardiac cells is expected to regulate the function of cardiac cells, tissues, or organs, for example, by inhibiting the phosphorylation of RYR2, inhibiting CaMKII activity, and / or regulating heart rhythm in other ways. Such nucleic acid molecules can be delivered to cells (e.g., cardiac cells) of subjects suffering from arrhythmias. Nucleic acid molecules are typically delivered to the cells of the subject in a form in which they can be ingested, thereby producing therapeutically effective levels of CaMKII inhibitory multimeric polypeptides (e.g., AIPx3, AIPx5) or fragments thereof.

[0140] Transduction virus (e.g., retrovirus, adenovirus, and adeno-associated virus (AAV)) vectors can be used for somatic gene therapy, particularly because of their high infection efficiency and stable integration and expression (see, e.g., Cayouette et al., Human Gene Therapy 8:423-430, 1997; Kido et al., Current Eye Research 15:833-844, 1996; Bloomer et al., Journal of Virology 71:6641-6649, 1997; Naldini et al., Science 272:263-267, 1996; and Miyoshi et al., Proc. Natl. Acad. Sci. USA 94:10319, 1997). For example, a polynucleotide encoding a CaMKII inhibitory multimeric polypeptide (e.g., an AIPx5 multimer, variant, or fragment thereof) can be cloned into a viral vector, and expression can be driven by a promoter (e.g., a promoter specific for the target cell type of interest). In one embodiment, a viral vector (eg, an AAV vector) is used to administer a polynucleotide encoding an AIP multimeric polypeptide (eg, AIPx3 or AIPx5) to cardiac tissue.

[0141] Transduction viral vectors have tissue tropism, allowing selective transduction of one cell type rather than another. For example, although CaMKII inhibition in cardiomyocytes can treat CPVT, AF or other forms of heart disease, its inhibition in other tissues (such as the brain) may not be ideal. In some embodiments, vectors targeting cardiomyocytes with high specificity compared to other cell types are used. This will allow the CaMKII inhibitor peptide molecules to express specific cardiac targeting. This is because CaMKII inhibition in other non-cardiac cells may be harmful. Potential adeno-associated virus candidates are AAV9, AAV6, AAV2i8, AAVrh74, AAVrh10, MyoAAV, Anc80 and Anc82. When the genome is "self-complementary", the transduction efficiency of the adeno-associated virus is enhanced. In some embodiments, self-complementary adeno-associated viruses are used to increase cardiac transduction of gene therapy vectors.

[0142] Other viral vectors that can be used include, for example, vaccinia virus, bovine papilloma virus, or herpes virus (such as Epstein-Barr virus) (see also, for example, the vectors of Miller, Human Gene Therapy 15-14, 1990; Friedman, Science 244:1275-1281, 1989; Eglitis et al., BioTechniques 6:608-614, 1988; Tolstoshev et al., Current Opinion in Biotechnology 1:55-61, 1990; Sharp, The Lancet 337:1277-1278, 1991; Cornetta et al., Nucleic Acid Research and Molecular Biology 36:311-322, 1987; Anderson, Science 226:401-409, 1984; Moen, Blood Cells 17:407-416, 1991; Miller et al., Biotechnology 7:980-990, 1989; Le Gal La Salle et al., Science 259:988-990, 1993; and Johnson, Chest 107:77S-83S, 1995). Retroviral vectors have been particularly well developed and used in clinical settings (Rosenberg et al., N. Engl. J. Med 323:370, 1990; Anderson et al., U.S. Pat. No. 5,399,346).

[0143] Non-viral approaches can also be used to introduce therapeutic agents into cardiac cells in patients in need of CaMKII inhibition. For example, nucleic acid molecules can be introduced into cells by administering the nucleic acid in the presence of lipofection (Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413, 1987; Ono et al., Neuroscience Letters 17:259, 1990; Brigham et al., Am. J. Med. Sci. 298:278, 1989; Staubinger et al., Methods in Enzymology 101:512, 1983), asialomucoid-polylysine conjugation (Wu et al., Journal of Biological Chemistry 263:14621, 1988; Wu et al., Journal of Biological Chemistry 264:16985, 1989), or by microinjection under surgical conditions (Wolff et al., Science 247:1465, 1990). Preferably, the nucleic acid is administered in combination with liposomes and protamine.

[0144] Gene transfer can also be achieved using non-viral methods involving in vitro transfection. Such methods include the use of calcium phosphate, DEAE dextran, electroporation and protoplast fusion. Liposomes or lipid nanoparticles may also be beneficial for DNA delivery to cells. Transplanting normal genes into the affected tissues of patients can also be accomplished by transferring normal nucleic acids to ex vivo culturable cell types (e.g., autologous or allogeneic primary cells or their progeny), and then injecting cells (or their progeny) into the target tissue.

[0145] The dosage of the administered vector disclosed herein depends on many factors, including the size and health of the individual patient. For any particular subject, the specific dosage regimen should be adjusted over time according to individual needs and the professional judgment of the person administering or supervising the administration of the composition. In some embodiments, the dosage is between about 1x10 10 Carrier units / kg to about 1x10 14 In some embodiments, the dose is about 1x10 10 Carrier units / kg, about 5x10 10 Carrier unit / kg, about 1x10 11 Carrier units / kg, about 5x10 11 Carrier unit / kg, about 1x10 12 Carrier units / kg, about 5x10 12 Carrier unit / kg, about 1x10 13 Carrier units / kg kg, about 5x10 13Carrier units / kg, or about 1x10 14 In an exemplary embodiment, the vector is a viral vector and the dose is between about 1 x 10 10 Viral genome / kg is about 1x10 14 In some embodiments, the dosage is based on the transfection, transformation or transduction efficiency. In some embodiments, the dosage is effective to transfect, transform or transduce at least about 20% of the cardiomyocytes in the subject, at least about 25% of the cardiomyocytes in the subject, at least about 30% of the cardiomyocytes in the subject, at least about 35% of the cardiomyocytes in the subject, or at least about 40% of the cardiomyocytes in the subject. In some embodiments, the dosage is effective to transfect, transform or transduce about 20% to about 40% of the myocytes in the subject.

[0146] Transcription of mRNA from a polynucleotide of the invention (e.g., a polynucleotide encoding a CaMKII inhibitory multimeric polypeptide (e.g., a multimer of AIP)) can be directed by any suitable promoter (e.g., human cytomegalovirus (CMV), simian virus 40 (SV40), CMV-chicken b-actin hybrid promoter ("CAG"), or metallothionein promoter) and regulated by any suitable mammalian regulatory element. For the treatment of CPVT or AF, it is ideal to selectively express CaMKII inhibitory multimeric polypeptides (e.g., multimers of AIP) in cardiomyocytes and minimize expression in other cell types. In some embodiments, a cardiomyocyte-selective promoter is used to express a CaMKII inhibitory multimeric polypeptide (e.g., a multimer of AIP). The promoter or enhancer used may include, but is not limited to, those characterized as tissue or cell specific enhancers. For example, the cardiac troponin T promoter, the α-myosin heavy chain (α-MHC) promoter, the myosin light chain-2v (MLC-2v) promoter, or the cardiac NCX1 promoter can be used for direct expression in cardiomyocytes. Alternatively, if a genomic clone is used as a therapeutic construct, regulation may be mediated by homologous regulatory sequences or, if desired, by regulatory sequences from a heterologous source, including any of the promoters or regulatory elements described above.

[0147] Another treatment method included in the invention of the present disclosure involves administering a recombinant CaMKII inhibitory multimeric polypeptide (e.g., a multimer of AIP), such as a recombinant AIP multimeric polypeptide (e.g., AIPx3 or AIPx5), a variant or fragment thereof directly to the site of a tissue potentially or actually affected by the disease or systemically (e.g., by any conventional recombinant protein administration technique). The dose of the peptide administered depends on many factors, including the size and health of the individual patient. For any particular subject, the specific dosage regimen should be adjusted over time according to individual needs and the professional judgment of the person administering or supervising the administration of the composition.

[0148] Stabilize mRNA

[0149] Stabilized mRNA encoding a CaMKII inhibitory multimeric polypeptide (e.g., by increasing or improving half-life, increasing or improving potency, increasing or improving resistance to endonuclease activity, reducing or reducing sensitivity to endonuclease activity to stabilize mRNA) can be used in some embodiments to prevent or improve CPVT, AF or other arrhythmias. Such stabilized mRNA can be delivered to cells (e.g., cardiac cells) of a subject suffering from an arrhythmia (e.g., CPVT or AF).

[0150] The expression of the CaMKII inhibitory multimeric polypeptide encoded by the stabilized mRNA described herein in cardiac cells is expected to regulate the function of cardiac cells, tissues or organs, for example, by inhibiting the phosphorylation of RYR2, inhibiting CaMKII activity and / or regulating heart rhythm in other ways. In some embodiments, as disclosed herein, the stabilized mRNA encodes a multimeric AIP polypeptide.

[0151] Treatment

[0152] CaMKII inhibitory multimeric polypeptides (e.g., multimers of AIP) and polynucleotides or vectors encoding them (e.g., AAV vectors) can be used to prevent or improve CPVT, AF or other arrhythmias. The methods and compositions of the present invention can be used to treat diseases and disorders characterized by arrhythmias (e.g., atrial fibrillation (AF), catecholamine-sensitive polymorphic ventricular tachycardia (CPVT), ischemic heart disease, arrhythmias, heart failure (e.g., heart failure associated with aortic entrapment), hypertensive heart disease and pulmonary hypertensive heart disease, myocardial infarction, valvular disease, congenital heart disease, myocardial hypertrophy, ventricular arrhythmias or Timothy syndrome).

[0153] In one treatment method, the agents identified herein are administered to the site of tissue potentially or actually affected by the disease or systemically. The dose of the agent administered depends on many factors, including the size and health of the individual patient. For any particular subject, the specific dosage regimen should be adjusted over time according to individual needs and the professional judgment of the person administering or supervising the administration of the composition.

[0154] Delivery or transfer of polypeptides, polynucleotides or vectors of the present invention can also be accomplished by using muscle targeting agents (e.g., agents that specifically target the myocardium). Exemplary muscle targeting agents, preferably myocardial targeting agents, can be found, for example, in U.S. Pat. No. 11,168,141, filed on August 2, 2019, the contents of which are incorporated herein by reference in their entirety. In some embodiments, muscle targeting agents (e.g., agents such as antibodies that target cardiac markers (e.g., caveolin-3)) can be bound to the polypeptides, polynucleotides or vectors disclosed herein so as to target such polypeptides, polynucleotides or vectors to targets in the myocardium.

[0155] Pharmaceutical composition

[0156] The present disclosure provides pharmaceutical compositions containing CaMKII inhibitory multimeric polypeptides (e.g., multimers of AIP) or polynucleotides and / or vectors encoding them. The administration of the pharmaceutical compositions disclosed herein for treating arrhythmias may be any suitable method for producing concentrations that effectively improve, reduce, or stabilize arrhythmias by inhibiting multimeric polypeptides (e.g., multimers of AIP) of CaMKII. The pharmaceutical compositions of the present invention may be used to treat diseases and disorders characterized by arrhythmias (e.g., atrial fibrillation (AF), catecholamine-sensitive polymorphic ventricular tachycardia (CPVT), ischemic heart disease, arrhythmias, heart failure (e.g., heart failure associated with aortic entrapment), hypertensive heart disease and pulmonary hypertensive heart disease, myocardial infarction, valvular disease, congenital heart disease, myocardial hypertrophy, ventricular arrhythmias, or Timothy syndrome). The composition may be contained in any suitable carrier substance in any suitable amount. The composition may be provided in a dosage form suitable for parenteral (e.g., subcutaneous, intravenous, intramuscular, or intraperitoneal) administration routes. Pharmaceutical compositions can be formulated according to conventional pharmaceutical practice (see, for example, Remington: The Science and Practice of Pharmacy (20th ed.), AR Gennaro, ed., Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, J. Swarbrick and JC Boylan, eds., 1988-1999, Marcel Dekker, New York).

[0157] For therapeutic use, the polypeptides, polynucleotides and / or vectors disclosed herein can be systemically administered, for example, formulated in a pharmaceutically acceptable buffer (such as saline). Preferred routes of administration include, for example, subcutaneous, intravenous, intraperitoneal, intramuscular or intradermal injections that can provide continuous, sustained levels of drugs in the patient. For AAV gene therapy, administration can be local or systemic, such as intravenous or intracoronary administration. The therapeutically effective amount of the therapeutic agent identified herein in a physiologically acceptable carrier will be used for the treatment of human patients or other animals. Suitable carriers and their formulations are described in, for example, Remington's Pharmaceutical Sciences by EWMartin. The amount of therapeutic agent administered depends on the mode of administration, the age and weight of the patient, and the clinical symptoms of arrhythmia. Typically, the dosage will be within the dosage range of other agents used to treat other diseases that require regulation of cardiac function, although in some cases the required amount is lower due to the increased specificity of the compound. Compositions comprising CaMKII inhibitory multimeric polypeptides (e.g., multimers of AIP) are administered at a dose that has CaMKII inhibitory activity or cardiac rhythm regulating activity as determined by methods known to those skilled in the art, or using any assay that measures the expression or biological activity of a CaMKII polypeptide.

[0158] The pharmaceutical composition of the present invention can be formulated to release the active compound substantially immediately after administration or at any predetermined time or time period after administration. The latter type of composition is generally referred to as a controlled release formulation, which includes (i) a formulation that produces a substantially constant drug concentration in the body over a long period of time; (ii) a formulation that produces a substantially constant drug concentration in the body over a long period of time after a predetermined lag time; (iii) a formulation that maintains the effect over a predetermined period of time by maintaining a relatively, constant, effective level in the body while minimizing undesirable side effects associated with fluctuations in the plasma level of the active substance (sawtooth kinetic pattern); (iv) a formulation that acts locally by, for example, placing the controlled release composition in a space near or in contact with the thymus; (v) a formulation that allows convenient administration, such as once a week or two weeks; and (vi) a formulation that targets arrhythmias by delivering the therapeutic agent to a specific cell type (e.g., cardiac cells) using a carrier or chemical derivative. For some applications, the controlled release formulation does not need to be frequently administered during the day to maintain the plasma concentration at a therapeutic level.

[0159] Any of a variety of strategies can be implemented to achieve controlled release, wherein the release rate exceeds the metabolic rate of the compound in question. In one embodiment, controlled release is achieved by appropriate selection of various formulation parameters and ingredients, including, for example, various types of controlled release compositions and coatings. Thus, the therapeutic agent is formulated with a suitable excipient as a pharmaceutical composition that releases the therapeutic agent in a controlled manner after administration. Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, molecular complexes, nanoparticles, patches, and liposomes.

[0160] Parenteral compositions

[0161] A pharmaceutical composition comprising a CaMKII inhibitory multimeric polypeptide (e.g., a multimer of AIP) or a polynucleotide or vector encoding the same (e.g., an AAV vector containing a polynucleotide encoding a multimeric polypeptide) of the present disclosure can be administered parenterally by injection, infusion, or implantation (subcutaneous, intravenous, intramuscular, intraperitoneal, intracoronary, etc.) in a dosage form, formulation, or by a suitable delivery device or implant containing conventional, non-toxic, pharmaceutically acceptable carriers and adjuvants. In an embodiment, the pharmaceutical composition contains a viral vector (e.g., an AAV vector) containing a polynucleotide encoding a CaMKII inhibitory multimeric polypeptide. The formulation and preparation of such compositions are well known to those skilled in the art of pharmaceutical preparations. The formulation can be found in Remington: The Science and Practice of Pharmacy, as above.

[0162] Compositions for parenteral use may be provided in unit dosage form (e.g., in single-dose ampoules) or in vials containing multiple doses, and suitable preservatives may be added thereto (see below). The composition may be in the form of a solution, suspension, emulsion, infusion device, or implantable delivery device, or it may be in the form of a dry powder, which is dissolved back in water or other suitable vehicles before use. In addition to the active agent that reduces or improves arrhythmias, the composition may include a suitable parenterally acceptable carrier and / or excipient. The active therapeutic agent may be incorporated into microspheres, microcapsules, nanoparticles, liposomes, etc. for controlled release. Furthermore, the composition may include a suspending agent, a solubilizing agent, a stabilizer, a pH adjuster, a tension adjuster, and / or a dispersant.

[0163] As mentioned above, the pharmaceutical composition according to the present invention can be in a form suitable for aseptic injection. In order to prepare this composition, the appropriate therapeutic agent is dissolved or suspended in a liquid vehicle acceptable to the parenteral. Acceptable vehicles and solvents that can be used are water, water or suitable buffer adjusted to an appropriate pH value by adding an appropriate amount of hydrochloric acid, sodium hydroxide, 1,3-butanediol, Ringer's solution and isotonic sodium chloride solution and glucose solution. Aqueous preparations may also contain one or more preservatives (e.g., methyl, ethyl or n-propyl paraben). Where one of the compounds is only slightly soluble in water or slightly soluble in water, a dissolution enhancer or solubilizer may be added, or the solvent may include 10 to 60 weight / weight % of propylene glycol, etc.

[0164] Controlled release parenteral compositions

[0165] Controlled release parenteral compositions can be in the form of aqueous suspensions, microspheres, microcapsules, magnetic microspheres, oil solutions, oil suspensions or emulsions. Alternatively, the active drug can be incorporated into biocompatible carriers, liposomes, nanoparticles, implants or infusion devices.

[0166] Materials used to prepare microspheres and / or microcapsules are, for example, biodegradable / bioerodible polymers such as polygalactose, polyisobutylcyanoacrylate, poly-2-hydroxyethyl-L-glutamine, and polylactic acid. Biocompatible carriers that can be used when formulating controlled-release parenteral formulations are carbohydrates (e.g., dextran), proteins (e.g., albumin), lipoproteins, or antibodies. Materials used for implants can be non-biodegradable (e.g., polydimethylsiloxane) or biodegradable (e.g., polycaprolactone, polylactic acid, polyglycolic acid, or poly(orthoesters) or combinations thereof).

[0167] Oral solid dosage forms

[0168] Oral formulations include tablets containing the active ingredient mixed with non-toxic pharmaceutically acceptable excipients. Such formulations are known to those skilled in the art. Excipients may be, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starch (including potato starch), calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate or sodium phosphate); granulating agents and disintegrants (e.g., cellulose derivatives (including microcrystalline cellulose), starch (including potato starch), cross-linked carboxymethyl cellulose sodium, alginates or alginic acid); binders (e.g., sucrose, glucose, sorbitol, gum arabic, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, sodium carboxymethyl cellulose, methylcellulose, hydroxypropyl methylcellulose, ethylcellulose, polyvinyl pyrrolidone or polyethylene glycol); and lubricants, glidants and anti-adherents (e.g., magnesium stearate, zinc stearate, stearic acid, silicon dioxide, hydrogenated vegetable oil or talc). Other pharmaceutically acceptable excipients may be colorants, flavoring agents, plasticizers, humectants, buffers, and the like.

[0169] Tablets may be uncoated or may be coated by known techniques, optionally postponing disintegration and absorption in the gastrointestinal tract, thereby providing a sustained action over a longer period of time. The coating may be suitable for releasing the active drug in a predetermined pattern (e.g., to achieve a controlled release formulation) or it may be suitable for releasing the active drug only after passing through the stomach (enteric coating). The coating may be a sugar coating, a film coating (e.g., based on hydroxypropyl methylcellulose, methylcellulose, methylhydroxyethylcellulose, hydroxypropyl cellulose, carboxymethylcellulose, acrylate copolymers, polyethylene glycol and / or polyvinyl pyrrolidone) or an enteric coating (e.g., based on methacrylic acid copolymers, cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, polyvinyl acetate phthalate, shellac and / or ethylcellulose). Furthermore, a time-delay material may be used, such as glyceryl monostearate or glyceryl distearate.

[0170] Solid tablet compositions may include coatings suitable for protecting the composition from unwanted chemical changes (e.g. chemical degradation prior to release of the active cardioactive therapeutic substance). Coatings may be applied to solid dosage forms in a manner similar to that described in Encyclopedia of Pharmaceutical Technology above.

[0171] In one embodiment, two or more cardiac therapeutic agents can be mixed together in the tablet or can be separated. In one example, the first active cardiac therapeutic agent is contained inside the tablet and the second active therapeutic agent is outside, so that most of the second therapeutic agent is released before the first active therapeutic agent is released.

[0172] Oral formulations may also be presented as chewable tablets, or as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent (e.g. potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate or kaolin), or as soft gelatin capsules in which the active ingredient is mixed with water or an oily medium (e.g. peanut oil, liquid paraffin or olive oil). Powders and granules may be prepared in a conventional manner using the ingredients mentioned for the above tablets and capsules, for example using a mixer, a fluidized bed apparatus or a spray drying device.

[0173] Controlled release oral dosage form

[0174] The controlled release composition for oral use can be constructed to release the AIP polymer or CaM-KNtide polymer, carrier and / or polynucleotide of the present disclosure by controlling its dissolution and / or diffusion. Dissolution or diffusion controlled release can be achieved by appropriately coating the tablet, capsule, pill or granule formulation of the compound, or by incorporating the compound into an appropriate matrix. The controlled release coating may include one or more of the above-mentioned coating materials and / or such as shellac, beeswax, sugar wax (glycowax), castor wax, carnauba wax, stearyl alcohol, glyceryl monostearate, glyceryl distearate, palmitic acid stearate, ethyl cellulose, acrylic resin, dl-polylactic acid, cellulose acetate butyrate, polyvinyl chloride, polyvinyl acetate, vinyl pyrrolidone, polyethylene, polymethacrylate, methyl methacrylate, 2-hydroxy methacrylate, methacrylate hydrogel, 1,3-butylene glycol, ethylene glycol methacrylate and / or polyethylene glycol. In controlled release matrix formulations, matrix materials may also include, for example, hydrated methylcellulose, carnauba wax and stearyl alcohol, carbomer 934, silicone, tristearin, methyl acrylate-methyl methacrylate, polyvinyl chloride, polyethylene and / or halogenated fluorocarbons.

[0175] Controlled release compositions containing one or more therapeutic compounds may also be in the form of buoyant tablets or capsules (i.e., tablets or capsules that float above the gastric contents for a period of time after oral administration). Buoyant tablet formulations of the compounds may be prepared by granulating a mixture of the compound with an excipient and 20 to 75 weight / weight % of a hydrocolloid (e.g., hydroxyethylcellulose, hydroxypropylcellulose, or hydroxypropylmethylcellulose). The resulting granules may then be compressed into tablets. Upon contact with gastric fluid, the tablet forms a substantially impermeable gel barrier around its surface. This gel barrier participates in maintaining the tablet density to less than 1, thereby keeping the tablet buoyant in gastric fluid.

[0176] Combination therapy

[0177] Optionally, the CaMKII-inhibiting multimeric polypeptides described herein (eg, multimers of AIP) can be administered in conjunction with any other standard treatment useful for modulating cardiac function; such methods are known to those of skill in the art and are described in Remington's Pharmaceutical Sciences by EW Martin.

[0178] Set

[0179] Also provided are kits for preventing or treating arrhythmias, cardiac disorders or pathologies (e.g., atrial fibrillation (AF), catecholamine-sensitive polymorphic ventricular tachycardia (CPVT), ischemic heart disease, arrhythmias, heart failure (e.g., heart failure associated with aortic tethering), hypertensive heart disease and pulmonary hypertensive heart disease, myocardial infarction, valvular disease, congenital heart disease, cardiac hypertrophy, ventricular arrhythmias, or Timothy syndrome) in a subject in need thereof. In one embodiment, the kit provides a therapeutic or preventive composition containing an effective amount of a CaMKII-inhibiting multimeric polypeptide (e.g., a multimer of AIP), a polynucleotide encoding such a polypeptide, and / or a vector comprising such a polynucleotide, wherein the kit is used to administer the multimeric polypeptide, polynucleotide, or vector to a subject.

[0180] In another embodiment, the kit provides a therapeutic or prophylactic composition comprising an effective amount of a CaMKII inhibitory multimeric polypeptide (eg, a multimer of AIP), a polynucleotide encoding the same, and / or a vector.

[0181] In some embodiments, the kit comprises a sterile container containing the therapeutic or prophylactic composition; such container can be in the form of a box, ampoule, bottle, vial, tube, bag, pouch, blister pack, or other suitable container known in the art. The container can be made of plastic, glass, laminated paper, metal foil, or other material suitable for containing drugs.

[0182] In an embodiment, the kit contains instructions for administering the composition to a subject having a disease (e.g., CPVT or AF) or at risk of developing the disease. The instructions typically include information about using the composition to treat the disease. In other embodiments, the instructions include at least one of: a description of the therapeutic agent; a dosage schedule and administration for treating or preventing a cardiac disease (e.g., CPVT or AF) or a symptom thereof; precautions; warnings; indications; contraindications; overdose information; adverse reactions; animal pharmacology; clinical studies; and / or references. The instructions may be printed directly on the container (if any), or as a label affixed to the container, as information stored on a remotely accessible server, or as a separate sheet, brochure, card, or binder placed in or provided with the container.

[0183] Unless otherwise indicated, the practice of the present invention employs conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the capabilities of those skilled in the art. Such techniques are fully explained in the literature, such as "Molecular Cloning: A Laboratory Manual", Second Edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzymology" "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction", (Mullis, 1994); "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of polynucleotides and polypeptides of the present invention, and therefore can be considered in making and practicing the present invention.Techniques that are particularly useful for specific embodiments will be discussed below.

[0184] The following examples are put forth so as to provide those skilled in the art with a complete disclosure and description of how to make and use the detection, screening and treatment methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.

[0185] Example

[0186] Example 1: Multimerization improves the potency of AIP against CaMKII inhibition in vivo, but does not improve the potency of CaMKII alternative potent inhibitors

[0187] Experiments were conducted to identify improved agents for the treatment of catecholamine-sensitive polymorphic ventricular tachycardia (CPVT). The peptide inhibitor autocamphin 2-related inhibitory peptide (AIP) is effective in treating CPVT (see Figures 1A to 1C To identify improved agents for the treatment of CPVT, multimeric peptides containing five consecutive repeats of AIP (i.e., AIPx5) were designed and evaluated for their efficacy in inhibiting the activity of the kinase CaMKII (see Figures 2A to 2G , 3, 4 and 5A to 5E).

[0188] Development of improved agents for the treatment of CPVT, A) containing multiple AIP repeats ( Figure 2C ) or the potent CaMKII inhibitor CN19o or B) containing FKBP12.6 (which binds to the CaMKII target RYR2 ( Figure 1A )) fused AIP or CN19o( Figure 2B The structural domains of the evaluated polypeptides are as follows Figure 2D As shown. CN19o is known to be a more potent CaMKII inhibitor than AIP ( Figure 2A ).like Figure 2D As shown, in RYR2 R4650I / WT The efficacy of peptides in treating catecholaminergic polymorphic ventricular tachycardia (CPVT) was evaluated in a mouse CPVT model. Among all the peptides evaluated, the reduction in ectopy ( Figure 2E and 2F ) were determined and found that the AIPx5 peptide was the most effective in treating CPVT. AIP-FKB12.6 was also effective in treating CPVT symptoms by reducing ectopic beats ( Figure 2E and 2F The polypeptides were administered to mice using AAV vectors and fluorescent imaging was used to confirm that the polynucleotide expressing the polypeptides was delivered to cardiac tissue ( Figure 2G ).

[0189] To confirm the inhibition of CaMKII activation in vivo, phosphorylation of phospholamban (PLB) at threonine-17, a known target of CaMKII, was assessed in cardiac tissue and whole heart lysates from mice exposed to isoproterenol (ISO). Figure 3 Exposure of cardiac cells to ISO induces CaMKII phosphorylation of PLB ( Figure 3 ). It was determined that exposure of cells to AIP or AIPx5 resulted in a statistically significant decrease in PLB phosphorylation in cells exposed to ISO.

[0190] Example 2: Multimerization improves the potency of AIP against CaMKII inhibition in vitro, but does not improve the potency of CaMKII alternative potent inhibitors

[0191] Experiments were performed to evaluate the in vitro inhibition of CaMKIIδ by the polypeptides evaluated in Example 1, whose amino acid sequences are provided in Table 1 below. Bioluminescence and homogeneous ADP kinase monitoring assays (ADP-GLO TM) to evaluate the inhibition of CaMKIIδ activity by polypeptides, which is described in Zegzouti et al., "ADP-Glo: A Bioluminescent and Homogeneous ADP Monitoring Assay for Kinases", ASSAY and Drug Development Technologies, December 2009, 560-572, doi.org / 10.1089 / adt.2009.0222, the disclosure of which is incorporated herein by reference in its entirety for all purposes ( Figure 4 It was found that multimerization (i.e., duplication of the AIP peptide or CN19oX5 peptide) increased the potency of AIP (i.e., decreased EC 50 ), but actually reduced the potency of CN19oX5 (i.e., increased EC 50 )( Figure 5 B to 5E). EC of AIP 50 The EC of AIPx3 multimer is 6149nM. 50 The EC of AIPx5 multimer was reduced to 543 nM. 50 It further decreased to 114 nM. However, the EC 50 The EC value of CN19o in CN19oX3 multimers was 1.6 nM. 50 The value increased to 2.1 nM. Thus, multimerization increases the potency of AIP but actually decreases the potency of CN19oX3.

[0192] Table 1. Peptide sequences

[0193]

[0194] Example 3: Administration of AIPx5 to animal models

[0195] Prepare AIPx5-expressing AAV vector or negative control AIV vector expressing only mScarlet (see Figure 6 ) and applied to RYR2 R176Q / QT and RYR2 R4560I / WT Mice (which is an animal model of catecholamine-sensitive polymorphic ventricular tachycardia (CPVT)). The efficacy of the vector in alleviating CPVT symptoms was evaluated in the animal model. Administration of the AIPx5 vector resulted in the expression of AIPx5 in mouse heart tissue and a reduction in CPVT symptoms (e.g., a reduction in ectopic beats and / or ventricular tachycardia).

[0196] Example 4: AAV9-AIPx5 inhibits arrhythmias in an animal model of atrial fibrillation (AF)

[0197] Experiments were performed to evaluate the efficacy of AIPx5 administration in treating atrial fibrillation. LKB Flox / Flox mice were dually injected with NPPA-Cre and AIPx5-mcherry using AAV vectors. The mice are a model of atrial fibrillation. In the mouse model, NPPA-Cre inactivates LKB in the atria, creating an atrial fibrillation (AF) model. It was evaluated whether co-injection with AAV-AIPx5 would reduce AF. Each RR interval (R) was plotted against the RR interval of the subsequent heartbeat (R1) (n = approximately 500 heartbeats) ( Fig. 7A ) to analyze the beat-to-beat variability of HR (an index of AF frequency). The RR interval is the time that elapses between two consecutive R waves of the QRS signal on the electrocardiogram and is the inverse of the heart rate. The beat-to-beat variability of HR was analyzed at weeks 2, 4, and 6. The RR interval showed little beat-to-beat variability at week 2, much variability at week 4, and then returned to a state of little variability at week 6 ( Fig. 7A and 7B ). Therefore, AIPx5 can effectively reduce AF symptoms.

[0198] Example 5: MyoAAV-AIPx5 inhibits arrhythmias in two different animal models of atrial fibrillation

[0199] Two different atrial fibrillation (AF) mouse models were used to test the efficacy of AIPx5 in treating AF of different origins. The two different AF mouse models were liver kinase B1 (LKB1) knockout mice and T-box transcription factor 5 (Tbx5) knockout mice.

[0200] LKB1 encodes a serine / threonine kinase that functions upstream of the AMP-activated protein kinase (AMPK) superfamily. LKB1 positively regulates AMP-activated protein kinase (AMPK) and other AMPK-related downstream kinases. LKB1 and AMPK are jointly involved in cell growth and metabolism and the cellular response to energy stress. Tbx5 is part of a gene set that specifies body regions. Tbx5 is involved in the development of the heart, forelimbs, and other structures of the body. It plays a crucial role in regulating the formation of the heart and its conduction system. Knocking out any of these genes in mice induces AF.

[0201] The efficacy of AIPx5 was tested using LKB1 conditional knockout or Tbx5 conditional knockout mice. LKB1 or Tbx5 was then conditionally knocked out from the atria of mice using AAV-NPPA-Cre. NPPA is an atrial-specific promoter, ensuring that Cre recombinase-directed knockout of LKB1 or Tbx5 is limited to the atria. AAV-NPPA-FP (fluorescent protein) was used as a control virus. AAV-NPPA-FP does not have Cre, so the target gene is not knocked out. Uninjected mice were also included as a control to account for any effects caused by the AAV injection. Either AAV-cTnT-AIPx5 or AAV-cTnT-CN19o was tested as a potential treatment for AF.

[0202] Figure 8 A schematic diagram of the treatment and testing protocols is provided. Briefly, P3 LKB1 conditional knockout or Tbx5 conditional knockout mice were injected (or not injected in uninjected controls), and then EKG measurements were taken every two weeks starting at P14. At week 12, mice were sacrificed, and the sacrificed mice were tested for protein expression.

[0203] EKG measurements of the mice were used to calculate whether the mice exhibited AF. AF was calculated using the standard deviation of the beat-to-beat time difference. High beat-to-beat variability during sedation indicates that the atria are not firing normally. High beat-to-beat variability was found to be absent in control mice, but present in LKB1 knockout mice, as shown in Figure 2. Fig. 9 shown.

[0204] Fig.10 This suggests that AIPx5 can effectively suppress beat-to-beat variability in LKB1 knockout mice, thereby suppressing symptoms of AF. When AIPx5 was present in LKB1 knockout mice, these mice did not show statistically significant differences in beat-to-beat variability compared to control (non-LKB1 knockout) mice. Surprisingly, when CN19o was provided to LKB1 knockout mice, beat-to-beat variability was not suppressed, and in fact, performed worse than LKB1 knockout mice alone in terms of beat-to-beat variability measures.

[0205] Fig.11 This suggests that AIPx5 can also effectively suppress the beat-to-beat variability of Tbx5 knockout mice, thereby suppressing the symptoms of AF. When AIPx5 was present in Tbx5 knockout mice, the beat-to-beat variability of these mice did not show statistically significant differences after the 4th week.

[0206] Fig.10 and 11 Statistical significance of the indicated tests was determined using two-way ANOVA with post hoc Dunnett's multiple comparisons compared to the control (FP) group.

[0207] Other Implementations

[0208] From the foregoing description it will be apparent that the invention described herein may be changed and modified to adapt it to various usages and conditions. Such embodiments are also within the scope of the appended claims.

[0209] The recitation of any defined variable in a list of elements herein includes the definition of that variable as any single element or combination (or subcombination) of the listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0210] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each individual patent and publication was specifically and individually indicated to be incorporated by reference.

Claims

1. A multimeric polypeptide that inhibits CaMKII.

2. The multimeric polypeptide according to claim 1, wherein The multimeric polypeptide comprises two or more AIP peptides.

3. The multimeric polypeptide according to claim 1, wherein The multimeric polypeptide comprises about 3 to about 20 AIP peptide repeats.

4. The multimeric polypeptide according to claim 3, wherein The multimeric polypeptide comprises 3, 4, 5 or 6 AIP peptide repeats.

5. The multimeric polypeptide according to claim 4, wherein The multimeric polypeptide comprises three AIP peptides.

6. The multimeric polypeptide according to claim 5, wherein The multimeric polypeptide comprises five AIP peptides.

7. The multimeric polypeptide according to any one of claims 2 to 6, wherein The AIP repeats are consecutive and / or separated by linkers.

8. The multimeric polypeptide according to claim 1, wherein The multimeric polypeptide comprises a sequence having at least 85% amino acid sequence identity to: AIPx3 YKKALHRQEAVDALYKKALHRQEAVDALYKKALHRQEAVDAL; or AIPx5 YKKALHRQEAVDALYKKALHRQEAVDALYKKALHRQEAVDALYKKALHRQEAVDAL YKKALHRQEAVDAL.

9. The multimeric polypeptide according to claim 1, wherein The multimeric polypeptide is fused to a 12.6-kDa FK506 binding protein (FKBP12.6) polypeptide.

10. The multimeric polypeptide according to claim 1, wherein Inhibition of CaMKII by the multimeric polypeptide 50 Less than the EC of AIP to inhibit CaMKII 50 10% of.

11. The multimeric polypeptide according to claim 1, wherein Inhibition of CaMKII by the multimeric polypeptide 50 Less than the EC of AIP to inhibit CaMKII 50 5%. 12 . An expression vector comprising a polynucleotide encoding the multimeric polypeptide according to claim 1 .

13. The expression vector according to claim 12, wherein The multimeric polypeptide is operably linked to a promoter suitable for driving expression of the multimeric polypeptide in mammalian heart cells.

14. The expression vector according to claim 13, wherein The promoter is selected from the group consisting of a cardiac troponin T promoter, an α-myosin heavy chain (α-MHC) promoter, a myosin light chain-2v (MLC-2v) promoter, and a cardiac NCX1 promoter.

15. The expression vector according to claim 12, wherein The vector is a lipid nanoparticle, a retroviral vector, an adenoviral vector or an adeno-associated virus vector (AAV).

16. The expression vector according to claim 15, wherein The AAV is selected from the group consisting of AAV9, AAV6, AAV2i8, AAVrh10, AAVrh74, MyoAAV, Anc80 and Anc82.

17. A pharmaceutical composition comprising an effective amount of the multimeric polypeptide according to any one of claims 1 to 11.

18. A pharmaceutical composition comprising an effective amount of the expression vector according to any one of claims 12 to 16.

19. A cell comprising the expression vector of any one of preceding claims 12 to 16.

20. A method of regulating arrhythmia in a subject, the method comprising contacting cells comprising a cardiac ryanodine channel (RYR2) in the subject with a multimeric polypeptide according to any one of claims 1 to 11 or a polynucleotide encoding the multimeric polypeptide.

21. A method for inhibiting phosphorylation of a ryanodine channel (RYR2) polypeptide in a cell, the method comprising contacting a cell comprising a cardiac ryanodine channel (RYR2) with a multimeric polypeptide according to any one of claims 1 to 11 or a polynucleotide encoding the multimeric polypeptide.

22. The method according to claim 20 or 21, wherein: The cell is in vivo or in vitro.

23. The method according to claim 22, wherein: The cells are in vivo human cells.

24. A method of treating a subject comprising a mutation associated with cardiac arrhythmia, the method comprising administering to the subject a multimeric polypeptide according to any one of claims 1 to 11 or a polynucleotide encoding the multimeric polypeptide.

25. The method according to claim 24, wherein: The mutation is located in the cardiac ryanodine channel (RYR2) 26. A method according to claim 24 or claim 25, wherein: The mutation is RYR2 R4651I .

27. The method according to any one of claims 20 to 26, wherein: The method suppresses cardiac arrhythmias.

28. The method according to claim 27, wherein: The arrhythmia is catecholamine-sensitive polymorphic ventricular tachycardia.

29. The method according to claim 27, wherein: The arrhythmia is atrial fibrillation.

30. The method according to any one of claims 20 to 29, wherein: The subject is a mammal and / or, wherein the cell is from a mammal.

31. The method according to claim 30, wherein: The mammal is a human.

32. A method of treating a subject suffering from a cardiac disease, condition or disorder characterized by cardiac arrhythmia, comprising administering to the subject a multimeric AIP polypeptide or a polynucleotide encoding the polypeptide.

33. The method of claim 32, wherein: The polynucleotide is DNA, RNA or a combination thereof.

34. The method of claim 33, wherein: The polynucleotide comprises one or more modified nucleobases.

35. The method of claim 32, wherein: The polynucleotide is present in a vector.

36. A method of treating a subject suffering from a cardiac disease, condition or disorder characterized by cardiac arrhythmia, comprising administering to the subject an adeno-associated viral vector comprising a polynucleotide encoding a multimeric AIP polypeptide.

37. The method of claim 33, wherein: The adeno-associated viral vector is an effective amount of adeno-associated viral vector.

38. The method of claim 34, wherein: The effective amount is between about 1x10 10 Viral genomes / kg is about 1x10 14 Between viral genomes / kg.

39. The method according to claim 34 or 35, wherein: The effective amount is effective to transfect about 20% to about 40% of the muscle cells in the subject.

40. The method according to any one of claims 32 to 36, wherein: The administration is effective to reduce heart rate variability in the subject.

41. The method according to any one of claims 32 to 37, wherein: The administering is effective to reduce atrial scarring in the subject.

42. The method of claim 32, wherein: The cardiac disease, condition or disorder is atrial fibrillation.

43. The method according to any one of claims 32 to 37, wherein: The multimeric AIP comprises between about 3 and 20 AIP repeats.

44. The method of claim 42, wherein: The multimeric AIP comprises between about 3 and 10 AIP repeats.

45. The method of claim 42, wherein: The multimeric AIP comprises 5 AIP repeats.

46. ​​A method of reducing cardiac variability in a subject with atrial fibrillation, comprising administering to the subject a multimeric AIP polypeptide or a polynucleotide encoding the multimeric AIP polypeptide.

47. The method of claim 46, wherein: The polynucleotide is DNA, RNA or a combination thereof.

48. The method of claim 47, wherein: The polynucleotide comprises one or more modified nucleobases.

49. The method of claim 46, wherein: The polynucleotide is present in a vector.

50. The method of claim 46, wherein: The vector is an adeno-associated virus vector.

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