Compositions and methods for treating cardiac disease

Abstract

The disclosed invention is 2+ -Featured are calmodulin-dependent kinase II (CaMKII) inhibitory multimeric polypeptides, polynucleotides encoding such polypeptides, and methods of using them to treat cardiac disorders (e.g., catecholaminergic polymorphic ventricular tachycardia (CPVT) or atrial fibrillation (AF)).

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a PCT international patent application claiming priority and benefit of U.S. Provisional Application No. 63 / 342,311, filed May 16, 2022, the entire contents of which are incorporated herein by reference.

[0002] STATEMENT OF RIGHTS TO INVETIONS MADE UNDER FEDERALLY SPONSORED RESEARCH This invention was made with Government support under Grant No. W81XWH1910473 awarded by the Department of Defense. The United States Government has certain rights in this invention. [Background technology]

[0003] 2. Background of the Invention Catecholamine-induced polymorphic ventricular tachycardia (CPVT) is a disorder characterized by an abnormal heart rhythm that affects as many as 1 in 10,000 people. Symptoms of CPVT include dizziness or fainting related to exercise or mental stress. Episodes of ventricular tachycardia can cause the heart to stop beating effectively (cardiac arrest) and lead to sudden death in children and young adults with no known cardiac abnormalities. Treatments for CPVT include exercise restriction, use of beta-blockers, and automatic implantable cardioverter-defibrillators. Other treatments include surgical sympathectomy and treatment with flecainide. Unfortunately, these treatments are not effective in all patients and are limited by patient compliance, side effects of medications, or the risk of adverse events such as fatal electrical storms caused by implantable cardioverter-defibrillators. Thus, there remains an unmet need for improved compositions and methods for treating CPVT and other cardiac disorders characterized by abnormal heart rhythms.

[0004] Atrial fibrillation (AF) currently affects over 2 million adults in the United States alone and is the most common type of sustained cardiac arrhythmia in clinical practice. Although treatments for AF exist, there is currently no cure, and AF is associated with reduced life expectancy. Furthermore, approximately 158,000 people die from AF each year in the United States alone. Thus, there is a need for improved compositions and methods for treating AF. Summary of the Invention

[0005] As described below, the invention of the present disclosure includes a method for producing a Ca 2+ - Calmodulin-dependent kinase II (CaMKII) inhibitory multimeric polypeptides (e.g., autocamtide 2-related inhibitory peptide ( a Utocamtide-2-related i nhibitory p The present invention features polynucleotides encoding CaMKII inhibitory multimeric polypeptides, including CaMKII inhibitory peptide (AIP) multimers, polynucleotides encoding CaMKII inhibitory multimeric polypeptides, and methods of using such polypeptides and polynucleotides to treat a cardiac disease, condition, or disorder characterized by cardiac arrhythmia (e.g., atrial fibrillation (AF), catecholamine-induced polymorphic ventricular tachycardia (CPVT), ischemic heart disease, cardiac arrhythmias, heart failure (e.g., heart failure associated with aortic binding), hypertensive heart disease and pulmonary hypertensive heart disease, myocardial infarction, valvular disease, congenital heart disease, myocardial hypertrophy, ventricular arrhythmia, or Timothy's syndrome).

[0006] In one aspect, the disclosed invention features multimeric polypeptides that inhibit CaMKII.

[0007] In one aspect, the disclosed invention features an expression vector including a polynucleotide encoding a multimeric polypeptide of any of the above aspects or embodiments thereof.

[0008] 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.

[0009] In one aspect, the disclosed invention features a pharmaceutical composition including an effective amount of an expression vector of any of the above aspects or embodiments thereof.

[0010] In one aspect, the disclosed invention features a cell that includes an expression vector of any of the above aspects or embodiments thereof.

[0011] In one aspect, the disclosed invention features a method for regulating cardiac arrhythmia in a subject, the method comprising contacting a cell of the subject that contains a cardiac ryanodine channel (RYR2) with a multimeric polypeptide of any of the above aspects or embodiments thereof, or a polynucleotide encoding the multimeric polypeptide.

[0012] In one aspect, the disclosed invention features a method for inhibiting phosphorylation of a ryanodine channel (RYR2) polypeptide in a cell, the method comprising contacting a cell containing a cardiac ryanodine channel (RYR2) with a multimeric polypeptide of any of the above aspects or embodiments thereof, or a polynucleotide encoding the multimeric polypeptide.

[0013] In one aspect, the disclosed invention features a method of treating a subject containing a mutation associated with cardiac arrhythmia, comprising administering to the subject a multimeric polypeptide of any of the above aspects or embodiments thereof, or a polynucleotide encoding the multimeric polypeptide.

[0014] 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.

[0015] In one aspect, the disclosed invention features a method of treating a subject having a cardiac disease, condition, or disorder characterized by cardiac arrhythmia, the method comprising administering to the subject an adeno-associated virus vector comprising a polynucleotide encoding a multimeric AIP polypeptide.

[0016] In one aspect, the disclosed invention features a method of reducing cardiac variability in a subject having atrial fibrillation, the method comprising administering to the subject a multimeric AIP polypeptide or a polynucleotide encoding a multimeric AIP polypeptide.

[0017] 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 repeats of the AIP peptide. In any of the above aspects or embodiments thereof, the multimeric polypeptide comprises 3, 4, 5, or 6 repeats of the AIP peptide. In any of the above aspects or embodiments thereof, the multimeric polypeptide comprises three AIP peptides. In any of the above aspects or embodiments thereof, the multimeric polypeptide comprises five AIP peptides. In any of the above aspects or embodiments thereof, the AIP repeats are consecutive and / or separated by a linker. In any of the above aspects or embodiments thereof, the multimeric polypeptide comprises Contains a sequence having at least 85% amino acid sequence identity to TIFF2025517333000001.tif20163.

[0018] 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.

[0019] In any of the above aspects or embodiments thereof, the EC50 of CaMKII inhibition by the multimeric polypeptide is less than 10% of the EC50 of AIP. In any of the above aspects or embodiments thereof, the EC50 of CaMKII inhibition by the multimeric polypeptide is less than 5% of the EC50 of AIP.

[0020] 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 a mammalian cardiac cell. In any of the above aspects or embodiments thereof, the promoter is selected from one or more of the following: 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.

[0021] In any of the above aspects or embodiments thereof, the vector is a retroviral vector, an adenoviral vector, or an adeno-associated viral vector (AAV). In some embodiments, the AAV is selected from one or more of AAV9, AAV6, AAV2i8, AAVrhlO, AAVrh74, MyoAAV, Anc80, and Anc82.

[0022] 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.

[0023] In any of the above aspects or embodiments thereof, the mutation is in the cardiac ryanodine channel (RYR2). R4651I It is.

[0024] In any of the above aspects or embodiments thereof, the method suppresses cardiac arrhythmia. In any of the above aspects or embodiments thereof, the arrhythmia is catecholaminergic polymorphic ventricular tachycardia. In any of the above aspects or embodiments thereof, the arrhythmia is atrial fibrillation.

[0025] 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.

[0026] 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.

[0027] In any of the above aspects or embodiments thereof, the adeno-associated viral vector is an effective amount of an adeno-associated viral vector. In any of the above aspects or embodiments thereof, the effective amount is about 1×10 10 Viral genome / kg ~ approx. 1 x 10 14 In any of the above aspects or embodiments thereof, the effective amount is effective to transfect about 20% to about 40% of muscle cells in the subject.

[0028] In any of the above aspects or embodiments thereof, the administration is effective to reduce heart rate variability in the subject. In any of the above aspects or embodiments thereof, the administration is effective to reduce atrial scarring in the subject.

[0029] In any of the above aspects or embodiments thereof, the cardiac disease, condition, or disorder is atrial fibrillation.

[0030] In any of the above aspects or embodiments thereof, the multimeric AIP comprises about 3 to 10 repeats of AIP.

[0031] The compositions and articles defined in the present invention are isolated or otherwise prepared in connection with the examples provided below. Other features and advantages of the present invention will become apparent from the detailed description and claims.

[0032] definition Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present invention belongs. The following references provide those skilled in the art with the general definitions of many of the terms used in the present invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). The following terms used herein have the following defined meanings unless otherwise specified.

[0033] "Multimer" or "multimeric polypeptide" refers to a polypeptide sequence that contains two or more repeats of an amino acid sequence. In one embodiment, the multimeric polypeptide inhibits CaMKII. Such a multimeric polypeptide is called a "CaMKII-inhibitory multimeric polypeptide".

[0034] "Autocamtide 2-related inhibitory peptide (AIP)" is AIP refers to a peptide or fragment thereof having at least about 85% amino acid sequence identity to TIFF2025517333000002.tif4128; comprising or consisting of at least about 9-14 contiguous amino acids of SEQ ID NO:1; and having cardioregulatory activity and / or CaMKII inhibitory activity. TIFF2025517333000003.tif4128. In some cases, the AIP has at least about 85% amino acid sequence identity to TIFF2025517333000003.tif4128. kkKlrrqeaFdal (AIPo), where the capital letters represent the amino acid sequence: TIFF2025517333000004.tif4128. In some cases, the AIP contains one or more of the modifications described in 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 disclosure of which is incorporated by reference in its entirety for all purposes, and / or the AIP contains modifications at one or more of the critical amino acid residues described therein (e.g., sites corresponding to the uppercase amino acid residues provided in the amino acid sequence kkKlrrqeaFdal). In some cases, the AIP contains one or more of the modifications described in 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 disclosure of which is incorporated by reference in its entirety for all purposes. and having at least 70%, 75%, 80%, 85%, or 90% amino acid sequence identity to TIFF2025517333000005.tif4128 and containing one or more or a combination of the following amino acid substitutions: 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-dimethyl-arginine), 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 some embodiments, AIPo has increased selectivity for CaMKII inhibition over inhibition of PKC. In one embodiment, the AIP peptide comprises one or more alterations 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 9-13 contiguous 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 9-13 contiguous 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 alterations in SEQ ID NO: 1.

[0035] "Autocamtide 2 associated inhibitory (AIP) multimeric polypeptide" refers to a polypeptide comprising two or more repeats of the AIP peptide. 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 repeats). In one embodiment, the AIP multimeric polypeptide comprises the sequence: TIFF2025517333000006.tif3128 or Array: TIFF2025517333000007.tif11163. In another embodiment, the repeats are contiguous.

[0036] "AIP multimer polynucleotide" means a polynucleotide that encodes an AIP multimer polypeptide.

[0037] By "calcium / calmodulin-dependent protein kinase II (CaMKII) polypeptide" is meant a polypeptide or fragment thereof having at least 85% sequence identity to the amino acid sequence of GenBank Accession No. AAH32784.1 provided below, or a fragment thereof that has kinase activity. >AAH32784.1 Calcium / calmodulin-dependent protein kinase II delta [Homo sapiens] TIFF2025517333000008.tif54163

[0038] "Calcium / calmodulin-dependent protein kinase II (CaMKII) polynucleotide" refers to a polynucleotide that encodes a CaMKII polypeptide. A representative CaMKII polynucleotide sequence is provided below (GenBank Accession No. BC032784.1). >BC032784.1:63-1499 Homo sapiens calcium / calmodulin-dependent protein kinase II delta, mRNA (cDNA clone MGC:44911 IMAGE:5178265), full length cds TIFF2025517333000009.tif161163

[0039] What is "CN19o peptide"? CN19o peptide or a fragment thereof having at least about 85% amino acid sequence identity to TIFF2025517333000010.tif5128; comprising or consisting of at least about 9-19 contiguous amino acids of SEQ ID NO:2; and having cardioregulatory activity and / or CaMKII inhibitory activity. In one embodiment, the CN19o peptide comprises one or more alterations in the peptide sequence.

[0040] "CN19o multimeric polypeptide" refers to a polypeptide or fragment thereof that includes two or more repeats of the CN19o peptide. In one embodiment, the CN19o multimer has the sequence: TIFF2025517333000011.tif3145 or Array: TIFF2025517333000012.tif11163. In another embodiment, the repeats are contiguous.

[0041] By "CN19o multimer polynucleotide" is meant a polynucleotide that encodes a CN19o multimer polypeptide.

[0042] By "12.6 kDa FK506 binding protein (FKBP12.6) polypeptide" is meant a polypeptide or fragment thereof having at least 85% sequence identity to the amino acid sequence of NCBI Ref. Seq. Accession No. NP_004107.1 provided below, or a fragment thereof capable of binding to an RYR2 polypeptide. >NP_004107.1 Peptidyl-prolyl cis-trans isomerase FKBP1B isoform a [Homo sapiens] TIFF2025517333000013.tif11163

[0043] "12.6 kDa FK506 binding protein (FKBP12.6) polynucleotide" refers to a polynucleotide that encodes an FKBP12.6 polypeptide. A representative FKBP12.6 polynucleotide sequence is provided below (NCBI Ref. Seq. Accession No. NM_004116.5). >NM_004116.5:110-436 Homo sapiens FKBP prolyl isomerase 1B (FKBP1B), transcript variant 1, mRNA TIFF2025517333000014.tif39163

[0044] By "ryanodine receptor 2 (RYR2) polypeptide" is meant a polypeptide or fragment thereof having at least 85% sequence identity to the amino acid sequence of GenBank Accession No. CAA62975.1 provided below, or a fragment thereof capable of forming a homotetramer that functions as a calcium channel. >CAA62975.1 Ryanodine receptor, partial [Homo sapiens] TIFF2025517333000015.tif18163

[0045] "Ryanodine receptor 2 (RYR2) polynucleotide" refers to a polynucleotide that encodes a RYR2 polypeptide. A representative RYR2 polynucleotide sequence is provided below (GenBank Accession No. X91869.1). >X91869.1 H. sapiens mRNA for ryanodine receptor TIFF2025517333000016.tif47163

[0046] "CaMKII inhibitor" refers to a peptide or small molecule that inhibits the activity of CaMKII. Exemplary inhibitors are known in the art (e.g., AIP, CN19, CN27, CN19o, CN21) and are described, for example, in Coultrap et al., PLOS One e25245, Vol 6, Issue 10, 2011 and Pellicena et al., Frontiers in Pharmacology 21:1-20, 2014. Other inhibitors include: TIFF2025517333000017.tif228157

[0047] By "agent" is meant a peptide, polypeptide, nucleic acid molecule, or small chemical compound.

[0048] By "ameliorate" is meant to reduce, suppress, attenuate, diminish, arrest, or stabilize the onset or progression of a disease. In some embodiments, the disease is a cardiac disease or disorder.

[0049] An "alteration" in reference to an amino acid sequence means a change in the identity of one or more amino acids in the amino acid sequence.

[0050] "Analog" refers to a molecule that is not identical but has similar functional or structural characteristics. For example, a polypeptide analog retains the biological activity of the corresponding naturally occurring polypeptide, but has certain biochemical modifications that enhance the function of the analog compared to the naturally occurring polypeptide. Such biochemical modifications may increase the analog's protease resistance, membrane permeability, or half-life, for example, without altering ligand binding. Analogs may include unnatural amino acids. A polynucleotide analog retains the biological activity of the corresponding naturally occurring polypeptide, but has certain biochemical modifications that enhance the analog's function compared to the naturally occurring polynucleotide. Such biochemical modifications may increase the analog's nuclease resistance, membrane permeability, or half-life, for example, without altering functional activity, such as the ability to encode a protein. Analogs may include modified nucleic acid molecules.

[0051] The term "cardiomyocyte" as used herein broadly refers to a cardiac muscle cell. In one aspect, a mammalian cardiac cell is a cardiomyocyte. In another aspect, a cardiomyocyte differentiated from an induced pluripotent stem cell is a cardiomyocyte.

[0052] The phrase "cardiac condition, disease, or disorder" as used herein is intended to include any disorder characterized by insufficient, undesirable, or abnormal cardiac function. Exemplary cardiac conditions, diseases, or disorders include, but are not limited to, atrial fibrillation (AF), catecholaminergic polymorphic ventricular tachycardia (CPVT), ischemic heart disease, cardiac arrhythmias, heart failure (e.g., heart failure associated with aortic attachment), hypertensive heart disease and pulmonary hypertensive heart disease, myocardial infarction, valvular disease, congenital heart disease, myocardial hypertrophy, ventricular arrhythmias, or Timothy's syndrome, and any condition that causes congestive heart failure in a subject, particularly a human subject. Insufficient or abnormal cardiac function can be the result of disease, injury, genetic mutation, and / or aging. By way of background, the response to myocardial injury follows a well-defined pathway in which some cells die while others enter a hibernation state and become dysfunctional while those that do not die yet do not. This is followed by infiltration of inflammatory cells and deposition of collagen as part of scar formation, all of which is paralleled by the in-growth of new blood vessels and some ongoing cell death.

[0053] "Effective amount" or "therapeutically effective amount" refers to the amount of agent required to improve the symptoms of a disease compared to an untreated patient. The effective amount of the active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending on the method of administration, the age, weight, and general health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate amount and dosing regimen. Such an amount is called an "effective" amount. Thus, the term "therapeutically effective amount" refers to an amount of a composition disclosed herein that is sufficient to therapeutically or prophylactically significantly reduce symptoms or clinical markers associated with cardiac dysfunction or disorder when administered to a typical subject with, for example, a cardiovascular condition, disease, or disorder.

[0054] With respect to the treatment of, for example, a cardiovascular condition or disease in a subject, the term "therapeutically effective amount" refers to an amount that is sufficient and safe to prevent or delay the onset of a cardiovascular disease or disorder (e.g., cardiac arrhythmia). Thus, the amount can cure or inhibit arrhythmia, or alleviate a cardiovascular disease or disorder, slow the progression of a cardiovascular disease, slow or inhibit the symptoms of a cardiovascular disease or disorder, slow or inhibit the establishment of secondary symptoms of a cardiovascular disease or disorder, or inhibit the onset of secondary symptoms of a cardiovascular disease or disorder. The amount effective for treating a cardiovascular disease or disorder will vary depending on the type of cardiovascular disease being treated, the severity of the symptoms, the subject being treated, the age and general condition of the subject, the method of administration, and the like. Therefore, an exact "effective amount" cannot be specified. However, in any given case, the appropriate "effective amount" can be determined by one of ordinary skill in the art using only routine experimentation. The efficacy of treatment can be determined by one skilled in the art, for example, efficacy can be assessed in the treatment of animal models of the cardiovascular diseases or disorders discussed herein, e.g., rodents having acute myocardial infarction or ischemia-reperfusion injury; any treatment or administration of a composition or formulation that leads to a reduction in at least one symptom of a cardiovascular disease or disorder disclosed herein, such as, for example, increased cardiac ejection fraction, reduced heart failure rate, reduced infarct size, reduced associated morbidity (pulmonary edema, renal failure, arrhythmias), improved exercise tolerance or other quality of life indicators, and reduced mortality, indicates effective treatment. In embodiments in which the compositions are used to treat cardiovascular diseases or disorders, the efficacy of the compositions can be determined using experimental animal models of cardiovascular disease, such as animal models of ischemia-reperfusion injury (Headrick JP, Am J Physiol Heart circ Physiol 285;H1797; 2003) and 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 experimental animal models, efficacy of treatment is demonstrated when a reduction in symptoms of a cardiovascular disease or disorder, e.g., one or more of dyspnea, chest pain, palpitations, dizziness, fainting, edema, cyanosis, pallor, fatigue and hypertension, occurs more quickly in treated animals compared to untreated animals.

[0055] Subjects suitable for treatment by the methods disclosed herein can be identified by any method of diagnosing cardiac arrhythmia. Methods of diagnosing such conditions are well known to those skilled in the art. As a non-limiting example, cardiac arrhythmia can be diagnosed by electrocardiogram (ECG or EKG), which is a graphical recording of the heart's activity on paper or computer monitor.

[0056] The terms "coronary artery disease" and "acute coronary syndrome" are used interchangeably herein and refer to myocardial infarction and to conditions, diseases or disorders of the cardiovascular system, including any disorder characterized by insufficient, undesirable or abnormal cardiac function, such as ischemic heart disease, hypertensive and pulmonary hypertensive heart disease, valvular disease, congenital heart disease, and any condition that results in congestive heart failure in a subject, particularly a human subject. Insufficient or abnormal cardiac function can be the result of disease, injury and / or aging. By way of background, the response to myocardial injury follows a well-defined pathway in which some cells die while others go into hibernation and become dysfunctional while those that do not die yet. This is followed by infiltration of inflammatory cells, deposition of collagen as part of scar formation, all of which occurs in parallel with the ingrowth of new blood vessels and some degree of ongoing cell death.

[0057] In this disclosure, "comprises, comprising, "containing", "having", and the like can have the meanings given them in U.S. Patent Law and can mean "includes, including", and the like; "consisting essentially of" or "consists essentially" likewise has the meaning given to it in U.S. Patent Law, and the term is open-ended, allowing for the presence of more than what is recited, but excluding prior art embodiments, so long as the basic or novel characteristics of what is recited are not altered by the presence of more than what is recited. Embodiments designated as "comprising" a particular component(s) or element(s) are also contemplated in some embodiments as "consisting of" or "consisting essentially of" the particular component(s) or element(s).

[0058] By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. The portion preferably comprises at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the full length of the reference nucleic acid molecule or polypeptide. A fragment may comprise 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.

[0059] "Heart rate variability" or "heart beat interval variability" refers to the variability of RR interval (the interval between two successive R waves measured by electrocardiogram), particularly the variability measured by the standard deviation of RR interval.In some embodiments, the increase in heart rate variability or heart beat interval variability in a subject compared to baseline indicates arrhythmia or is a symptom of a cardiovascular disease, condition, or disorder.In some embodiments, administering an agent disclosed herein to a subject with increased heart rate variability or heart beat interval variability compared to baseline is effective to reduce heart rate variability or heart beat interval variability, and preferably, such administration is effective to reduce heart rate variability or heart beat interval variability to baseline level.

[0060] "Hybridization" refers to hydrogen bonding between complementary nucleobases, which may be Watson-Crick, Hoogsteen, or reversed Hoogsteen. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.

[0061] The terms "isolated," "purified," or "biologically pure" refer to material that is free, to a greater or lesser extent, from components that normally accompany it as found in its natural state. "Isolated" means some degree of separation from the original source or surrounding environment. "Purified" means a degree of separation greater than isolation. A "purified" or "biologically pure" protein has been sufficiently free of other materials so that the impurities do not significantly affect the biological properties of the protein or cause other deleterious consequences. That is, the nucleic acids or peptides of the invention are purified when they are substantially free of cellular material, viral material, or culture medium if produced by recombinant DNA technology, or substantially free of chemical precursors or other chemicals if chemically synthesized. Purity and homogeneity are usually confirmed using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can mean that the nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. In the case of proteins that can be modified, such as phosphorylation or glycosylation, different modifications can give rise to different isolated proteins, which can be purified separately.

[0062] By "isolated polynucleotide" is meant a nucleic acid (e.g., DNA) that is free of the genes that flank it in the naturally occurring genome of the organism from which the nucleic acid molecule of the invention is derived. Thus, the term includes recombinant DNA that is incorporated, for example, into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule independent of other sequences (e.g., cDNA or genomic or cDNA fragments generated by PCR or restriction endonuclease digestion). In addition, the term also includes RNA molecules transcribed from a DNA molecule, as well as recombinant DNA that is part of a hybrid gene that encodes additional polypeptide sequences.

[0063] By "isolated polypeptide" is meant a polypeptide of the invention separated from components that naturally accompany it. Generally, a polypeptide is isolated when it is at least 60%, by weight, free from proteins and naturally occurring organic molecules with which it is naturally associated. Preferably, a preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention. An isolated polypeptide of the invention can be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding the polypeptide, or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0064] As used herein, "obtaining," as in "obtaining an agent," includes synthesizing, purchasing, or otherwise acquiring the agent.

[0065] A "polypeptide" or "amino acid sequence" refers to a chain of amino acids, 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 can be made to a polypeptide to provide a functionally equivalent variant or homologue of the polypeptide. In some aspects, the invention encompasses sequence changes that result in conservative amino acid substitutions. In some embodiments, "conservative amino acid substitutions" refer to amino acid substitutions that do not change the relative charge or size characteristics of the protein in which the conservative amino acid substitution is made. Mutants can be prepared according to methods of modifying polypeptide sequences known to those skilled in the art, such as those found in references that summarize them, such as Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; or Current Protocols in Molecular Biology, FM Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Non-limiting examples of conservative amino acid substitutions include substitutions made 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.

[0066] "Reduce" refers to a negative change relative to a baseline. In the context of cardiac arrhythmia, the term "reduce" refers to a significant reduction in the symptom, e.g., a reduction in the incidence of the arrhythmia or symptom, or the severity of the symptom, 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%).

[0067] "Reference" refers to a corresponding control condition. In one embodiment, the reference is an untreated control. In another embodiment, the reference is a healthy wild-type control. In another embodiment, a subject with a cardiac disease or disorder is treated with a multimeric polypeptide described herein, and the effect of such treatment is evaluated in comparison to the subject before treatment, or in comparison to an untreated subject that also has a cardiac disease or disorder.

[0068] A "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence can be a subset or the entirety of a specified sequence; for example, a segment 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 generally at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, 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 generally at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, even more preferably about 100 nucleotides or about 300 nucleotides, or any integer number therebetween.

[0069] Nucleic acid molecules useful in the method of the present invention include any nucleic acid molecule that encodes a polypeptide of the present invention or a fragment thereof. Such nucleic acid molecules do not need to be 100% identical to an endogenous nucleic acid sequence, but will usually show substantial identity. A polynucleotide that has "substantial identity" to an endogenous sequence can usually hybridize with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the method of the present invention include any nucleic acid molecule that encodes a polypeptide of the present invention or a fragment thereof. Such nucleic acid molecules do not need to be 100% identical to an endogenous nucleic acid sequence, but will usually show substantial identity. A polynucleotide that has "substantial identity" to an endogenous sequence can usually hybridize with at least one strand of a double-stranded nucleic acid molecule. "Hybridize" means pairing to form a double-stranded molecule between complementary polynucleotide sequences (e.g., genes described herein) or portions thereof under various conditions of stringency (see, e.g., Wahl, GM and SL Berger (1987) Methods Enzymol. 152:399; Kimmel, AR (1987) Methods Enzymol. 152:507).

[0070] For example, stringent salt concentrations are usually less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvents, such as formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, more preferably at least about 50% formamide. Stringent temperature conditions usually include a temperature of at least about 30°C, more preferably at least about 37°C, and most preferably at least about 42°C. Various additional parameters, such as hybridization time, concentration of detergent such as sodium dodecyl sulfate (SDS), 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 various conditions as necessary. In a preferred embodiment, hybridization is performed in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS at 30° C. In a more preferred embodiment, hybridization is performed 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 is performed in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA at 42° C. Beneficial modifications to these conditions will be readily apparent to those of skill in the art.

[0071] In most applications, the washing steps following hybridization also vary in stringency. Wash stringency conditions can be defined by salt concentration and temperature. As mentioned above, washing stringency can be increased by decreasing salt concentration or increasing temperature. For example, stringent salt concentrations for the washing steps are preferably less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the washing steps usually 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 steps are performed at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, the washing steps are performed at 42°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, the washing steps are performed at 68° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Further modifications to these conditions will be readily 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., USA 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.

[0072] "Substantially identical" refers to a polypeptide or nucleic acid molecule that exhibits at least 85% identity to a reference amino acid or nucleic acid sequence. In some embodiments, such a sequence is at least 90%, 95%, or even 99% identical to the reference sequence at the amino acid or nucleic acid level.

[0073] 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, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other alterations and 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 approach for determining the degree of identity, the BLAST program can be used, e.g. -3 and e -100 Probability scores between 0 and 1 indicate closely related sequences.

[0074] As used herein, the term "modulate" means to regulate or adjust to some degree.

[0075] As used herein, the terms "pharmacologically acceptable," "physiologically tolerable," and grammatical variations thereof, when referring to compositions, carriers, diluents, and reagents, are used interchangeably to indicate that the material can be administered to a mammal without producing undesirable physiological effects, such as nausea, dizziness, upset stomach, etc. A pharma- ceutically acceptable carrier does not promote a mounting immune response to the agent with which it is mixed, unless such desire exists. The preparation of pharmacological compositions containing an active ingredient dissolved or dispersed therein is well understood in the art and need not be limited based on formulation. Typically, such compositions are prepared as injectables, either as solutions or suspensions, but may also be prepared as solid dosage forms suitable for dissolving or suspending in liquid prior to use. The preparations may also be emulsified or presented as liposomal compositions. The active ingredient may be mixed with excipients that are pharma- ceutically acceptable and compatible with the active ingredient, in amounts suitable for use in the therapeutic methods described herein. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In addition, if necessary, the composition may contain minor amounts of auxiliary substances, such as wetting agents, emulsifying agents, pH buffering agents, and the like, which enhance the effectiveness of the active ingredient. The therapeutic composition of the present invention may contain pharma-ceutically acceptable salts of the components therein. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the polypeptide) formed with inorganic acids, such as, for example, hydrochloric acid, phosphoric acid, or organic acids, such as acetic acid, tartaric acid, mandelic acid, and the like. Salts formed with free carboxyl groups may also be derived from inorganic bases, such as, for example, sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, ferric hydroxide, and the like, and organic bases, such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like. Physiologically tolerable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions, which contain no substances or ingredients other than the active ingredient and water, or contain a buffer solution, such as sodium phosphate, saline, or both, for example phosphate buffered saline, at physiological pH values.Additionally, aqueous carriers can include salts such as sodium chloride, potassium chloride, as well as multiple buffer salts, dextrose, polyethylene glycol, and other solutes. Liquid compositions can also include liquid phases in addition to or to the exclusion of water. Examples of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions. The amount of active agent used in the methods described herein that is effective in treating 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 text in this field. For example, a parenteral composition suitable for administration by injection is prepared by dissolving 1.5% by weight of the active ingredient in 0.9% sodium chloride solution.

[0076] In one aspect, "pharmaceutically acceptable" carriers do not include in vitro cell culture media.

[0077] In one aspect, the term "pharmaceutical acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias for use in animals, and more specifically in humans. Specifically, it refers to compounds, substances, compositions, and / or dosage forms that are suitable for use in contact with the tissues of humans and animals, within the scope of sound medical judgment, without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0078] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions, aqueous dextrose, and aqueous glycerol solutions can 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, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, and the like. The composition can also contain minor amounts of wetting agents, emulsifying agents, or pH buffering agents, if desired. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, etc. The compositions can be formulated as suppositories, using traditional binders and carriers, such as triglycerides. Oral formulations can include standard carriers, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, 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 method of administration.

[0079] "Subject" means a mammal, including, but not limited to, a human or a non-human mammal, such as a cow, horse, dog, sheep, rodent, cat, etc. In one aspect, the subject suffers from or is prone to develop a monogenic disease, disorder, or condition that can be treated using gene therapy vectors, cell-based therapeutics, and methods disclosed elsewhere herein.

[0080] Ranges provided herein are understood to be shorthand for all values ​​within that range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0081] The term "tissue" refers to a group or layer of similarly specialized cells that together perform some specialized function. The term "tissue-specific" refers to the origin of cells from a particular tissue, or the distinct characteristics of the cells.

[0082] As used herein, the terms "treat," "treating," "treatment," and the like refer to reducing or ameliorating a disorder and / or symptoms associated therewith. It will be understood that treating a disorder or condition does not require, but does not preclude, the complete elimination of the disorder, condition, or symptoms associated therewith.

[0083] As used herein, the term "or" is understood to be inclusive unless otherwise stated or clear from context. As used herein, the terms "a," "an," and "the" are understood to be singular or plural unless otherwise stated or clear from context.

[0084] Unless otherwise specified or clear from the context, the term "about" as used herein is understood to be within the normal acceptance in the art.

[0085] The recitation of a listing of a chemical group in a definition of a variable herein includes definitions of that variable as a single group or as any combination of the listed groups. The recitation of an embodiment of a variable or aspect herein includes that embodiment as a single embodiment or in combination with other embodiments or portions thereof.

[0086] Any composition or method provided herein can be combined with any one or more of the other compositions and methods provided herein. [Brief description of the drawings]

[0087] [Figure 1A]Figures 1A-1C provide schematics, plots, and bar graphs illustrating how the peptide inhibitor autocamtide 2-related inhibitory peptide (AIP) can be used as part of a gene therapy for catecholamine-induced polymorphic ventricular tachycardia (CPVT). Figure 1A is a schematic illustrating how a clinical vector (e.g., adeno-associated virus (AAV)) can be used to deliver AIP to cells (e.g., mouse cells) to inhibit or prevent phosphorylation of CPVT mutant RYR2 (mouse polypeptide R4650I is shown as an example) by CaMKII. In addition to the inhibitory peptide cargo, this clinical vector contains a promoter and capsid, either of which can be optimized to increase the efficacy of the vector in therapy. In Figure 1A, RYR2 stands for "cardiac ryanodine receptor 2." Figure 1B shows electrocardiograms of mice treated with a negative control (green fluorescent protein (GFP)) or AAV expressing AIP. Mice treated with AIP showed a reduction in ventricular tachycardia (VT) after pacing compared to mice treated with GFP. Figure 1C is a bar graph showing the percentage of mice exhibiting inducible ventricular tachycardia (VT) in wild-type or RYR2-R176Q+ / - mice (CPVT mice with the RyR2 R176Q mutation) treated with AIP or GFP. The fraction within the bar graph represents the number of mice exhibiting inducible VT relative to the total number of mice tested. Administration of AIP resulted in a reduction in inducible VT in RYR2-R176Q+ / - mice. [Figure 1B] See legend to Figure 1A. [Figure 1C] See legend to Figure 1A. [Figure 2A]Figures 2A-2G provide plots, protein structures, schematics, bar graphs, and images showing how therapeutic cargos, including CaMKII inhibitors, were optimized to treat catecholamine-induced polymorphic ventricular tachycardia (CPVT) by altering the potency, binding, and / or multimerization of the CaMKII inhibitors. FIG. 2A is a plot showing the IC50 concentrations of autocamtide 2-related inhibitory peptide (AIP) and CN19o (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 by reference in their entirety for all purposes). CN19o is a more potent inhibitor of CaMKII activity in the in vitro system used for these assays, as it has a lower IC50 than AIP. FIG. 2B provides an EM map of RYR2 at 6 angstrom resolution. In Figure 2B, FKBP12.6 stands for "FK506 binding protein," a protein that stabilizes RyR2 to prevent abnormal activation of the channel during the resting phase of the cardiac cycle. FKBP12.6 stabilizes RyR2 in the closed state.FKBP12.6 functions as a stabilizer to increase the rigidity of the HD2 domain (HD2 and HD2') and the P2 domain (P2 and P2') (Chi, et al. "Molecular basis for allosteric regulation of the type 2 ryanodine receptor channel gating by key modulators," PNAS, 116:25575-25582 (2019); the disclosure of which is incorporated by reference in its entirety for all purposes). Figure 2C is a schematic diagram showing AIPx5 multimers. Figure 2D is a schematic diagram showing the experimental design to evaluate the efficacy of various candidate therapeutic cargoes to reduce arrhythmias in the CPVT mouse model (RYR2-R46450I). The left portion of Figure 2D shows a schematic diagram of different polypeptides that can be included within the therapeutic cargo delivered to the subject. In FIG. 2D, P2A represents a self-cleaving peptide, AIP represents an example of an autocamtide 2-associated inhibitory peptide (AIP), mCherry represents the fluorescent protein mCherry, CN19o represents the CaMKII inhibitor CN19o, FKBP12.6 represents the FKBP12.6 polypeptide that binds to RYR2, and the polygonal shape represents the viral capsid. The rightmost portion of FIG. 2D (FIG. 2D-2) shows electrocardiograms of WT and RYR2R4650 / WT mice. RYR2R4650 / WT mice show irregularities in their heart beat. FIG. 2E is a plot showing the percentage of ectopy in RYR2R4650I / WT mice administered the polypeptides described in FIG. 2D shown. Administration of AIPx5 resulted in the greatest reduction in the number of ectopy events observed in the mice. Figure 2F is a bar graph showing the percentage of RYR2R4650I / WT mice administered the polypeptides shown in Figure 2D that exhibited inducible ventricular tachycardia (VT). Administration of AIPx5 was associated with a reduction in the number of inducible VT episodes in the mice. Figure 2G is an image of cardiac tissue showing mCherry expression in the tissue, thereby confirming that the constructs described in Figure 2D were delivered to and expressed in the cardiac tissue of RYR2R4650I / WT mice. [Figure 2B] See legend to Figure 2A. [Figure 2C] See legend to Figure 2A. [Figure 2D-1] See legend to Figure 2A. [Figure 2D-2] See legend to Figure 2A. [Figure 2E] See legend to Figure 2A. [Figure 2F] See legend to Figure 2A. [Figure 2G] See legend to Figure 2A. [Diagram 3] Figure 3 provides charts, capillary western images, and plots showing the effect of constructs described in the indicated Figure 2D on CaMKII activity in vivo. 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. The center panel of Figure 3 provides capillary western images showing expression of glyceraldehyde-3-phosphate dehydrogenase polypeptide (GAPDH) and levels of phosphorylated PLB (pPLB) and nonphosphorylated PLB (PLB) in cells exposed (+) or not (-) to isoproterenol (ISO). The right panel of Figure 3 is a plot showing the ratio of pPLB to PLB in hearts treated without ISO, or with ISO and mCherry ("ISO" sample), AIP, AIPx5, AIP-FKBP12.6, CN19o, CN19oX3, or CN19o-FKBP12.6 (as described in Figure 2D). [Figure 4]FIG. 4 is a schematic diagram showing the design of an in vitro experiment to evaluate the effect of the purified polypeptides described in FIG. 2D and shown in the left portion of FIG. 4 on CaMKIIδ (an isomer of CaMKII) activity. The sequences of the polypeptides used in the in vitro experiments are shown in Table 1. The activity of CaMKIIδ was measured using a bioluminescent homogeneous ADP monitoring assay for kinases (ADP-GLO™); this assay is described in Zegzouti, et al. “ADP-Glo: A Bioluminescent and Homogeneous ADP Monitoring Assay for Kinases,” ASSAY and Drug Development Technologies, Dec. 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. [Diagram 5] Figure 5 provides plots from an in vitro bioluminescence homogeneous ADP monitoring assay showing that multimerization of AIP improved the potency of CaMKII inhibition (i.e., decreased EC50), whereas multimerization of CN19o did not improve the potency of CaMKII inhibition. Multimerization of CN19o resulted in decreased CaMKII inhibition (i.e., increased EC50). The sequences of these peptides are shown in Table 1. [Figure 6]Figure 6 is a schematic diagram showing administration of AAV vectors to RYR2R176Q / WT and RYR2R4560I / WT mice. The top of Figure 6 provides a schematic diagram showing the polynucleotide components of the AAV vector. In Figure 6, ITR stands for "inverted terminal repeat", CASQ2 stands for calsequestrin 2 enhancer, cTnT stands for "cardiac troponin" promoter, INT stands for hemoglobin enhancer, AIPx5 stands for multimer containing five autocamtide 2-associated inhibitory peptide (AIP) units, mScarlet stands for 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. [Figure 7A] Figures 7A and 7B are plots showing the effect of AIPx5 administration on atrial fibrillation in a mouse model. LKB Flox / Flox mice were double-injected with AAV-NPPA-Cre and AAV-AIPx5-mcherry (see Figure 6). Figure 7A is a plot showing results from an experiment in which mice positive for LKB1 flanked by LoxP sites (LKB1 flox / flox) were injected on postnatal day 3 (P3) with either AAV9-NPPA-RFP, AAV9-NPPA-Cre, or a double injection of AAV9-NPPA-Cre+AAV9-cTnT-AIPx5. At 2-week intervals, 3-minute ECG recordings were performed under anesthesia. The time difference between the two QRS complexes (RR interval) was plotted as a function of the next interval (RR+1). Figure 7B provides a plot of the standard deviation of the RR interval for each minute of recording, calculated for each animal at the indicated time points. [Figure 7B] See legend to Figure 7A. [Figure 8] FIG. 8 shows a schematic diagram of the treatment and testing plan for Example 5 disclosed herein. [Figure 9]Figure 9 shows plots and graphs showing beat-to-beat variability in control mice compared to LKB1 knockout mice. (A) shows that control mice (floxed LKB1 mice in the absence of Cre) had very little beat-to-beat variability. (B) shows that LKB1 knockout mice (floxed LKB1 mice in the presence of Cre) had significantly increased beat-to-beat variability compared to control mice. [Figure 10] FIG. 10 is a plot showing that AIPx5 was associated with reduced beat-to-beat variability in LKB1 knockout mice. [Figure 11] FIG. 11 is a plot showing that AIPx5 was associated with reduced beat-to-beat variability in Tbx5 knockout mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0088] Detailed Description of the Invention The disclosed invention generally relates to a method for producing a Ca 2+ The present invention features multimeric polypeptides that inhibit calmodulin-dependent kinase II (CaMKII) (e.g., AIP multimeric polypeptides), polynucleotides encoding the CaMKII inhibitory multimeric polypeptides, and methods of using same to treat a cardiac disease or disorder (e.g., catecholaminergic polymorphic ventricular tachycardia (CPVT) or atrial fibrillation (AF)).

[0089] As reported in detail below, the invention of the present disclosure is based at least in part on the discovery that AIP multimeric polypeptides exhibit increased CaMKII inhibition and increased arrhythmia suppression compared to non-multimerized AIP. CaMKII activation and subsequent inhibition of downstream signaling significantly alleviated catecholamine-stimulated potential arrhythmias associated with mutations in calcium ryanodine channel RYR2. Thus, the invention of the present disclosure provides compositions comprising AIP multimers or polynucleotides encoding same, and methods of use thereof in treating cardiac diseases and disorders (e.g., CPVT or AF). In some embodiments, AIP multimeric polypeptides are delivered to subjects using expression vectors (e.g., AAV vectors).

[0090] Catecholamine-induced polymorphic ventricular tachycardia (CPVT) Catecholamine-induced polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmia caused primarily by autosomal dominant mutations in the gene encoding the cardiac ryanodine receptor (RYR2), the major intracellular calcium release channel in cardiomyocytes. Patients with CPVT are usually asymptomatic at rest, but develop potentially fatal ventricular tachycardias during exercise or emotional distress. In wild-type cardiomyocytes, cardiac action potentials are initiated by voltage-sensitive L-type Ca2+ receptors located in the cell membrane. 2+ Opening the channel results in localized Ca 2+ Ca influx from the sarcoplasmic reticulum via RYR2 2+ The resulting cytoplasmic Ca 2+ An increase in Ca leads to sarcomere contraction. As the cell enters diastole, RYR2 closes and cytoplasmic Ca 2+ Sarcoplasmic reticulum Ca 2+ In cells with CPVT-associated mutations, RYR2 is released into the cytoplasm, resulting in diastolic Ca 2+This increases the level of catecholamines in the plasma membrane, promoting the exchange of sodium and calcium across the plasma membrane via the sodium-calcium exchanger 1 (NCX1), resulting in after-depolarization that can trigger further action potentials. The molecular mechanism by which catecholamine stimulation unmasks the arrhythmic nature of CPVT mutations is unclear. The mechanism by which RYR2 mutations result in the clinical phenotype of ventricular tachycardia is also uncertain.

[0091] CPVT mutations inhibit the uptake of diastolic Ca by RYR2 from the sarcoplasmic reticulum to the cytoplasm. 2+ In individual cardiomyocytes, it increases diastolic Ca release. 2+ Elevated sodium-calcium exchange induces reverse sodium-calcium exchange via NCX1 in the plasma membrane, resulting in afterdepolarization that can potentially trigger further action potentials. Although the molecular mechanism by which catechol stimulation unmasks the arrhythmic nature of CPVT mutants is unclear, catechol-induced Ca 2+ -Activation of calmodulin-dependent protein kinase II (CaMKII) is thought to be involved. The mechanism by which RYR2 mutations result in the clinical phenotype of ventricular tachycardia is also uncertain, but one theory is that activation induced by cardiomyocytes produces ventricular tachycardia.

[0092] CPVT is associated with recurrent atrial or ventricular arrhythmias that occur during exercise or emotional distress. The average age of onset is approximately 10 years, and 30% of patients present with cardiac arrest. Approximately 60% to 70% of patients with CPVT have mutations in RYR2. CPVT is commonly treated with medications, left cardiac sympathetic denervation (LCSD), or an implantable cardioverter defibrillator (ICD) if episodes of atrial or ventricular arrhythmias recur.

[0093] The advent of induced pluripotent stem cell (iPSC) technology and efficient methods to differentiate iPSCs into cardiomyocytes (iPSC-CMs) has created a great opportunity to study inherited arrhythmias. iPSC-CMs have been generated from patients with CPVT as well as other inherited arrhythmias and have been shown to capture key features of these diseases, including abnormal action potential duration and drug response.

[0094] Atrial fibrillation (AF) Atrial fibrillation (AF or A-fib) is an abnormal heart rhythm (arrhythmia) characterized by rapid, irregular beating of the atrial chambers of the heart. It often begins as a short period of abnormal beats that become longer or more continuous over time. It may also begin as other forms of arrhythmia, such as atrial flutter, and then change into AF. Episodes may be asymptomatic. Symptomatic episodes may include palpitations, fainting, lightheadedness, 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.

[0095] Atrial fibrillation is the most common severe abnormal heart rhythm, affecting more than 33 million people worldwide as of 2020. As of 2014, about 2–3% of the population in Europe and North America were affected by it. Around 2005, this increased from 0.4% to 1% of the population. In developing countries, about 0.6% of men and 0.4% of women are affected. The proportion of people with AF increases with age, from 0.1% under 50 years old, to 4% of those aged 60–70 years, to 14% of those aged 80 years or older. Deaths due to A-fib and atrial flutter increased from 29,000 in 1990 to 193,300 in 2015.

[0096] Atrial fibrillation can be diagnosed using electrocardiograms, blood tests, Holter monitors, event recorders, echocardiograms, stress tests, chest x-rays, combinations of these, etc. In general, treatments for AF include drug therapy (e.g., beta-blockers, calcium channel blockers, digoxin, antiarrhythmics, and / or blood thinners), defibrillation therapy (e.g., electrical or pharmacological cardioversion), or surgery or catheterization procedures (e.g., atrioventricular (AV) node ablation, or maze surgery).

[0097] Multimeric Polypeptides A CaMKII inhibitory multimeric polypeptide (eg, a multimeric polypeptide containing AIP repeats) significantly reduces CaMKII activity (eg, CaMKII kinase activity, such as phosphorylation of RYR2 by CaMKII).

[0098] In some embodiments, the multimeric polypeptide of the present invention comprises 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 comprising 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 some embodiments, the repeats are consecutive and adjacent to each other. In some cases, one or more repeats are separated by a linker, which can be a stretch of amino acid residues that is about, or at least about, and / or only about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues in length.

[0099] In some embodiments, the CaMKII inhibitory 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 inhibitory multimeric polypeptide (e.g., a multimeric polypeptide comprising AIP repeats) renders CaMKII activity undetectable.

[0100] In some embodiments, the CaMKII inhibitory multimeric polypeptide (e.g., a multimeric polypeptide comprising AIP repeats) has a lower EC50 for CaMKII inhibition than that of a non-multimeric CaMKII inhibitory polypeptide. In some embodiments, the CaMKII inhibitory multimeric polypeptide (e.g., a multimeric polypeptide comprising AIP repeats) has a lower EC50 for CaMKII inhibition than that of a non-multimeric CaMKII inhibitory polypeptide. 50 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 that of the reference peptide. In some examples, the CaMKII inhibitory multimeric polypeptide (e.g., a multimeric polypeptide comprising AIP repeats) has an EC 50 is less than 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 1%, or 0.1% of that of a non-multimeric CaMKII inhibitory polypeptide (e.g., AIP). In some cases, the EC50 is measured using a bioluminescent homogeneous ADP monitoring assay for kinases (ADP-GLO™); this assay is described in Zegzouti, et al. “ADP-Glo: A Bioluminescent and Homogeneous ADP Monitoring Assay for Kinases,” ASSAY and Drug Development Technologies, Dec. 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.

[0101] In some embodiments, a CaMKII inhibitory multimeric polypeptide (e.g., a multimeric polypeptide comprising AIP) is altered or modified to enhance its ability to regulate cardiac rhythm or not inhibit its ability. In one embodiment, the present invention provides a method for optimizing an amino acid sequence or a nucleic acid sequence by generating modifications. Such changes may include specific mutations, deletions, insertions, post-translational modifications, and tandem duplications. In one preferred embodiment, the amino acid sequence of a CaMKII inhibitory multimeric polypeptide (e.g., a multimeric polypeptide comprising AIP repeats) is altered or modified to enhance protease resistance, particularly metalloprotease resistance. Thus, the present invention further includes analogs of the naturally occurring polypeptides of the present invention. Analogs may differ from the naturally occurring polypeptides of the present invention by differences in amino acid sequence, post-translational modifications, or both. Analogs of the present invention will generally exhibit at least 85%, more preferably 90%, most preferably 95%, or even 99% identity with all or a portion of a naturally occurring amino acid sequence of the present invention. The length of the sequence comparison is at least 10, 13, 15 amino acid residues. Again, an exemplary approach to determining the degree of identity can use the BLAST program, e.g. -3 and e -100A probability score between indicates a closely related sequence. Modifications include in vivo and in vitro chemical derivatization of the polypeptide, e.g., acetylation, carboxylation, phosphorylation, or glycosylation; such modifications may occur during synthesis or processing of the polypeptide or after treatment with an isolated modifying enzyme. Analogs may also differ from naturally occurring polypeptides of the invention by changes in primary sequence. These include genetic variants, both naturally occurring and induced (e.g., variants resulting from random mutagenesis by exposure to radiation or ethanemethylsulfate or from site-directed mutagenesis; see Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual (2d ed.), CSH Press, 1989, or Ausubel et al., supra). Also included are cyclized peptides, molecules, and analogs containing residues other than L-amino acids, e.g., D-amino acids or non-naturally occurring or synthetic amino acids (e.g., β- or γ-amino acids).

[0102] In addition to full-length polypeptides, the present invention also includes fragments of any one of the polypeptides of the present invention. As used herein, the term "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 can be generated by methods known to those skilled in the art, or can result from normal protein processing (e.g., removal of amino acids that are not required for biological activity from a nascent polypeptide, or removal of amino acids by alternative mRNA splicing or alternative protein processing events).

[0103] Analogs of CaMKII inhibitory multimeric polypeptides (e.g., multimeric polypeptides containing AIP) may exceed the physiological activity of native CaMKII inhibitory polypeptides (e.g., non-multimeric AIP). Methods for analog design are well known in the art, and analog synthesis may be performed according to such methods by modifying the chemical structure such that the resulting analog exhibits the activity of the unmodified CaMKII inhibitory multimeric polypeptide. Such chemical modifications include, but are not limited to, substituting alternative R groups and changing the degree of saturation at specific carbon atoms of the CaMKII inhibitory multimeric polypeptide. Preferably, analogs of CaMKII inhibitory multimeric polypeptides are relatively resistant to degradation in vivo, resulting in a more prolonged therapeutic effect after administration. Assays for measuring functional activity include, but are not limited to, those described in the Examples below.

[0104] Polynucleotide Therapy Polynucleotide therapy featuring polynucleotides encoding CaMKII inhibitory multimeric polypeptides (e.g., multimers of AIP), analogs, variants, or fragments thereof is another therapeutic approach for treating cardiac arrhythmias (e.g., CPVT or AF). Expression of such proteins in cardiac cells is expected to regulate the function of cardiac cells, tissues, or organs, for example, by inhibiting phosphorylation of RYR2, suppressing CaMKII activity, and / or otherwise controlling cardiac rhythm. Such nucleic acid molecules can be delivered to cells (e.g., cardiac cells) of a subject with cardiac arrhythmias. The nucleic acid molecules are generally delivered to cells of a subject in a form that allows them to be incorporated and produce therapeutically effective levels of CaMKII inhibitory multimeric polypeptides (e.g., AIPx3, AIPx5) or fragments thereof.

[0105] Transducing viral (e.g., retroviral, adenoviral, and adeno-associated viral (AAV)) vectors can be used in somatic cell gene therapy, particularly due to 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, a variant or fragment thereof) can be cloned into a viral vector and expression can be driven from a promoter (e.g., a promoter specific to the target cell type of interest). In one embodiment, a viral vector (e.g., an AAV vector) is used to administer a polynucleotide encoding an AIP multimeric polypeptide (e.g., AIPx3 or AIPx5) to cardiac tissue.

[0106] Viral vectors for transduction have tissue tropism that allows selective transduction of certain cell types over other cell types. For example, inhibition of CaMKII in cardiomyocytes may be effective in treating CPVT, AF, or other types of cardiac disease, but its inhibition in other tissues, such as the brain, may be undesirable. In some embodiments, vectors are used that target cardiomyocytes with high specificity over other cell types. This would allow specific cardiac targeting of expression of CaMKII inhibitory peptide molecules, since inhibition of CaMKII in cells other than the heart may be harmful. Potential adeno-associated virus candidates are AAV9, AAV6, AAV2i8, AAVrh74, AAVrh10, MyoAAV, Anc80, and Anc82, among others. The transduction efficiency of adeno-associated viruses is improved when their genomes are "self-complementary." In some embodiments, self-complementary adeno-associated viruses are used to enhance transduction of the heart with gene therapy vectors.

[0107] Other viral vectors which can be used include, for example, vaccinia virus, bovine papilloma virus, or herpes viruses such as Epstein-Barr virus (see, e.g., 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 are used in clinical settings (Rosenberg et al., N. Engl. J. Med 323:370, 1990; Anderson et al., U.S. Patent No. 5,399,346).

[0108] Non-viral approaches can also be employed to deliver therapeutic agents to cardiac cells in patients in need of CaMKII inhibition. For example, nucleic acid molecules can be introduced into the retina by 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), by administration of the nucleic acid in the presence of asialoorosomucoid-polylysine conjugates (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, The nucleic acid can be introduced into cells by the methods described above (Kubota et al., 1990). Preferably, the nucleic acid is administered in combination with liposomes and protamine.

[0109] Gene transfer can also be achieved using non-viral means, including in vitro transfection. Such methods include the use of calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes or lipid nanoparticles can also potentially be beneficial in delivering DNA to cells. Transplantation of normal genes into diseased tissues of patients can also be achieved by ex vivo transfer of normal nucleic acid into culturable cell types (e.g., autologous or heterologous primary cells or their progeny), and then injecting these cells (or their progeny) into target tissues.

[0110] The dosage of the vectors disclosed herein depends on many factors, including the size and health of each individual patient. For a particular subject, the specific dosage regimen should be adjusted over time according to the individual's needs and the professional judgment of the person administering or supervising the administration of the composition. In some embodiments, the dosage is about 1×10 10 Vector units / kg to approximately 1 x 10 14 In some embodiments, the dosage is between about 1×10 vector units / kg. 10 Vector units / kg, approximately 5 × 10 10 Vector units / kg, approximately 1 x 10 11 Vector units / kg, approximately 5 × 10 11 Vector units / kg, approximately 1 x 10 12 Vector units / kg, approximately 5 × 10 12 Vector units / kg, approximately 1 x 10 13 Vector units / kg, approximately 5 × 10 13 Vector units / kg, or approximately 1 × 10 14 In an exemplary embodiment, the vector is a viral vector and the dosage is about 1×10 10 Viral genomes / kg to approximately 1 × 10 14 Between about 100 and 150 viral genomes / kg. In some embodiments, the dosage is based on the efficiency of transfection, transformation, or transduction. In some embodiments, the dosage is effective to transfect, transform, or transduce at least about 20% of the subject's cardiomyocytes, at least about 25% of the subject's cardiomyocytes, at least about 30% of the subject's cardiomyocytes, at least about 35% of the subject's cardiomyocytes, or at least about 40% of the subject's cardiomyocytes. In some embodiments, the dosage is effective to transfect, transform, or transduce about 20% to about 40% of the subject's cardiomyocytes.

[0111] 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 driven from any suitable promoter (e.g., human cytomegalovirus (CMV), Simian Virus 40 (SV40), CMV-chicken β-actin hybrid promoter ("CAG"), or metallothionein promoter) and regulated by any suitable mammalian regulatory element. In the case of treating CPVT or AF, it is desirable to selectively express the CaMKII inhibitory multimeric polypeptide (e.g., a multimer of AIP) in cardiomyocytes to minimize expression in other cell types. In some embodiments, a cardiomyocyte-selective promoter is used for expression of the CaMKII inhibitory multimeric polypeptide (e.g., a multimer of AIP). Promoters or enhancers used include, but are not limited to, those characterized as tissue-specific or cell-specific enhancers. For example, the cardiac troponin T promoter, the alpha-myosin heavy chain (alpha-MHC) promoter, the myosin light chain-2v (MLC-2v) promoter, or the cardiac NCX1 promoter can be used to induce expression in cardiomyocytes. Alternatively, when using genomic clones as therapeutic constructs, regulation can be mediated by cognate regulatory sequences or, optionally, by regulatory sequences from heterologous sources, including any of the promoters or regulatory elements listed above.

[0112] Another therapeutic approach encompassed by the disclosed invention includes administering recombinant CaMKII inhibitory multimeric polypeptides (e.g., multimers of AIP), such as recombinant AIP multimeric polypeptides (e.g., AIPx3 or AIPx5), variants or fragments thereof, directly to potential or actual affected tissue sites or systemically (e.g., by conventional recombinant protein administration techniques). The amount of peptide administered will depend on many factors, including the size and health of the individual patient. For a particular subject, specific dosing regimens should be adjusted over time according to the individual's needs and the professional judgment of the person administering or supervising the administration of the compositions.

[0113] Stabilized mRNA Stabilized mRNAs encoding CaMKII inhibitory multimeric polypeptides (e.g., mRNAs stabilized by increased or improved half-life, increased or improved potency, increased or improved resistance to endonuclease activity, decreased or reduced susceptibility to endonuclease activity) are useful in some embodiments for preventing or ameliorating CPVT, AF, or another cardiac arrhythmia. Such stabilized mRNAs can be delivered to cells (e.g., cardiac cells) of a subject having a cardiac arrhythmia (e.g., CPVT or AF).

[0114] Expression in cardiac cells of CaMKII inhibitory multimeric polypeptides encoded by the stabilized mRNAs described herein is expected to regulate the function of cardiac cells, tissues, or organs, for example, by inhibiting phosphorylation of RYR2, suppressing CaMKII activity, and / or otherwise controlling cardiac rhythm. In some embodiments, the stabilized mRNA encodes the multimeric AIP polypeptides disclosed herein.

[0115] Treatment method CaMKII inhibitory multimeric polypeptides (e.g., multimers of AIP) and polynucleotides or vectors (e.g., AAV vectors) encoding same are useful for preventing or ameliorating CPVT, AF, or another cardiac arrhythmia. Diseases and disorders characterized by cardiac arrhythmias can be treated using the methods and compositions of the invention (e.g., atrial fibrillation (AF), catecholaminergic polymorphic ventricular tachycardia (CPVT), ischemic heart disease, cardiac arrhythmias, heart failure (e.g., heart failure associated with aortic attachment), hypertensive heart disease and pulmonary hypertensive heart disease, myocardial infarction, valvular disease, congenital heart disease, myocardial hypertrophy, ventricular arrhythmias, or Timothy's syndrome).

[0116] In one treatment approach, the identified active substance as described herein is administered to the potential or actual affected tissue site or administered systemically.The amount of the active substance administered depends on many factors, including the size and health of each patient.For a particular subject, the specific administration schedule needs to be adjusted over time according to the individual's needs and the professional judgment of the person who manages or supervises the administration of the composition.

[0117] Delivery or introduction of the polypeptides, polynucleotides, or vectors of the present invention can also be achieved by the use of muscle targeting agents (e.g., agents that specifically target myocardium). Exemplary muscle targeting agents, preferably myocardium targeting agents, are described, for example, in U.S. Patent No. 11,168,141, issued August 2, 2019, the disclosure of which is incorporated herein by reference in its entirety. In some embodiments, muscle targeting agents (e.g., agents such as antibodies that target cardiac markers, such as Caveolin-3) can be attached to the polypeptides, polynucleotides, or vectors disclosed herein to target such polypeptides, polynucleotides, or vectors to the myocardium.

[0118] Pharmaceutical Compositions The present disclosure provides pharmaceutical compositions containing CaMKII inhibitory multimeric polypeptides (e.g., AIP multimers) or polynucleotides and / or vectors encoding same. The administration of the pharmaceutical compositions of the present disclosure for treating cardiac arrhythmias can be performed by any suitable means that results in a concentration of CaMKII inhibitory multimeric polypeptides (e.g., AIP multimers) effective to improve, reduce, or stabilize cardiac arrhythmias. Diseases and disorders characterized by cardiac arrhythmias (e.g., atrial fibrillation (AF), catecholamine-induced polymorphic ventricular tachycardia (CPVT), ischemic heart disease, cardiac arrhythmias, heart failure (e.g., heart failure associated with aortic attachment), hypertensive heart disease and pulmonary hypertensive heart disease, myocardial infarction, valvular disease, congenital heart disease, myocardial hypertrophy, ventricular arrhythmias, or Timothy's syndrome) can be treated using the pharmaceutical compositions of the present invention. The compositions can be contained in any suitable amount in any suitable carrier substance. The composition can be provided in a dosage form suitable for parenteral (e.g., subcutaneous, intravenous, intramuscular, or intraperitoneal) administration. Pharmaceutical compositions can be formulated according to conventional pharmaceutical practice (see, for example, Remington: The Science and Practice of Pharmacy (20th ed.), ed. AR Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and JC Boylan, 1988-1999, Marcel Dekker, New York).

[0119] In therapeutic use, the polypeptides, polynucleotides, and / or vectors disclosed herein may be administered systemically, for example, formulated in a pharma- ceutically acceptable buffer, such as saline. Preferred routes of administration include, for example, subcutaneous, intravenous, intraperitoneal, intramuscular, and intradermal injections, which provide a continuous and sustained drug level to the patient. For AAV gene therapy, administration may be local or systemic, for example, intravenous or intracoronary. Treatment of human patients or other animals is carried out with a therapeutically effective amount of the therapeutic agents identified herein in a physiologically acceptable carrier. Suitable carriers and their formulations are described, for example, in Remington's Pharmaceutical Sciences by EW Martin. The amount of therapeutic agent administered will vary depending on the method of administration, the age and weight of the patient, and the clinical symptoms of cardiac arrhythmia. Generally, the amount will be within the range of other agents used to treat other diseases requiring modulation of cardiac function, although in certain cases, lower amounts may be required due to the increased specificity of the compounds. A composition comprising a CaMKII inhibitory multimeric polypeptide (e.g., an AIP multimer) is administered at a dose that has CaMKII inhibitory activity or cardiac rhythm regulating activity, which dose is determined by methods known to those of skill in the art or using any assay that measures the expression or biological activity of a CaMKII polypeptide.

[0120] The pharmaceutical compositions according to the present invention can be formulated to release the active compound substantially immediately after administration or at a predetermined time or period after administration. The latter type of composition is generally known as a controlled release formulation, and includes the following formulations: (i) formulations that provide substantially constant drug concentration in the body for an extended period of time; (ii) formulations that provide substantially constant drug concentration in the body for an extended period of time after a predetermined lag time; (iii) formulations that sustain the action for a predetermined period of time by maintaining a relatively constant effective level in the body, while minimizing the undesirable side effects associated with fluctuations in the plasma level of the active substance (sawtooth kinetic pattern); (iv) formulations that localize the action, such as by spatially placing the controlled release composition adjacent to or in contact with the thymus; (v) formulations that allow for convenient dosing, such as once every week or two weeks; and (vi) formulations that target cardiac arrhythmias by using carriers or chemical derivatives to deliver the therapeutic agent to a specific cell type (e.g., cardiac cells). In some applications, controlled release formulations avoid the need for frequent daytime dosing to maintain plasma levels at therapeutic levels.

[0121] Any of many strategies can be pursued to obtain controlled release, such that the release rate exceeds the metabolic rate of the compound in question.In one example, controlled release can be obtained by appropriately selecting various formulation parameters and ingredients, including, for example, various types of controlled release compositions and coatings.The therapeutic agent is thus formulated into a pharmaceutical composition using suitable excipients, which releases the therapeutic agent in a controlled manner after administration.Examples include single-unit or multiple-unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, molecular complexes, nanoparticles, patches, and liposomes.

[0122] Parenteral Compositions Pharmaceutical compositions comprising the CaMKII inhibitory multimeric polypeptides (e.g., multimers of AIP), the polynucleotides encoding them, or vectors (e.g., AAV vectors comprising polynucleotides encoding multimeric polypeptides) of the present disclosure can be administered parenterally by injection, infusion (infusion) or implantation (subcutaneous, intravenous, intramuscular, intraperitoneal, intracoronary, etc.) via dosage forms, formulations, or suitable delivery devices or implants containing conventional non-toxic pharmaceutically acceptable carriers and adjuvants. In some embodiments, the pharmaceutical composition contains a viral vector (e.g., AAV vector) comprising 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 formulation. Formulation and formulation can be found in Remington: The Science and Practice of Pharmacy, supra.

[0123] Compositions for parenteral use may be provided in unit dosage form (e.g., single-dose ampoules) or in vials containing multiple doses, to which suitable preservatives may be added (see below). The compositions may be in the form of a solution, suspension, emulsion, injection (infusion) device, or delivery device for implantation, or may be presented as a dry powder to be reconstituted with water or other suitable vehicle before use. Apart from the active substance that reduces or improves cardiac arrhythmia, the composition may contain suitable parenterally acceptable carriers and / or excipients. The active therapeutic agent(s) may be incorporated into microspheres, microcapsules, nanoparticles, liposomes, etc. for controlled release. In addition, the composition may contain suspending agents, solubilizing agents, stabilizing agents, pH adjusting agents, tonicity agents, and / or dispersing agents.

[0124] As mentioned above, the pharmaceutical compositions according to the present invention may be in a form suitable for sterile injection. To prepare such compositions, the appropriate therapeutic agent(s) are dissolved or suspended in a parenterally acceptable liquid vehicle. Acceptable vehicles and solvents that can be used include water, water adjusted to an appropriate pH by the addition of an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer's solution, isotonic sodium chloride solution and glucose solution. Aqueous formulations may contain one or more preservatives (e.g., methyl, ethyl or n-propyl p-hydroxybenzoate). In cases where one of the compounds is poorly or sparingly soluble in water, a solubility enhancer or solubilizer may be added, or the solvent may contain 10-60% w / w propylene glycol, etc.

[0125] Controlled Release Parenteral Compositions 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 agent may be incorporated into biocompatible carriers, liposomes, nanoparticles, implants, or injection (infusion) devices.

[0126] Materials used in the preparation of microspheres and / or microcapsules are, for example, biodegradable / bioerodible polymers, such as polygalactin, poly(isobutylcyanoacrylate), poly(2-hydroxyethyl-L-glutamine), and poly(lactic acid). Biocompatible carriers that can be used in 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., poly(caprolactone), poly(lactic acid), poly(glycolic acid), poly(orthoesters), or combinations thereof).

[0127] Oral solid dosage forms Oral formulations include tablets containing the active ingredient(s) in a mixture with non-toxic pharma- ceutically acceptable excipients. Such formulations are known to those skilled in the art. The excipients may be, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starches including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate); granulating and disintegrating agents (e.g., cellulose derivatives including microcrystalline cellulose, starches including potato starch, croscarmellose sodium, alginates, or alginic acid); binders (e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol); lubricants, flow agents, and antiadherents (e.g., magnesium stearate, zinc stearate, stearic acid, silica, hydrogenated vegetable oils, or talc). Other pharma- ceutically acceptable excipients can be colorants, flavoring agents, plasticizers, humectants, buffers, and the like.

[0128] The tablets may be uncoated or may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained action over a longer period of time. The coating may be adapted to release the active agent in a predetermined pattern (e.g., to achieve a controlled release formulation) or not release the active agent until after passing through the stomach (enteric coating). The coating may be a sugar coating, a film coating (e.g., based on hydroxypropylmethylcellulose, methylcellulose, methylhydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose, acrylate copolymers, polyethylene glycol and / or polyvinylpyrrolidone), or an enteric coating (e.g., based on methacrylic acid copolymers, cellulose acetate phthalate, hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate succinate, polyvinyl acetate phthalate, shellac, and / or ethylcellulose). In addition, a time delay material may be used, such as, for example, glyceryl monostearate or glyceryl distearate.

[0129] The solid tablet composition may include a coating adapted to protect the composition from undesired chemical changes (e.g., chemical degradation prior to release of the cardioactive therapeutic substance). Coatings can be applied to solid dosage forms in a manner similar to that described in the Encyclopedia of Pharmaceutical Technology, supra.

[0130] In one embodiment, two or more cardiac therapeutics may be mixed together in a tablet or may be compartmentalized. In one example, a first active cardiac therapeutic is contained on the inside of the tablet and a second active therapeutic is contained on the outside; such that the majority of the second therapeutic is released prior to the release of the first active therapeutic.

[0131] Oral formulations can 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 oil medium (e.g., peanut oil, liquid paraffin, or olive oil). Powders and granules can be prepared with the ingredients mentioned above for tablets and capsules in a conventional manner, for example using a mixer, a fluidized bed apparatus, or a spray drying apparatus.

[0132] Controlled-release oral dosage forms Oral controlled release compositions can be constructed to release, for example, the AIP multimers or CaM-KNtide multimers, vectors, and / or polynucleotides of the present disclosure by controlling their dissolution and / or diffusion. Dissolution or diffusion controlled release can be achieved by suitable coating of tablets, capsules, pellets, or granules of the compound or by incorporating the compound into a suitable matrix. The controlled release coating can include one or more of the coating materials described above and / or, for example, shellac, beeswax, glycowax, castor wax, carnauba wax, stearyl alcohol, glyceryl monostearate, glyceryl distearate, glycerol palmitostearate, ethylcellulose, acrylic resins, dl-polylactic acid, cellulose acetate butyrate, polyvinyl chloride, polyvinyl acetate, vinylpyrrolidone, polyethylene, polymethacrylate, methyl methacrylate, 2-hydroxymethacrylate, methacrylate hydrogel, 1,3 butylene glycol, ethylene glycol methacrylate, and / or polyethylene glycol. Matrix materials in controlled release matrix formulations can also include, for example, hydrated methylcellulose, carnauba wax and stearyl alcohol, carbopol 934, silicone, glyceryl tristearate, methyl acrylate-methyl methacrylate, polyvinyl chloride, polyethylene, and / or halogenated fluorocarbons.

[0133] The controlled release composition containing one or more therapeutic compounds may be in the form of a buoyant tablet or capsule (i.e., a tablet or capsule that, when administered orally, floats on top of the stomach contents for a period of time). A buoyant tablet of the compound(s) may be prepared by granulating a mixture of the compound(s) with excipients and 20-75% w / w of a hydrocolloid (e.g., hydroxyethylcellulose, hydroxypropylcellulose, or hydroxypropylmethylcellulose). The resulting granules can then be compressed into a tablet. Upon contact with gastric fluid, the tablet forms a substantially water-impermeable gel barrier around its surface. This gel barrier helps maintain a density of less than 1, thereby allowing the tablet to remain floating in the gastric fluid.

[0134] Combination therapy Optionally, the CaMKII inhibitory multimeric polypeptides described herein (e.g., AIP multimers) can be administered in combination with other standard therapies useful for regulating cardiac function; such methods are known to those of skill in the art and are described in Remington's Pharmaceutical Sciences by EW Martin.

[0135] kit Also provided are kits for preventing or treating a cardiac arrhythmia, cardiac condition, or pathology in a subject (e.g., atrial fibrillation (AF), catecholaminergic polymorphic ventricular tachycardia (CPVT), ischemic heart disease, cardiac arrhythmia, heart failure (e.g., heart failure associated with aortic attachment), hypertensive heart disease and pulmonary hypertensive heart disease, myocardial infarction, valvular disease, congenital heart disease, myocardial hypertrophy, ventricular arrhythmia, or Timothy's syndrome). In one aspect, the kit provides a therapeutic or prophylactic composition containing an effective amount of a CaMKII inhibitory multimeric polypeptide (e.g., a multimer of AIP), a polynucleotide encoding the polypeptide, and / or a vector comprising the polynucleotide; the kit is used in administering the multimeric polypeptide, polynucleotide, or vector to a subject.

[0136] In another embodiment, the kit provides a therapeutic or prophylactic composition containing an effective amount of a CaMKII inhibitory multimeric polypeptide (e.g., a multimer of AIP), polynucleotide, and / or vector encoding same.

[0137] In some embodiments, the kit comprises a sterile container that contains the therapeutic or prophylactic composition; such container may be a box, an ampoule, a bottle, a vial, a tube, a bag, a pouch, a blister pack, or other suitable container form known in the art. These containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medications.

[0138] In some embodiments, the kit includes instructions for administering the composition to a subject having or at risk of developing a disease (e.g., CPVT or AF). The instructions will generally include information about using the composition to treat the disease. In other embodiments, the instructions include at least one of the following: description of the therapeutic agent; dosing schedule and administration for treating or preventing a cardiac disease (e.g., CPVT or AF) or symptoms 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 present), or may be provided as a label affixed to the container, as information stored on a remotely accessible server, or as a separate sheet, pamphlet, card, or folder provided in or with the container.

[0139] The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the knowledge of those skilled in the art. Such techniques are fully explained in such references 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 the polynucleotides and polypeptides of the invention and therefore may be considered in making and practicing the invention. Techniques that are particularly useful in certain embodiments will be described in the following sections.

[0140] The following examples are intended to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assays, screening and treatment methods of the present invention, and are not intended to limit the scope of what the inventors regard as their invention. EXAMPLES

[0141] Example 1: Multimerization improved the inhibitory potency of CaMKII inhibition by autocamtide 2-related inhibitory peptide (AIP) in vivo, but not of alternative potent inhibitors of CaMKII. Experiments were undertaken to identify improved agents for the treatment of catecholamine-induced polymorphic ventricular tachycardia (CPVT). The peptide inhibitor autocamtide 2-related inhibitory peptide (AIP) is effective in treating CPVT (see Figures 1A-1C). To identify improved agents for the treatment of CPVT, a multimeric peptide containing five consecutive repeats of AIP (i.e., AIPx5) was designed and its efficacy in inhibiting the activity of the kinase CaMKII was evaluated (see Figures 2A-2G, 3, 4, and 5A-5E).

[0142] Development of improved agents for the treatment of CPVT. A) A polypeptide containing multiple repeats of AIP (FIG. 2C) or multiple repeats of the potent CaMKII inhibitor CN19o, or B) a polypeptide containing AIP or CN19o fused to FKBP12.6 (FIG. 2B), which binds to RYR2, a target of CaMKII (FIG. 1A), for the treatment of CPVT. The domain structures of the evaluated polypeptides are shown in FIG. 2D. CN19o is known to be a more potent CaMKII inhibitor than AIP (FIG. 2A). The efficacy of the polypeptides in the treatment of catecholamine-induced polymorphic ventricular tachycardia (CPVT) was demonstrated by the binding of RYR2, a target of CPVT, to the CPVT domain shown in FIG. 2D. R4650I / WT The peptides were evaluated in a mouse model. Among all peptides evaluated, the AIPx5 peptide was found to be the most effective in treating CPVT, as judged by the reduction in observed ectopy (Figures 2E and 2F). AIP-FKB12.6 was also effective in treating CPVT symptoms by reducing ectopy (Figures 2E and 2F). These polypeptides were administered to mice using AAV vectors, and delivery of polynucleotides expressing the polypeptides to cardiac tissue was confirmed using fluorescent imaging (Figure 2G).

[0143] To confirm inhibition of CaMKII activation in vivo, phosphorylation of phospholamban (PLB), a known target of CaMKII, at threonine-17 was assessed in cardiac tissue and whole heart lysates from mice exposed to isoproterenol (ISO) (Fig. 3). Exposure of cardiac cells to ISO induced phosphorylation of PLB by CaMKII (Fig. 3). Exposure of cells to AIP or AIPx5 was found to result in a statistically significant decrease in PLB phosphorylation in cells exposed to ISO.

[0144] Example 2: Multimerization improved the inhibitory potency of CaMKII inhibition by autocamtide 2-related inhibitory peptide (AIP) in vitro, but not of alternative potent inhibitors of CaMKII. Experiments were undertaken to assess the inhibition of CaMKIIδ in vitro by the polypeptides evaluated in Example 1, the amino acid sequences of which are shown in Table 1 below. Inhibition of CaMKIIδ activity by these polypeptides was assessed using a bioluminescent homogeneous ADP monitoring assay for kinases (ADP-GLO™); this assay is described in Zegzouti, et al. “ADP-Glo: A Bioluminescent and Homogeneous ADP Monitoring Assay for Kinases,” ASSAY and Drug Development Technologies, Dec. 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 (FIG. 4). Multimerization (i.e., repeats of the AIP peptide or the CN19oX5 peptide) was found to increase the potency of AIP (i.e., lower the EC50) but unexpectedly lower the potency of CN19oX5 (i.e., raise the EC50) (Figures 5B-5E). The EC50 of AIP was 6149 nM, which was reduced to 543 nM for AIPx3 multimers and further reduced to 114 nM for AIPx5 multimers. However, the EC50 value of 1.6 nM for CN19o was increased to 2.1 nM for CN19oX3 multimers. Thus, multimerization increased the potency of AIP but unexpectedly reduced the potency of CN19oX3.

[0145] Table 1 Peptide sequences TIFF2025517333000018.tif97165

[0146] Example 3: Administration of AIPx5 to an animal model We prepared AAV vectors expressing AIPx5 or only mScarlet, a negative control (see Figure 6), and transduced them into the RYR2 mouse model of catecholamine-induced polymorphic ventricular tachycardia (CPVT). R176Q / QT Mice and RYR2 R4560I / WTThe AIPx5 vector is administered to mice. The efficacy of these vectors in reducing symptoms of CPVT is evaluated in the animal model. Administration of the AIPx5 vector results in expression of AIPx5 in cardiac tissue of the mice and reduction of symptoms of CPVT (e.g., reduction of ectopic excitation and / or ventricular tachycardia).

[0147] Example 4: AAV9-AIPx5 suppressed arrhythmias in an animal model of atrial fibrillation (AF). We undertook experiments to evaluate the efficacy of AIPx5 administration in the treatment of atrial fibrillation. LKB Flox / Flox mice were double-injected with NPPA-Cre and AIPx5-mcherry using AAV vectors. This mouse was a model of atrial fibrillation. In this mouse model, NPPA-Cre inactivates LKB in the atrium to create a model of atrial fibrillation (AF). We evaluated whether co-injection with AAV-AIPx5 alleviates AF. Beat-to-beat variability in HR, an index of AF frequency, was analyzed by plotting each RR interval (R) versus the RR interval of the next beat (R1) (n = approximately 500 heartbeats) (Figure 7A). The RR interval is the elapsed time between two successive R waves of the QRS signal on the electrocardiogram and is the reciprocal of the heart rate. Beat-to-beat variability in HR was analyzed at 2 weeks, 4 weeks, and 6 weeks. The RR interval showed little beat-to-beat variability at week 2, high variability at week 4, and then returned to a low variability state at week 6 (Figures 7A and 7B). Thus, AIPx5 was effective in reducing AF symptoms.

[0148] Example 5: MyoAAV-AIPx5 suppressed arrhythmias in two different animal models of atrial fibrillation. We tested the efficacy of AIPx5 in treating AF of different origins using two different atrial fibrillation (AF) mouse models: liver kinase B1 (LKB1) knockout mice and T-box transcription factor 5 (Tbx5) knockout mice.

[0149] 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 additional AMPK-related downstream kinases. Together, LKB1 and AMPK are involved in cell growth and metabolism and the cellular response to energy stress. Tbx5 is part of a group of genes that specify body parts. Tbx5 is involved in the development of the heart, forelimbs, and other structures in the body. It plays a key role in regulating the formation of the heart and its conduction system. Knockout of either of these genes in mice induces AF.

[0150] To test the efficacy of AIPx5, we used LKB1-floxed or Tbx5-floxed mice. We then conditionally knocked out LKB1 or Tbx5 from the atria of the mice using AAV-NPPA-Cre. NPPA is an atrium-specific promoter, ensuring that knockout of LKB1 or Tbx5 by Cre recombinase is restricted to the atrium. AAV-NPPA-FP (fluorescent protein) was used as a control virus. AAV-NPPA-FP does not carry Cre, and therefore does not knock out the gene of interest. Uninjected mice were also included as a control to consider the effects of AAV injection. We tested both AAV-cTnT-AIPx5 and AAV-cTnT-CN19o as potential treatments for AF.

[0151] A schematic of the treatment and testing regimen is shown in Figure 8. Briefly, LKB1 floxed or Tbx5 floxed mice were injected (uninjected controls were not injected) at P3 and EKG measurements were taken of the mice every 2 weeks thereafter starting at P14. Mice were sacrificed at 12 weeks and sacrificed mice were tested for protein expression.

[0152] EKG measurements of the mice were used to calculate whether the mice actually exhibited AF. AF was calculated using the standard deviation of the difference in beat-to-beat timing. High beat-to-beat variability during sedation indicated atrial misfiring. As shown in Figure 9, high beat-to-beat variability was found to be absent in control mice, but was present in LKB1 knockout mice.

[0153] Figure 10 shows that AIPx5 suppressed beat-to-beat variability in LKB1 knockout mice and was therefore effective in suppressing the symptoms of AF. When AIPx5 was present in LKB1 knockout mice, these mice did not show a statistically significant difference in beat-to-beat variability compared to control (non-LKB1 knockout) mice. Surprisingly, CN19o, when administered to LKB1 knockout mice, did not suppress beat-to-beat variability and actually performed worse in measuring beat-to-beat variability than LKB1 knockout mice alone.

[0154] Figure 11 shows that AIPx5 also suppressed beat-to-beat variability in Tbx5 knockout mice and was therefore effective in suppressing AF symptoms. When AIPx5 was present in Tbx5 knockout mice, these mice did not show statistically significant differences in beat-to-beat variability from the fourth week onwards.

[0155] Statistical significance of the tests shown in Figures 10 and 11 was performed using a 2-way Anova with post-hoc Dunnett's multiple comparisons against the control (FP) group.

[0156] Other Aspects From the foregoing description, it will be apparent that variations and modifications can be made to the invention described herein to adapt it to various usages and conditions, such variations also falling within the scope of the following claims.

[0157] The recitation of a list of elements in a definition of a variable herein includes definitions of that variable as a single element or as a combination (or subcombination) of the listed elements. The recitation of an embodiment herein includes that embodiment as a single embodiment or in combination with other embodiments or portions thereof.

[0158] 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 multimer polypeptide according to claim 1, comprising approximately 2 to approximately 20 repeats of the AIP peptide.

3. The polymer polypeptide according to claim 2, wherein the repeats of the AIP are continuous and / or separated by a linker.

4. AIPx3 YKKALHRQEAVDALYKKALHRQEAVDALYKKALHRQEAVDAL (SEQ ID NO: 3); or AIPx5 YKKALHRQEAVDALYKKALHRQEAVDALYKKALHRQEAVDALYKKALHRQEAVDALYKKALHRQEAVDAL (SEQ ID NO: 4) The polymer polypeptide according to claim 1, comprising a sequence having at least 85% amino acid sequence identity with respect to the given sequence.

5. The multimer polypeptide according to claim 1, which is fused to a 12.6 kDa FK506-binding protein (FKBP12.6) polypeptide.

6. The multimer polypeptide according to claim 1, wherein the EC50 of CaMKII inhibition by the multimer polypeptide is less than 10% of the EC50 of AIP.

7. An expression vector comprising a polynucleotide encoding a polymer polypeptide according to any one of claims 1 to 6.

8. The expression vector according to claim 7, wherein a polynucleotide encoding the multimer polypeptide is functionally linked to a promoter suitable for driving the expression of the multimer polypeptide in mammalian cardiac cells.

9. A pharmaceutical composition comprising an effective amount of the polymer polypeptide described in any one of claims 1 to 6.

10. A pharmaceutical composition comprising an effective amount of the expression vector described in claim 7.

11. A cell comprising the expression vector according to claim 7.

12. A pharmaceutical composition for use in the regulation of a target cardiac arrhythmia, comprising a polymerized polypeptide according to any one of claims 1 to 6 or a polynucleotide encoding the polymerized polypeptide.

13. A pharmaceutical composition for use in inhibiting the phosphorylation of intracellular ryanodine channel (RYR2) polypeptides, comprising a polymerized polypeptide according to any one of claims 1 to 6 or a polynucleotide encoding the polymerized polypeptide.

14. A pharmaceutical composition for use in the treatment of a subject including a mutation associated with cardiac arrhythmia, comprising a polymerized polypeptide according to any one of claims 1 to 6 or a polynucleotide encoding the polymerized polypeptide.

15. The pharmaceutical composition according to claim 14, wherein the mutation is located in the cardiac ryanodine channel (RYR2).

16. The aforementioned mutation is RYR2 R4651I The pharmaceutical composition according to claim 15.

17. A pharmaceutical composition for use in the treatment of a subject having a cardiac disease, condition, or disorder characterized by cardiac arrhythmia, the pharmaceutical composition comprising a polymerized AIP polypeptide or a polynucleotide encoding the polypeptide.

18. A pharmaceutical composition for use in the treatment of a subject having a cardiac disease, condition, or disorder characterized by cardiac arrhythmia, comprising an adeno-associated virus vector containing a polynucleotide encoding a multimeric AIP polypeptide.

19. A pharmaceutical composition for use in the treatment of a subject having a cardiac disease, condition, or disorder characterized by cardiac arrhythmia, comprising an effective amount of an adeno-associated virus vector comprising a polynucleotide encoding a multimer AIP polypeptide, wherein the effective amount is about 1 × 10 10 Viral genome / kg ~ approximately 1 × 10⁻⁶ 14 The pharmaceutical composition having a viral genome / kg.

20. A pharmaceutical composition for use in reducing cardiac variability in a person having atrial fibrillation, comprising a multimer AIP polypeptide or a polynucleotide encoding the multimer AIP polypeptide.

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