WO2018090036A1 - Method of protection for cardiac tissue - Google Patents

Abstract

The present invention is based on methods for preventing cardiac tissue damage following a myocardial infarction, protecting cardiomyocytes and/or restoring cardiomyocyte function, preventing or reversing age related loss of heart functionality, and treating arrhythmia by the administration of a cardiac tissue protective nucleic acid. The present invention also provides vectors and kits comprising a cardiac tissue protective nucleic acid sequence.

Description

METHOD OF PROTECTION FOR CARDIAC TISSUE

CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application claims benefit of priority under 35 U.S.C. §119(e) of U.S. Serial No. 62/421,918 filed November 14, 2016; U.S. Serial No. 62/510,659 filed May 24, 2017; and US 62/483,251 filed April 7, 2017, the entire contents of which is incorporated herein by reference in its entirety.

INCORPORATION OF SEQUENCE LISTING

[002] The material in the accompanying sequence listing is hereby incorporated by reference into this application. The accompanying sequence listing text file, name RENOVA1160_3WO_Sequence_Listing, was created on November 14, 2017, and is 59 kb. The file can be assessed using Microsoft Word on a computer that uses Windows OS.

FIELD OF THE INVENTION

[003] The present invention relates generally to the protection of cardiac cells and more specifically to the use of cardiac tissue protective nucleic acid sequences for the protection of cardiac cells prior to or following an event that results in damage or aging of such cells.

BACKGROUND INFORMATION

[004] It has been reported by the American Heart Association (1995 Statistical Supplement), that about 60 million adults in the United States suffer from cardiovascular disease. Cardiovascular diseases are responsible for almost a million deaths annually in the United States representing over 40% of all deaths. Cardiovascular disease (CVD) is a class of diseases that involve the heart or blood vessels including myocardial infarction (MI), ischemic heart disease (IHD), stroke, hypertensive heart disease, rheumatic heart disease (RHD), aortic aneurysms, cardiomyopathy, atrial fibrillation, congenital heart disease, endocarditis, and peripheral artery disease (PAD), among others.

[005] Overall, 60-70% of all heart failure cases are secondary to acute myocardial infarction

(MI). In patients with MI, the treatment of choice for reducing acute myocardial ischemic injury and limiting MI size is timely and effective myocardial reperfusion using either thombolytic therapy or primary percutaneous coronary intervention (PPCI). However, the process of reperfusion can itself induce cardiomyocyte death, known as myocardial reperfusion injury, for which there is still no effective therapy. There is a need for new strategies for the prevention and treatment of heart damage caused by Mis.

[006] One such strategy is the use of gene therapy to deliver cardiac tissue protective nucleic acid sequences to the heart. Enhanced adenylate cyclase 6 (AC6) expression has been shown to improve myocardial performance in models of pre-existing heart failure. AC6 has also been shown to protect against the development of HF, be effective in heart failure associated with aging and in pressure-overloaded animal models of heart failure. It has been shown that AC6 over-expression can also improve the function of all surviving cardiac myocytes leading to the expectation that the maximum improvement cardiac function in a patient will be limited only by the magnitude of infarction at the commencement of treatment. Therefore, early gene therapy with nucleic acid sequences encoding cardiac tissue protective proteins is advocated in the prevention and treatment of cardiovascular disease or cardiac cell damage.

SUMMARY OF THE INVENTION

[007] The present invention is based on methods for preventing cardiac tissue damage following a myocardial infarction (MI); for protecting cardiomyocytes and/or restoring cardiomyocyte function; preventing or reversing age related loss of heart functionality, and treating or preventing arrhythmia by the administration of a cardiac tissue protective nucleic acid sequences to a subject in need thereof. The present invention also provides vectors and kits comprising a cardiac tissue protective nucleic acid for use in the methods of the invention.

[008] In one embodiment, the present invention provides a method of protecting cardiac tissue in a subject having or previously having had a myocardial infarction (MI) comprising administering a cardiac tissue protective nucleic acid sequence to a subject in need thereof after suffering from an MI, thereby protecting the cardiac tissue from damage caused by the MI. In one aspect, the subject is at risk of heart failure or arrhythmia following the MI. In a further aspect, the heart failure is systolic heart failure, diastolic heart failure, reduced ejection fraction heart failure or preserved ejection fraction heart failure. Preferably, the nucleic acid sequence is administered within about twenty four hours; about 2 days; about 3 days; about 4 days; about 5 days; about 6 days or about 7 days after the myocardial infarction.

[009] In another aspect, the nucleic acid sequence comprises a transgene and an operably linked promoter, preferably a heterologous promoter to the nucleic acid sequence. In an aspect, the nucleic acid sequence is in a vector. In one aspect, the vector is an adenovirus or an adeno-associated virus (AAV). In a further aspect, the vector is Ad5, AAV1, AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhlO, AAV1 1 and AAV12 or pseudotyped vectors comprising combinations thereof (e.g., AAV2/9 for cardiac tissue). In a one aspect, the vector is AAV2/6. In another aspect, the transgene is adenylyl cyclase 6 (AC6), urocortin or stresscopin. In a specific aspect, the urocortin transgene is urocortin 1, urocortin 2 or urocortin 3. [0010] In an additional aspect, following administration of the cardiac tissue protective nucleic acid sequence left ventricle (LV) contractility is increased. In one aspect, the nucleic acid sequence is a calcium channel regulator. In an aspect, the nucleic acid sequence reduces diastolic sarcoplasmic reticulum (SR) Ca2+ leakage. In another aspect, following administration of the cardiac tissue protective nucleic acid sequence diastolic function is improved, Ca2+ uptake is increased and LV dilation is decreased. In a further aspect, following administration of the cardiac tissue protective nucleic acid sequence sodium- calcium exchanger 1 (NCX1) and protein phosphatase 1 (PP1) expression is reduced.

[0011] In another aspect, the method further comprises the administration of a therapeutic agent. In certain aspects, the therapeutic agent is selected from the group consisting of: tissue plasminogen activator (tPA), tenecteplase (TNKase), alteplase (Activase), urokinase (abbokinase), reteplase (Retavase), streptokinase (Kabikinase, Streptase), anistreplase (Eminase), chlorothiazide (Diuril), chlorthalidone (Hygroton), indapamide (Lozol), hydrochlorothiazide (Hydrodiuril), methyclothiazide (Enduron), metolazone (Zaroxolyn, Diulo, Mykrox), bumetanide (Bumex), furosemide (Lasix), ethacrynate (Edecrin), torsemide (Demadex), Amiloride hydrochloride, spironolactone (Aldactone), triamterene (Dyrenium), Acetazol amide, Methazol amide, glycerin (Glycerol), Isosorbide, Mannitol and Urea. In an additional aspect, the therapeutic agent is administered prior to, simultaneously with or following administration of the nucleic acid sequence. In one aspect, the nucleic acid sequence is administered during angioplasty or stent insertion.

[0012] In an additional aspect, the nucleic acid sequence is administered after reperfusion. In a further aspect, the protein encoded by the nucleic acid is coated on a stent. In certain aspects, the nucleic acid sequence is administered within about twenty four hours; about 2 days; about 3 days; about 4 days; about 5 days; about 6 days or about 7 days after the myocardial infarction.

[0013] In an additional embodiment, the present invention provides a method of protecting cardiomyocytes and/or restoring cardiomyocyte function in a subject comprising administering a cardiac tissue protective nucleic acid sequence to a subject in need thereof. In one aspect, the cardiomyocytes are protected from hypertrophy and/ or apoptosis. In another aspect, following administration of the cardiac tissue protective nucleic acid sequence LV contractility is increased and Ca2+ uptake is improved. In certain aspects, the nucleic acid sequence is administered within about 0 to 24 hours; about 0 to 12 hours; about 0 to 8 hours; about 0 to 6 hours; about 0 to 4 hours; about 0 to 3 hours; about 0 to 2 hours; or about 0 to 1 hours after a myocardial infarction. In an additional aspect, the nucleic acid sequence is administered within about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days or about 7 days after a myocardial infarction.

[0014] In an additional embodiment, the present invention provides a method of preventing or reversing age related loss of heart functionality comprising administering a cardiac tissue protective nucleic acid sequence to a subject in need thereof. In one aspect, the subject has low ejection fraction. In an additional aspect, following administration of the cardiac tissue protective nucleic acid sequence LV contractility is increased and Ca2+ uptake is improved.

[0015] In another embodiment, the present invention provides a method of treating arrhythmia comprising administering a cardiac tissue protective nucleic acid sequence to a subject in need thereof. In one aspect, the nucleic acid sequence comprises a transgene and an operably linked promoter. In another aspect, the nucleic acid sequence is in a vector. In a further aspect, the vector is an adenovirus or an adeno-associated virus (AAV). In certain aspects, the vector is Ad5, AAV1, AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhlO, AAV11 or AAV12 or pseudotyped vectors comprising combinations thereof (e.g., AAV2/9 for cardiac tissue)In another aspect, the transgene is adenylyl cyclase 6 (AC6), urocortin or stresscopin. In a further aspect, the urocortin transgene is urocortin 1, urocortin 2 or urocortin 3. In an additional aspect, following administration of the cardiac tissue protective nucleic acid sequence LV contractility is increased. In an aspect, the nucleic acid sequence is a calcium channel regulator. In one aspect, the arrhythmia is not associated with a heart condition.

[0016] In one embodiment, the present invention provides a kit comprising a stent coated with a protein encoded by a cardiac specific nucleic acid sequence. In one aspect, the nucleic acid sequence comprises a transgene and an operably linked promoter. In another aspect, the nucleic acid sequence is in a vector. In an additional aspect, the vector is an adenovirus or an adeno-associated virus (AAV).

[0017] In one embodiment, the present invention provides for a method for the treatment of diabetes comprising administering a vector comprising a transgene to a subject in need thereof thereby treating diabetes. In one aspect, the vector is an adenovirus or an adeno- associated virus (AAV). In certain aspects the vector is Ad5, AAV1, AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or AAV12. In a specific aspect, the vector is AAV6. In another aspect, transgene is adenylyl cyclase 6 (AC6), urocortin or stresscopin. In a further aspect, the transgene is urocortin 1, urocortin 2 or urocortin 3. DETAILED DESCRIPTION OF THE INVENTION

[0018] The present invention is based on methods for preventing cardiac tissue damage following a myocardial infarction (MI); for protecting cardiomyocytes and/or restoring cardiomyocyte function; preventing or reversing age related loss of heart functionality, and treating or preventing arrhythmia by the administration of a cardiac tissue protective nucleic acid sequences to a subject in need thereof. The present invention also provides vectors and kits comprising a cardiac tissue protective nucleic acid for use in the methods of the invention.

[0019] Before the present compositions and methods are described, it is to be understood that this invention is not limited to particular compositions, methods, and experimental conditions described, as such compositions, methods, and conditions may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only in the appended claims.

[0020] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, references to "the method" includes one or more methods, and/or steps of the type described herein which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

[0021] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods and materials are now described.

[0022] Heart disease encompasses many disorders related to the heart, such as coronary heart disease, heart attack (i.e. myocardial infarction), heart failure, congenital heart disease and arrhythmias. Myocardial ischemia is a condition in which the heart muscle does not receive adequate levels of oxygen and nutrients, which is typically due to inadequate blood supply to the myocardium and can damage heart muscle, reducing its ability to pump efficiently.

Myocardial infarction (MI) occurs when cardiac ischemia lasts too long leading to often irreversible damage to the cells of the heart as well as scarring. A MI may lead to secondary heart problems, such as heart failure and/or arrhythmia. In patients with MI, the treatment of choice for reducing acute myocardial ischemic injury and limiting MI size is timely and effective myocardial reperfusion using either thombolytic therapy or primary percutaneous coronary intervention (PPCI). However, the process of reperfusion can itself induce cardiomyocyte death, known as myocardial reperfusion injury, for which there is still no effective therapy.

[0023] Heart failure (HF) is clinically defined as a condition in which the heart does not provide adequate blood flow to the body to meet metabolic demands. Types of heart failure include, but are not limited to, systolic heart failure, diastolic heart failure, reduced ejection fraction heart failure or preserved ejection fraction heart failure. Typically 60-70% of all heart failure cases are secondary to acute myocardial infarction. Patients with severe heart failure suffer a high mortality; typically 50% of the patients die within two years of developing the condition. In some cases, heart failure is associated with severe coronary artery disease ("CAD"), typically resulting in myocardial infarction and either progressive chronic heart failure or an acute low output state. Heart failure is treated with therapeutic agents such as ACE inhibitors and beta blockers, and surgical procedures such as bypass surgery, left ventricular assist device and heart valve surgery.

[0024] Cardiac arrhythmia is a group of conditions in which the heartbeat is irregular, i.e. tachycardia or bradycardia. Many arrhythmias have no symptoms. When symptoms are present these may include palpitations or feeling a pause between heartbeats. More seriously there may be lightheadedness, passing out, shortness of breath, or chest pain. Most arrhythmias are not serious, however, some predispose a person to complications such as stroke or heart failure, while others may result in cardiac arrest. Treatments for arrhythmias include therapeutic agents such as blood thinners or implantable devices such as a pace maker.

[0025] Currently available options for treating heart disease are directed to restoring heart function but not to the prevention or treatment of the damage caused by heart disease. One such strategy is the use of gene therapy to deliver cardiac tissue protective nucleic acid sequences to the heart. A cardiac tissue protective nucleic acid sequence is a nucleic acid sequence which encodes for a protein that prevents cardiac tissue damage following a myocardial infarction, protects cardiomyocytes and/or restoring cardiomyocyte function, prevents or reverses age related loss of heart functionality and treats arrhythmia. Examples of cardiac tissue protective nucleic acid sequences include, but is not limited to, adenylate cyclase 6 (AC6), urocortin 1, urocortin 2, urocortin 3 and stresscopin.

[0026] Adenylate cyclase (AC) is a catalyst in the conversion of adeonsine triphosphate (ATP) to 3'5'-cylclic AMP (cAMP) which is critical for intracellular signal transduction. There are ten different AC proteins, including adenlyate cycles 6 (AC6). It has been shown that chronic over expression of AC6 leads to increased left ventricle (LV) function and increased cAMP levels. Enhanced AC6 expression has been shown to improve the intracellular signaling, including calcium handling, in isolated cardiac myocytes and protect cardiac myocytes against hypertrophy and apoptosis. Consequently, enhanced AC6 expression has been shown to improve myocardial performance in models of pre-existing heart failure. AC6 has also been shown to protect against the development of HF, be effective in heart failure associated with aging and in pressure-overloaded animal models of heart failure

[0027] Additionally, the existing treatment regimen for MI could be applied immediately so that blood flow is restored to offer the optimal benefit of AC6 gene therapy to those heart cells that have survived the primary MI and those that may be protected during the critical period following reperfusion. Further, AC6 would be a viable the treatment to be given post- clot busting drugs and routinely during heart related surgical procedures (i.e. during angioplasty, stent insertion, drug-eluting stent insertion). Improved calcium handling, of the type observed with enhanced AC6 over-expression, has also been shown in pre-clinical models to reduce arrhythmias.

[0028] AC6 therapy has also been shown to be effective in pre-clinical models of heart failure with preserved ejection fraction (HFpEF). LV contractility, as reflected in the end- systolic pressure volume relationship (Emax) was increased by activation of AC6 expression. In addition, diastolic function was improved and LV dilation reduced. LV samples from AC6-on mice exhibited a profile consistent with improved calcium handling, namely, reduced expression of sodium/calcium exchanger (NCXl), protein phosphatase 1 (PPl), and increased phosphorylation of phosphlamban (PLN) at position Ser 12. Sarcoplasmic reticulum (SR) Ca2+ content was also increased in isolated cardiac myocytes from AC6-on mice.

[0029] Urocortin 1 is a member of the sauvagine/corticotropin-releasing factor/urotensin I family. It is structurally related to the corticotropin-releasing factor (CRF) gene and the encoded product is an endogenous ligand for CRF type 2 receptors. Urocortin- 1 has been shown in animal studies to have effects on the pituitary-adrenal axis, the cardiovascular system, circulating neurohormones, and renal function and to suppress appetite. Urocortin 2 is an endogenous peptide in the corticotrophin-releasing factor (CRF) family.

Immunohistochemistry analysis of human myocytes has shown greater immunoreactivity of

Urocortin 2 in myocytes of the failing heart compared to those of the healthy heart.

Researchers suggest this is a result of an innate mechanism in which Ucn2 acts to improve function of the failing heart. The pathophysiology of heart failure is often a consequence of improper calcium handling and relaxation resulting in a lower cardiac output, decreased blood flow and overall decreased heart function. Infusion of Ucn2 in healthy humans has shown a dose dependent increase in cardiac output, heart rate and left ventricle ejection fraction and a decrease in systemic vascular resistance. Urocortin 3 is a 38 amino acid peptide that is a member of the CRF family of peptides and differs from a similar protein, stresscopin, by three amino acids (see Table 1). Unlike urocortin 1, and similar to urocortin 2, urocortin 3 is highly selective for the CRF2 receptor and does not show affinity for the CRF binding protein. Stresscopin is a protein very similar to urocortin 3 and differs by only 2 amino acids.

[0030] In one embodiment, the stresscopin-like peptide is SEQ ID NO:2 (h-SCP). In other embodiments, it comprises a modified h-SCP, wherein h-SCP has been modified by covalent attachment of a reactive group, by conservative amino acid substitution, deletion or addition, by pegylation or a combination of such modifications. For example, one modification includes cys-variant stresscopin-like peptides or nucleic acid sequences encoding such peptides. Tables 2-5 illustrate such peptides.

[0031] Table 1

[0032] As discussed above, it has been shown that AC6, urocortin 1, urocortin 2 and urocortin 3 over-expression can improve the function of all surviving cardiac myocytes leading to the expectation that the maximum improvement cardiac function in a patient will be limited only by the magnitude of the MI at the commencement of treatment. Therefore, early AC6, urocortin 1, urocortin 2 and urocortin 3 gene therapy is advocated in progressive heart disease regardless of its etiology, stage or type.

[0033] Recently it has been found that urocortins, specifically urocortin 1, increase insulin secretion and promote the division of insulin producing beta cells in the pancreas. Diabetes mellitus (DM), commonly referred to as diabetes, is a group of metabolic diseases in which there are high blood sugar levels over a prolonged period. Diabetes is due to either the pancreas not producing enough insulin or the cells of the body not responding properly to the insulin produced. There are three main types of diabetes mellitus: Type 1 DM results from the pancreas's failure to produce enough insulin; Type 2 DM begins with insulin resistance, a condition in which cells fail to respond to insulin properly. As the disease progresses a lack of insulin may also develop; and Gestational diabetes, is the third main form and occurs when pregnant women without a previous history of diabetes develop high blood-sugar levels. Prevention and current treatment involve a healthy diet, physical exercise, maintaining a normal body weight, and avoiding use of tobacco. Control of blood pressure and maintaining proper foot care are important for people with the disease. Type 1 DM must be managed with insulin injections. Type 2 DM may be treated with medications with or without insulin. Insulin and some oral medications can cause low blood sugar. Gestational diabetes usually resolves after the birth of the baby. Urorcortin 1 has been shown to increase beta cell mass and increase insulin production indicating that urocortin 1 gene therapy is a potential therapy for the treatment of diabetes type 1.

[0034] Additionally, it has recently been shown that urocortin 2 may be useful for treating diabetes. Delivery of an AAV 8 vector encoding urocortin 2 resulted in reduced plasma insulin, increased glucose disposal rates and increased insulin sensitivity in mouse models. Further, delivery of urocortin 2 in an AAV 8 vector in an insulin resistance model resulted in increased glucose disposal. It was shown that urocorin 2 gene transfer reduced fatty infiltration of the liver and increases GLu4 translocation to the plasma membrane in skeletal myotubes similar to insulin. Gene delivery of urocortin 2 results in insulin sensitization and this effect was found to las for several months following a single injection.

Nucleic Acids

[0035] As used herein, the terms "nucleic acids" or "nucleic acid sequences" refer to oligonucleotide, nucleotide, polynucleotide, or any fragment of any of these; and include DNA or RNA (e.g., mRNA, rRNA, tRNA, iRNA) of genomic or synthetic origin which may be single-stranded or double-stranded; and can be a sense or antisense strand, or a peptide nucleic acid (PNA), or any DNA-like or RNA-like material, natural or synthetic in origin, including, e.g., iRNA, ribonucleoproteins (e.g., e.g., double stranded iRNAs, e.g., iRNPs), nucleic acids, i.e., oligonucleotides, containing known analogues of natural nucleotides.

[0036] In some aspects, this invention includes nucleic acid sequences or any segment of DNA encoding AC6, urocortin 1, urocortin 2, urocortin 3 and/or stresscopin; it can include regions preceding and following the coding region (leader and trailer) as well as, where applicable, intervening sequences (introns) between individual coding segments (exons). The nucleic acid and amino acid sequences of urocortin 1, urocortin 2, urocortin 3 and/or stresscopin are well known in the art, for example the sequences are disclosed in U.S. Patent Nos. 6,838,274; 6,953,838; 8,481,686 and 7,829, 330.

[0037] The nucleic acids can be made, isolated and/or manipulated by, e.g., cloning and expression of cDNA libraries, amplification of message or genomic DNA by PCR, and the like. Nucleic acids, including DNA, RNA, iRNA, antisense nucleic acid, cDNA, genomic

DNA, vectors, viruses or hybrids thereof, can be isolated from a variety of sources, genetically engineered, amplified, and/or expressed/ generated recombinantly. Any recombinant expression system or gene therapy delivery vehicle can be used, including e.g., viral (e.g., AAV constructs or hybrids) bacterial, fungal, mammalian, yeast, insect or plant cell expression systems or expression vehicles.

[0038] As used herein, the terms "operatively linked" or "operatively associated" refer to the functional relationship of the nucleic acid sequences with regulatory sequences of nucleotides, such as promoters, enhancers, transcriptional and translational stop sites, and other signal sequences. For example, operative linkage of nucleic acid sequences, typically DNA, to a regulatory sequence or promoter region refers to the physical and functional relationship between the DNA and the regulatory sequence or promoter such that the transcription of such DNA is initiated from the regulatory sequence or promoter, by an RNA polymerase that specifically recognizes, binds and transcribes the DNA. In order to optimize expression and/or in vitro transcription, it may be necessary to modify the regulatory sequence for the expression of the nucleic acid or DNA in the cell type for which it is expressed. In some aspects the regulatory region or promoter is a heterologous sequence with respect to the nucleic acid sequence being regulated.

[0039] In certain aspects, "expression cassettes" comprising a cardiac tissue protective nucleic acid sequence is used, which can be capable of affecting expression of the nucleic acid, e.g., a structural gene or a transcript (e.g., encoding an AC6, urocortin 1, urocortin 2, urocortin 3 and/or stresscopin protein) in a host compatible with such sequences. Expression cassettes can include at least a promoter operably linked with the polypeptide coding sequence or inhibitory sequence; and, optimally, with other sequences, e.g., transcription termination signals. Additional factors necessary or helpful in effecting expression may also be used, e.g., enhancers.

[0040] As used herein, the term "regulatory sequences" and "regulatory elements" and refer to an element of a segment of nucleic acid that modulates the transcription of the nucleic acid sequence to which it is operatively linked, and thus act as transcriptional modulators. Regulatory sequences modulate the expression of gene and/or nucleic acid sequence to which they are operatively linked. Typical regulatory sequences include, but are not limited to, transcriptional promoters, an optional operate sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding sites, and sequences to control the termination of transcription and/or translation.

[0041] As used herein, the terms "promoter" or "promoter region" or "promoter element" refer to a segment of a nucleic acid sequence that controls the transcription of the nucleic acid sequence to which it is operatively linked. The promoter region includes specific sequences that are sufficient for RNA polymerase recognition, binding and transcription initiation. This portion of the promoter region is referred to as the promoter. In addition, the promoter region includes sequences which modulate this recognition, binding and transcription initiation activity of RNA polymerase. These sequences may be cis-acting or may be responsive to trans-acting factors. Promoters, depending upon the nature of the regulation may be constitutive or regulated.

[0042] The term "constitutively active promoter" refers to a promoter of a gene which is expressed at all times within a given cell. Exemplary promoters for use in mammalian cells include cytomegalovirus (CMV), and for use in prokaryotic cells include the bacteriophage T7 and T3 promoters, and the like.

[0043] As used herein, the term "vector" refers to a nucleic acid construct, designed for delivery to a host cell or transfer between different host cells or a liposome/micelle encapsulating nucleic acids for delivery to a host cell or transfer between different host cells. As used herein, a vector may be viral or non-viral vector. The vector can also be a plasmid. The vector may be an expression vector for the purpose of expressing the encoded protein in the transfected cell. A viral vector can be any viral vector known in the art including but not limited to those derived from adenovirus, adeno-associated virus (AAV), retrovirus, and lentivirus. Recombinant viruses provide a versatile system for gene expression studies, gene transfer and genome integration, and therapeutic applications. AAV vectors include, but are not limited to, Ad5, AAV1, AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhlO, AAV11 and AAV12 or combinations thereof to produce pseudotyped vectors.

[0044] Further, it is envisioned that an initial treatment or gene delivery may use one vector, e.g., adenovirus, and a subsequent treatment may use a different vector, e.g., AAV, with the same or different transgenes being delivered to the subject. While not wanting to be bound by a theory, it is believed that alternating the vector may reduce immunogenicity and provide a more effective treatment to the subject who requires multiple administrations of transgenes.

[0045] As sued herein, the terms "gene delivery" and "gene transfer" refer to the introduction of an exogenous polynucleotide (sometimes referred to as a "transgene") into a host cell, irrespective of the method used for the introduction. Such methods include a variety of well- known techniques such as vector-mediated gene transfer (by, e.g., viral infection/transfection, or various other protein-based or lipid-based gene delivery complexes) as well as techniques facilitating the delivery of "naked" polynucleotides. The introduced polynucleotide may be stable or transiently maintained in the host cell. Stable maintenance typically requires that the introduced polynucleotide either contains an origin of replication compatible with the host cell or integrates into a replicon of the host cell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome. A number of vectors are known to be capable of mediating transfer of genes to mammalian cells.

[0046] As used herein, the terms "gene" or "transgene" refer to a polynucleotide or portion of a polynucleotide comprising a sequence that encodes a protein. For most situations, it is desirable for the gene to also comprise a promoter operably linked to the coding sequence in order to effectively promote transcription. Enhancers, repressors and other regulatory sequences may also be included in order to modulate activity of the gene, as is well known in the art.

[0047] In certain embodiments, methods of the invention comprise use of nucleic acid (e.g., gene or polypeptide encoding nucleic acid) delivery systems to deliver a payload of an AC6, urocortin 1, urocortin 2, urocortin 3 and/or stresscopin encoding nucleic acid to a cell or cells in vitro, ex vivo, or in vivo, e.g., as gene therapy delivery vehicles.

[0048] Expression vehicle, vector, recombinant virus, or equivalents comprise: an adeno- associated virus (AAV), a lentiviral vector or an adenovirus vector; an AAV serotype Ad5, AAV1, AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhlO, AAV11 and AAV12 or combinations thereof to produce pseudotyped vectors. The AAV may be engineered to increase efficiency in targeting a specific cell type that is non-permissive to a wild type (wt) AAV and/or to improve efficacy in infecting only a cell type of interest. In alternative embodiments, the hybrid AAV is retargeted or engineered as a hybrid serotype by one or more modifications comprising: 1) a transcapsidation, 2) adsorption of a bi-specific antibody to a capsid surface, 3) engineering a mosaic capsid, and/or 4) engineering a chimeric capsid. It is well known in the art how to engineer an adeno-associated virus (AAV) capsid in order to increase efficiency in targeting specific cell types that are non-permissive to wild type (wt) viruses and to improve efficacy in infecting only the cell type of interest.

[0049] As used herein, the term "treatment" refers to any method of preventing, treating or ameliorating the damage caused by heart disease, i.e. myocardial infarction, arrhythmia and/or heart failure or the prevention of secondary heart problems (i.e. heart failure or arrhythmia) following a myocardial infarction or for the treatment of diabetes, specifically diabetes type 1. One such method is the administration of a therapeutic agent comprising a vector and a transgene, wherein the transgene is a cardiac tissue protective nucleic acid sequence (i.e. AC6, urocortin 1, urocortin 2, urocortin 3 and/or stresscopin). Treatment may also include the administration of additional therapeutic agents prior to, simultaneously with or following the administration of the cardiac tissue protective nucleic acid sequence. The additional therapeutic agents include, but are not limited to, tissue plasminogen activator (tPA), tenecteplase (TNKase), alteplase (Activase), urokinase (abbokinase), reteplase (Retavase), streptokinase (Kabikinase, Streptase), anistreplase (Eminase), chlorothiazide (Diuril), chlorthalidone (Hygroton), indapamide (Lozol), hydrochlorothiazide (Hydrodiuril), methyclothiazide (Enduron), metolazone (Zaroxolyn, Diulo, Mykrox), bumetanide (Bumex), furosemide (Lasix), ethacrynate (Edecrin), torsemide (Demadex), Amiloride hydrochloride, spironolactone (Aldactone), triamterene (Dyrenium), Acetazolamide, Methazol amide, glycerin (Glycerol), Isosorbide, Mannitol and Urea. Further, the administration of the cardiac tissue protective agent may occur prior to, simultaneously with or following surgical procedures such as during angioplasty, stent insertion and/or drug-eluting stent insertion.

[0050] The treatment of heart disease often requires rapid response to the onset of symptoms to prevent or treat the damage to the heart. In particular, the administration of a cardiac tissue protective nucleic acid sequence following reperfusion is performed within about twenty four hours; about 2 days; about 3 days; about 4 days; about 5 days; about 6 days or about 7 days after the myocardial infarction. Further, for the protection of cardiomyocytes and/or restoration cardiomyocyte function the cardiac tissue protective nucleic acid sequence is administered within about 0 to 24 hours; about 0 to 12 hours; about 0 to 8 hours; about 0 to 6 hours; about 0 to 4 hours; about 0 to 3 hours; about 0 to 2 hours; or about 0 to 1 hour after a myocardial infarction or within about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days or about 7 days after a myocardial infarction.

[0051] As used herein, the term "therapeutically effective amount" refers to an amount that is sufficient to effect a therapeutically significant reduction in heart failure, vascular dysfunction, endothelial dysfunction, diabetes, and hypertension symptoms as well as slow the progression of these ailments over time. The term also refers to that amount necessary to attain, at least partly, the desired effect, of reducing, ameliorating, stopping, abating, alleviating, and inhibiting the symptoms associated with heart failure, vascular dysfunction, endothelial dysfunction, diabetes, and hypertension, and also control and prevent further progression of the ailments. Such amounts will depend, of course, the severity of the condition and individual patient parameters including age, physical condition, size, weight and concurrent treatment. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation.

[0052] The cardiac tissue protective nucleic acid mat be administered by oral, subcutaneous, topical, rectal, nasal, intraarterial, intravenous, intramuscular, intracardiac, or transdermal routes.

[0053] Treatment with a cardiac tissue protective nucleic acid sequence in a subject having or previously having a MI might be evidenced by improvement in diastolic function, increased Ca2+ uptake, increased LV dilation, and reduced expression of sodium-calcium exchanger 1 (NXC1) and protein phosphatase 1 (PP1). Protection of cardiomyocytees and/or restoring cardiomyocyte by the administration of with a cardiac tissue protective nucleic acid sequence function might be evidenced by increase LV contractility and improved Ca2+ uptake. Preventing or reversing age related loss of heart functionality by the administration of with a cardiac tissue protective nucleic acid sequence might be evidenced by increased LV contractility and improved Ca2+ uptake. Treatment with a cardiac tissue protective nucleic acid sequence in a subject having an arrhythmia, associated or not associated with a heart condition might be evidenced by increased LV contractility.

[0054] In one embodiment, the present invention provides a method of protecting cardiac tissue in a subject having or previously having had a myocardial infarction (MI) comprising administering a cardiac tissue protective nucleic acid sequence to a subject in need thereof after suffering from an MI, thereby protecting the cardiac tissue from damage caused by the MI. In one aspect, the subject is at risk of heart failure or arrhythmia following the MI. In a further aspect, the heart failure is systolic heart failure, diastolic heart failure, reduced ejection fraction heart failure or preserved ejection fraction heart failure.

[0055] In another aspect, the nucleic acid sequence comprises a transgene and an operably linked promoter. In an aspect, the nucleic acid sequence is in a vector. In one aspect, the vector is an adenovirus or an adeno-associated virus (AAV). In a further aspect, the vector is Ad5, AAV1, AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12. In a specific aspect, the vector is AAV6. In another aspect, the transgene is adenylyl cyclase 6 (AC6), urocortin or stresscopin. In a specific aspect, the transgene is urocortin 1, urocortin 2 or urocortin 3.

[0056] In an additional aspect, following administration of the cardiac tissue protective nucleic acid sequence left ventricle (LV) contractility is increased. In one aspect, the nucleic acid sequence is a calcium channel regulator. In an aspect, the nucleic acid sequence reduces diastolic sarcoplasmic reticulum (SR) Ca2+ leakage. In another aspect, following administration of the cardiac tissue protective nucleic acid sequence diastolic function is improved, Ca2+ uptake is increased and LV dilation is decreased. In a further aspect, following administration of the cardiac tissue protective nucleic acid sequence sodium- calcium exchanger 1 (NCX1) and protein phosphatase 1 (PP1) expression is reduced.

[0057] In another aspect, the method further comprises the administration of a therapeutic agent. In certain aspects, the therapeutic agent is selected from the group consisting of: tissue plasminogen activator (tPA), tenecteplase (TNKase), alteplase (Activase), urokinase (abbokinase), reteplase (Retavase), streptokinase (Kabikinase, Streptase), anistreplase (Eminase), chlorothiazide (Diuril), chlorthalidone (Hygroton), indapamide (Lozol), hydrochlorothiazide (Hydrodiuril), methyclothiazide (Enduron), metolazone (Zaroxolyn, Diulo, Mykrox), bumetanide (Bumex), furosemide (Lasix), ethacrynate (Edecrin), torsemide (Demadex), Amiloride hydrochloride, spironolactone (Aldactone), triamterene (Dyrenium), Acetazol amide, Methazol amide, glycerin (Glycerol), Isosorbide, Mannitol and Urea. In an additional aspect, the therapeutic agent is administered prior to, simultaneously with or following administration of the nucleic acid sequence. In one aspect, the nucleic acid sequence is administered during angioplasty or stent insertion.

[0058] In an additional aspect, the nucleic acid sequence is administered after reperfusion. In a further aspect, the protein encoded by the nucleic acid is coated on a stent. In certain aspects, the nucleic acid sequence is administered within about twenty four hours; about 2 days; about 3 days; about 4 days; about 5 days; about 6 days or about 7 days after the myocardial infarction, the nucleic acid sequence is administered within about 0 to 24 hours; about 0 to 12 hours; about 0 to 8 hours; about 0 to 6 hours; about 0 to 4 hours; about 0 to 3 hours; about 0 to 2 hours; or about 0 to 1 hours after a myocardial infarction.

[0059] In an additional embodiment, the present invention provides a method of protecting cardiomyocytes and/or restoring cardiomyocyte function in a subject comprising administering a cardiac tissue protective nucleic acid sequence to a subject in need thereof. In one aspect, the cardiomyocytes are protected from hypertrophy and/ or apoptosis. In another aspect, following administration of the cardiac tissue protective nucleic acid sequence LV contractility is increased and Ca2+ uptake is improved. In certain aspects, the nucleic acid sequence is administered within about 0 to 24 hours; about 0 to 12 hours; about 0 to 8 hours; about 0 to 6 hours; about 0 to 4 hours; about 0 to 3 hours; about 0 to 2 hours; or about 0 to 1 hours after a myocardial infarction. In an additional aspect, the nucleic acid sequence is administered within about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days or about 7 days after a myocardial infarction.

[0060] In an additional embodiment, the present invention provides a method of preventing or reversing age related loss of heart functionality comprising administering a cardiac tissue protective nucleic acid sequence to a subject in need thereof. In one aspect, the subject has low ejection fraction. In an additional aspect, following administration of the cardiac tissue protective nucleic acid sequence LV contractility is increased and Ca2+ uptake is improved.

[0061] In another embodiment, the present invention provides a method of treating arrhythmia comprising administering a cardiac tissue protective nucleic acid sequence to a subject in need thereof. In one aspect, the nucleic acid sequence comprises a transgene and an operably linked promoter. In another aspect, the nucleic acid sequence is in a vector. In a further aspect, the vector is an adenovirus or an adeno-associated virus (AAV). In an additional aspect, following administration of the cardiac tissue protective nucleic acid sequence LV contractility is increased. In an aspect, the nucleic acid sequence is a calcium channel regulator. In one aspect, the arrhythmia is not associated with a heart condition.

[0062] In one embodiment, the present invention provides a kit comprising a stent coated with a protein encoded by a cardiac specific nucleic acid sequence. In one aspect, the nucleic acid sequence comprises a transgene and an operably linked promoter. In another aspect, the nucleic acid sequence is in a vector. In an additional aspect, the vector is an adenovirus or an adeno-associated virus (AAV).

[0063] In one embodiment, the present invention provides for a method for the treatment of diabetes comprising administering a vector comprising a transgene to a subject in need thereof thereby treating diabetes. In one aspect, the vector is an adenovirus or an adeno- associated virus (AAV). In certain aspects the vector is Ad5, AAV1, AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or AAV12. In a specific aspect, the vector is AAV6. In another aspect, transgene is adenylyl cyclase 6 (AC6), urocortin or stresscopin. In a further aspect, the transgene is urocortin 1, urocortin 2 or urocortin 3.

TABLE 2

Human stresscopin with amidated C-tenninus and

Cys-variant stresscopin-like peptides

TKFTL SLDVP TNIMN LLFNI AKAKN LRAQA AA AH LMAQI-NH₂ SEQ ID O.: 1

CKFTL SLDVP TNIMN LLFNI AKAKN LRAQA AA AH LMAQI-NH₂ SEQ ID O.: 2

TCFTL SLDVP TNIMN LLFNI AKAKN LRAQA AANAH LMAQI-NH₂ SEQ ID O.: 3

TKCTL SLDVP TNIMN LLFNI AKAKN LRAQA AANAH LMAQI-NH₂ SEQ ID O.: 4

TKFCL SLDVP TNIMN LLFNI AKAKN LRAQA AANAH LMAQI-NH₂ SEQ ID O.: 5

TKFTC SLDVP TNIMN LLFNI AKAKN LRAQA AANAH LMAQI-NH₂ SEQ ID O.: 6

TKFTL CLDVP TNIMN LLFNI AKAKN LRAQA AANAH LMAQI-NHj SEQ ID O.: 7

TKFTL SCDVP TNIMN LLFNI AKAKN LRAQA AANAH LMAQI-NHj SEQ ID O.: 8

TKFTL SLCVP TNIMN LLFNI AKAKN LRAQA AANAH LMAQI-NHj SEQ ID O.: 9

TKFTL SLDCP TNIMN LLFNI AKAKN LRAQA AANAH LMAQI-NHj SEQ ID O.: 10

TKFTL SLDVC TNIMN LLFNI AKAKN LRAQA AANAH LMAQI-NHj SEQ ID O.: 11

TKFTL SLDVP CNIMN LLFNI AKAKN LRAQA AANAH LMAQI-NHj SEQ ID O.: 12

TKFTL SLDVP TCIMN LLFNI AKAKN LRAQA AANAH LMAQI-NHj SEQ ID O.: 13

TKFTL SLDVP TNCMN LLFNI AKAKN LRAQA AANAH LMAQI-NHj SEQ ID O.: 14

TKFTL SLDVP TNICN LLFNI AKAKN LRAQA AANAH LMAQI-NHj SEQ ID O.: 15

TKFTL SLDVP TNIMC LLFNI AKAKN LRAQA AANAH LMAQI-NHj SEQ ID O.: 16

TKFTL SLDVP TNIMN CLFNI AKAKN LRAQA AANAH LMAQI-NHj SEQ ID O.: 17

TKFTL SLDVP TNIMN LCFNI AKAKN LRAQA AANAH LMAQI-NHj SEQ ID O.: 18

TKFTL SLDVP TNIMN LLCNI AKAKN LRAQA AANAH LMAQI-NHj SEQ ID O.: 19 TABLE 2

Human stresscopin with amidated C-terminus and

Cys-variant stresscopin-like peptides

TKFTL SLDVP TNIMN LLFCI AKAKN LRAQA AANAH LMAQI-NH₂ SEQIDNO.: 20

TKFTL SLDVP TNIMN LLFNC AKAKN LRAQA AANAH LMAQI-NH₂ SEQIDNO.: 21

TKFTL SLDVP TNIMN LLFNI CKAKN LRAQA AANAH LMAQI-NH₂ SEQIDNO.: 22

TKFTL SLDVP TNIMN LLFNI ACAKN LRAQA AANAH LMAQI-NH₂ SEQIDNO.: 23

TKFTL SLDVP TNIMN LLFNI AKCKN LRAQA AANAH LMAQI-NH₂ SEQIDNO.: 24

TKFTL SLDVP TNIMN LLFNI AKACN LRAQA AANAH LMAQI-NH₂ SEQIDNO.: 25

TKFTL SLDVP TNIMN LLFNI AKAKC LRAQA AANAH LMAQI-NH₂ SEQIDNO.: 26

TKFTL SLDVP TNIMN LLFNI AKAKN CRAQA AANAH LMAQI-NHj SEQIDNO.: 27

TKFTL SLDVP TNIMN LLFNI AKAKN LCAQA AANAH LMAQI-NHj SEQIDNO.: 28

TKFTL SLDVP TNIMN LLFNI AKAKN LRCQA AANAH LMAQI-NHj SEQIDNO.: 29

TKFTL SLDVP TNIMN LLFNI AKAKN LRACA AANAH LMAQI-NHj SEQIDNO.: 30

TKFTL SLDVP TNIMN LLFNI AKAKN LRAQC AANAH LMAQI-NHj SEQIDNO.: 31

TKFTL SLDVP TNIMN LLFNI AKAKN LRAQA CAN AH LMAQI-NHj SEQIDNO.: 32

TKFTL SLDVP TNIMN LLFNI AKAKN LRAQA ACNAH LMAQI-NHj SEQIDNO.: 33

TKFTL SLDVP TNIMN LLFNI AKAKN LRAQA AACAH LMAQI-NHj SEQIDNO.: 34

TKFTL SLDVP TNIMN LLFNI AKAKN LRAQA AANCH LMAQI-NHj SEQIDNO.: 35

TKFTL SLDVP TNIMN LLFNI AKAKN LRAQA AANAC LMAQI-NHj SEQIDNO.: 36

TKFTL SLDVP TNIMN LLFNI AKAKN LRAQA AANAH CMAQI-NHj SEQIDNO.: 37

TKFTL SLDVP TNIMN LLFNI AKAKN LRAQA AANAH LCAQI-NHj SEQIDNO.: 38

TKFTL SLDVP TNIMN LLFNI AKAKN LRAQA AANAH LMCQI-NHj SEQIDNO.: 39

TKFTL SLDVP TNIMN LLFNI AKAKN LRAQA AANAH LMACI-NHj SEQIDNO.: 40

TKFTL SLDVP TNIMN LLFNI AKAKN LRAQA AANAH LMAQC-NHj SEQIDNO.: 41

TABLE 3

Cys-variant of stresscopin peptide with

N-Ethylsuccinimide (NES) reactive group

TKFTL SLDVP TNIMN LLFNI SEQID

AKAKN LRAQA AANAH LMAQC (-NES)-NH;

NO.: 42

TKFTL SLDVP TNIMN LLFNI SEQID

AKAKN LRAQA AANAC (-NES) LMAQI-NH;

NO.: 43

TKFTL SDL VP TNIMN LLFNI SEQID

AKAKN LRAQA AAC (-NES) AH LMAQI-NH;

NO.: 44

TKFTL SLDVP TNIMN LLFNI SEQID

AKAKN LRAQA AC (-NES) NAH LMAQI-NH;

NO.: 45

TKFTL SLDVP TNIMN LLFNI SEQID

AKAKN LRAQA C (-NES) ANAH LMAQI-NH;

NO.: 46

TKFTL SLDVP TNIMN LLFNI SEQID

AKAKN LRC (-NES) QA AANAH LMAQI-NH;

NO.: 47

TKFTL SLDVP TNIMN LLFNI SEQID

AKAKN C (-NES) RAQA AANAH LMAQI-NH;

NO.: 48

TKFTL SLDVP TNIMN LLFNI SEQID

AKAKC (-NES) LRAQA AANAH LMAQI-NH;

NO.: 49 TKFTL SLDVP TNIMN LLr Nl SEQ ID

AKAC (-NES) N LRAQA AANAH LMAQI-NH;

NO.: 50 TKFTL SLDVP TNIMN LLFNC (-NES) SEQ ID

AKAKN LRAQA AANAH LMAQI-NH;

NO.: 5 !

TKFTL SLDVP TNIMN LLFC (-NES) I SEQ ID

AKAKN LRAQA AANAH LMAQI-NH;

NO.: 52

TKFTL SLDVP TNIMN LC (-NES) FNI SEQ ID

AKAKN LRAQA AANAH LMAQI-NH;

NO.: 53 TKFTL SLDVP TNIMN C (-NES) LFNI SEQ ID

AKAKN LRAQA AANAH LMAQI-NH;

NO.: 54

TABLE 4

Pegylated Cye-variant stresscopin-like peptides with N- Ethlysuccinimide (NES) linker and PEG weighing about 20 kDa

C (-NES-PEG) KFTL SLDVP TNIMN LLFNI AKAKN LRAQA AANAH LMAQI-NH2 SEQ ID NO. : 55

TC (-NES-PEG) FTL SLDVP TNIMN LLFNI AKAKN LRAQA AANAH LMAQI-NH2 SEQ ID NO. : 56

TKC (-NES-PEG) TL SLDVP TNIMN LLFNI AKAKN LRAQA AANAH LMAQI-NH2 SEQ ID NO. : 57

TKFC (-NES-PEG) L SLDVP TNIMN LLFNI AKAKN LRAQA AANAH LMAQI-NH2 SEQ ID NO. : 58

TKFTC (-NES-PEG) SLDVP TNIMN LLFNI AKAKN LRAQA AANAH LMAQI-NH2 SEQ ID NO. : 59

TKFTL C (-NES-PEG) LDVP TNIMN LLFNI AKAKN LRAQA AANAH LMAQI-NH2 SEQ ID NO. : 60

TKFTL SC (-NES-PEG) DVP TNIMN LLFNI AKAKN LRAQA AANAH LMAQI-NH2 SEQ ID NO. : 61

TKFTL SLC (-NES-PEG)VP TNIMN LLFNI AKAKN LRAQA AANAH LMAQI-NH2 SEQ ID NO. : 62

TKFTL SLDC (-NES-PEG) P TNIMN LLFNI AKAKN LRAQA AANAH LMAQI-NH2 SEQ ID NO. : 63

TKFTL SLDVC (-NES-PEG) TNIMN LLFNI AKAKN LRAQA AANAH LMAQI-NH2 SEQ ID NO. : 64

TKFTL SLDVP C (-NES-PEG) NIMN LLFNI AKAKN LRAQA AANAH LMAQI-NH2 SEQ ID NO. : 65

TKFTL SLDVP TC (-NES-PEG) IMN LLFNI AKAKN LRAQA AANAH LMAQI-NH2 SEQ ID NO. : 66

TKFTL SLDVP TNC (-NES- PEG) MN LLFNI AKAKN LRAQA AANAH LMAQI-NH2 SEQ ID NO. : 67

TKFTL SLDVP TNIC (-NES-PEG) N LLFNI AKAKN LRAQA AANAH LMAQI-NH2 SEQ ID NO. : 68

TKFTL SLDVP TNTMC (-NES-PEG) LLFNI AKAKN LRAQA AANAH LMAQI-NH2 SEQ ID NO. : 69

TKFTL SLDVP TNIMN C (-NES-PEG) LFNI AKAKN LRAQA AANAH LMAQI-NH2 SEQ ID NO. : 70

TKFTL SLDVP TNIMN LC (-NES-PEG) FNI AKAKN LRAQA AANAH LMAQI-NH2 SEQ ID NO. : 71

TKFTL SLDVP TNIMN LLC (-NES-PEG) NI AKAKN LRAQA AANAH LMAQI-NH2 SEQ ID NO. : 72

TKFTL SLDVP TNIMN LLFC (-NES-PEG) I AKAKN LRAQA AANAH LMAQI-NH2 SEQ ID NO. : 73

TKFTL SLDVP TNIMN LLFNC (-NES-PEG) AKAKN LRAQA AANAH LMAQI-NH2 SEQ ID NO. : 74

TKFTL SLDVP TNIMN LLFNI C (-NES-PEG) KAKN LRAQA AANAH LMAQI-NH2 SEQ ID NO. : 75

TKFTL SLDVP TNIMN LLFNI AC (-NES-PEG) AKN LRAQA AANAH LMAQI-NH2 SEQ ID NO. : 76

TKFTL SLDVP TNIMN LLFNI AKC (-NES-PEG) KN LRAQA AANAH LMAQI-NH2 SEQ ID NO. : 77 TABLE 4

Pegylated Cye-variant stresscopin-like peptides with N- Ethlysuccinimide (NES) linker and PEG weighing about 20 kDa

TKFTL SLDVP TNIMN LLFNI AKAC (-NES-PEG) N LRAQA AANAH LMAQI-NH₂ SEQ ID NO. : 78

TKFTL SLDVP TNIMN LLFNI AKAKC (-NES-PEG) LRAQA AANAH LMAQI-NH₂ SEQ ID NO. : 79

TKFTL SLDVP TNIMN LLFNI AKAKN C (-NES-PEG) RAQA AANAH LMAQI-NH₂ SEQ ID NO. : 80

TKFTL SLDVP TNIMN LLFNI AKAKN LC (-NES-PEG) AQA AANAH LMAQI-NH₂ SEQ ID NO. : 81

TKFTL SLDVP TNIMN LLFNI AKAKN LRC (-NES-PEG) QA AANAH LMAQI-NH₂ SEQ ID NO. : 82

TKFTL SLDVP TNIMN LLFNI AKAKN LRAC (-NES-PEG) A AANAH LMAQI-NH₂ SEQ ID NO. : 83

TKFTL SLDVP TNIMN LLFNI AKAKN LRAQC (-NES-PEG) AANAH LMAQI-NH₂ SEQ ID NO. : 84

TKFTL SLDVP TNIMN LLFNI AKAKN LRAQA C (-NES-PEG) ANAH LMAQI-NH₂ SEQ ID NO. : 85

TKFTL SLDVP TNIMN LLFNI AKAKN LRAQA AC (-NES-PEG) NAH LMAQI-NH₂ SEQ ID NO. : 86

TKFTL SLDVP TNIMN LLFNI AKAKN LRAQA AAC (-NES-PEG) AH LMAQI-NH₂ SEQ ID NO. : 87

TKFTL SLDVP TNIMN LLFNI AKAKN LRAQA AANC (-NES-PEG) H LMAQI-NH₂ SEQ ID NO. : 88

TKFTL SLDVP TNIMN LLFNI AKAKN LRAQA AANAC (-NES-PEG) LMAQI-NH₂ SEQ ID NO. : 89

TKFTL SLDVP TNIMN LLFNI AKAKN LRAQA AANAH C(-NES-PEG)MAQI-NH₂ SEQ ID NO. : 90

TKFTL SLDVP TNIMN LLFNI AKAKN LRAQA AANAH LC(-NES-PEG) AQI-NH₂ SEQ ID NO. : 91

TKFTL SLDVP TNIMN LLFNI AKAKN LRAQA AANAH LMC(-NES-PEG) QI-NH₂ SEQ ID NO. : 92

TKFTL SLDVP TNIMN LLFNI AKAKN LRAQA AANAH LMAC(-NES-PEG) I-NH₂ SEQ ID NO. : 93

TKFTL SLDVP TNIMN LLFNI AKAKN LRAQA AANAH LMAQC(-NES-PEG) -NH SEQ ID NO. : 94

Table 5 List of Stresscopin (STR) Analogues

All molecules are variations of the structure of C-terminal amidated human STR (1).

TKFTL SLDVP TNF N LLFNI AKAKN LRAQA AANAH LMAQI-NH2 (1)

NB: PEG-IA compounds are prepared from PEG-iodoacetamide reagent

H 0

MeO-PEG (20,000)-IA-A28C-STR (2)

MeO-PEG (30,000)-IA-A28C-STR (3)

MeO-PEG (5,000)-IA-A28C-STR (4)

Any other bioconjugates such as XTEN, Fc fragments to be considered L16A- MeO-PEG (20,000)-IA-A28C- STR

L16S- MeO-PEG (20,000)-IA-A28C- STR ¾) L16N- MeO-PEG (20,000)-IA-A28C- STR 7) L5A- MeO-PEG (20,000)-IA-A28C- STR ¾) L5S- MeO-PEG (20,000)-IA-A28C-STR

L5N- MeO-PEG (20,000)-IA-A28C-STR ;io)

MeO -PEG (20,000)-IA-K2N-A28C-STR

MeO -PEG (20,000)-IA-K24N-A28C- ■STR ;i2)

MeO -PEG (20,000)-IA-R27N-A28C- STR ;i3)

MeO -PEG (20,000)-IA-H35N-A28C- ■STR ;i )

MeO -PEG (20,000)-IA-N12K-A28C- ■STR ;i5)

MeO -PEG (20,000)-IA-N12R-A28C- STR ;i6)

MeO -PEG (20,000)-IA-N25K-A28C- ■STR ;i7)

MeO -PEG (20,000)-IA-N25R-A28C- STR ;i8)

2MeO-PEG (20,000)-IA-A21C-A28C- STR Λ9) 2MeO-PEG (20,000)-IA-A23C-A28C- STR [20) 2MeO-PEG (20,000)-IA-A28C-A31C- STR ;2i) 2MeO-PEG (20,000)-IA-A28C-A32C- STR [22) L3 A- MeO-PEG (20,000)-IA-A28C-STR [23) L3S- MeO-PEG (20,000)-IA-A28C-STR 24) L3N- MeO-PEG (20,000)-IA-A28C-STR [25) L20A- MeO-PEG (20,000)-IA-A28C-STR 26) L20S- MeO-PEG (20,000)-IA-A28C-STR 21) L20N- MeO-PEG (20,000)-IA-A28C-STR ;28) L26A- MeO-PEG (20,000)-IA-A28C-STR ;29) L26S- MeO-PEG (20,000)-IA-A28C-STR 30) L26N-MeO-PEG (20,000)-IA-A28C-STR ;3 i) [0064] Although the invention has been described with reference to the above example, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.

Claims

What is claimed is:

1. A method of protecting cardiac tissue in a subject having or previously having had a myocardial infarction (MI) comprising administering a cardiac tissue protective nucleic acid sequence to a subject in need thereof after suffering from an MI, thereby protecting the cardiac tissue from damage caused by the MI.

2. The method of claim 1, wherein the subject is at risk of heart failure or arrhythmia following the MI.

3. The method of claim 2, wherein the heart failure is systolic heart failure, diastolic heart failure, reduced ejection fraction heart failure or preserved ejection fraction heart failure.

4. The method of claim 1, wherein the nucleic acid sequence comprises a transgene and an operably linked promoter.

5. The method of claim 4, wherein the nucleic acid sequence is in a vector.

6. The method of claim 5, wherein the vector is an adenovirus or an adeno-associated virus (AAV).

7. The method of claim 6, wherein the vector is selected from the group consisting of: Ad5, AAV1, AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12.

8. The method of claim 7, wherein the vector is AAV6.

9. The method of claim 4, wherein the transgene is adenylyl cyclase 6 (AC6), urocortin or stresscopin.

10. The method of claim 9, wherein the transgene is urocortin 1, urocortin 2 or urocortin 3.

11. The method of claim 1, wherein following administration left ventricle (LV) contractility is increased.

12. The method of claim 1, wherein the nucleic acid sequence is a calcium channel regulator.

13. The method of claim 1, wherein the nucleic acid sequence reduces diastolic sarcoplasmic reticulum (SR) Ca2+ leakage.

14. The method of any of claims 1 - 3, wherein following administration diastolic function is improved, Ca2+ uptake is increased and LV dilation is decreased.

15. The method of any of claims 1 - 3, wherein following administration sodium-calcium exchanger 1 (NCX1) and protein phosphatase 1 (PP1) expression is reduced.

16. The method of claim 1 further comprising the administration of a therapeutic agent.

17. The method of claim 16, wherein the therapeutic agent is selected from the group consisting of: tissue plasminogen activator (tPA), tenecteplase (T Kase), alteplase

(Activase), urokinase (abbokinase), reteplase (Retavase), streptokinase (Kabikinase,

Streptase), anistreplase (Eminase), chlorothiazide (Diuril), chlorthalidone (Hygroton), indapamide (Lozol), hydrochlorothiazide (Hydrodiuril), methyclothiazide (Enduron), metolazone (Zaroxolyn, Diulo, Mykrox), bumetanide (Bumex), furosemide (Lasix), ethacrynate (Edecrin), torsemide (Demadex), Amiloride hydrochloride, spironolactone (Aldactone), triamterene (Dyrenium), Acetazolamide, Methazol amide, glycerin (Glycerol), Isosorbide, Mannitol and Urea.

18. The method of claim 16, wherein the therapeutic agent is administered prior to, simultaneously with or following administration of the nucleic acid sequence.

19. The method of claim 1, wherein the nucleic acid sequence is administered during angioplasty or stent insertion.

20. The method of claim 1, wherein the nucleic acid sequence is administered after reperfusion.

21. The method of claim 1, wherein the protein encoded by the nucleic acid is coated on a stent.

22. The method of claim 1, wherein the nucleic acid sequence is administered within about twenty four hours; about 2 days; about 3 days; about 4 days; about 5 days; about 6 days or about 7 days after the myocardial infarction.

23. A method of protecting cardiomyocytes and/or restoring cardiomyocyte function in a subject comprising administering a cardiac tissue protective nucleic acid sequence to a subject in need thereof.

24. The method of claim 23, wherein the cardiomyocytes are protected from hypertrophy and/ or apoptosis.

25. The method of claim 23, wherein following administration LV contractility is increased and Ca2+ uptake is improved.

26. The method of claim 23, wherein the nucleic acid sequence is administered within about 0 to 24 hours; about 0 to 12 hours; about 0 to 8 hours; about 0 to 6 hours; about 0 to 4 hours; about 0 to 3 hours; about 0 to 2 hours; or about 0 to 1 hours after a myocardial infarction.

27. The method of claim 23, wherein the nucleic acid sequence is administered within about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days or about 7 days after a myocardial infarction.

28. A method of preventing or reversing age related loss of heart functionality comprising administering a cardiac tissue protective nucleic acid sequence to a subject in need thereof.

29. The method of claim 28, wherein the subject has low ejection fraction.

30. The method of claim 28, wherein following administration LV contractility is increased and Ca2+ uptake is improved.

31. A method of treating arrhythmia comprising administering a cardiac tissue protective nucleic acid sequence to a subject in need thereof.

32. The method of claim 31, wherein the nucleic acid sequence comprises a transgene and an operably linked promoter.

33. The method of claim 32, wherein the nucleic acid sequence is in a vector.

34. The method of claim 33, wherein the vector is an adenovirus or an adeno-associated virus (AAV).

35. The method of claim 34, wherein the vector is selected from the group consisting of: Ad5, AAVl, AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12.

36. The method of claim 35, wherein the vector is AAV6.

37. The method of claim 32, wherein the transgene is adenylyl cyclase 6 (AC6), urocortin or stresscopin.

38. The method of claim 37, wherein the transgene is urocortin 1, urocortin 2 or urocortin 3.

39. The method of claim 31, wherein following administration LV contractility is increased.

40. The method of claim 31, wherein the nucleic acid sequence is a calcium channel regulator.

41. The method of claim 31, wherein the arrhythmia is not associated with a heart condition.

42. A kit comprising a stent coated with a protein encoded by a cardiac specific nucleic acid sequence.

43. The kit of claim 42, wherein the nucleic acid sequence comprises a transgene and an operably linked promoter.

44. The kit of claim 43, wherein the nucleic acid sequence is in a vector.

45. The method of claim 44, wherein the vector is an adenovirus or an adeno-associated virus (AAV).

46. The kit of claim 45, wherein the vector is selected from the group consisting of: Ad5, AAVl, AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12.

47. The kit of claim 46, wherein the vector is AAV6.

48. The kit of claim 42, wherein the transgene is adenylyl cyclase 6 (AC6), urocortin or stresscopin.

49. The kit of claim 48, wherein the transgene is urocortin 1, urocortin 2 or urocortin 3.

50. A method for the treatment of diabetes comprising administering a vector comprising a transgene to a subject in need thereof thereby treating diabetes.

51. The method of claim 50, wherein the vector is an adenovirus or an adeno-associated virus (AAV).

52. The method of claim 51, wherein the vector is selected from the group consisting of: Ad5, AAVl, AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12.

53. The method of claim 52, wherein the vector is AAV6.

54. The method of claim 50, wherein the transgene is adenylyl cyclase 6 (AC6), urocortin or stresscopin.

55. The method of claim 54, wherein the transgene is urocortin 1, urocortin 2 or urocortin 3.

56. The method of any of claims 9, 37 or 54, or the kit of claim 48 wherein the stresscopin is cysteine-modified and/or pegylated.

57. The method or kit of claim 56, wherein the stresscopin is selected from peptides of Tables 2-5.