WO2018090042A1 - Methods of treating heart failure - Google Patents

Abstract

The present invention is based on methods for treating heart failure comprising administering a cardiac tissue protective nucleic acid sequence and a stresscopin like peptide. The present invention also provides vectors and kits comprising a cardiac tissue protective nucleic acid and stresscopin like peptides for use in the methods of the invention.

Description

METHODS OF TREATING HEART FAILURE

CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application claims benefit of priority under 35 U.S.C. §119(e) of U.S. Serial No. 62/421,935 filed November 14, 2016; U.S. Serial No. 62/510,675 filed May 24, 2017; and US 62/483,263 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 RENOVA1180_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 treatment of heart failure and more specifically to the use of cardiac tissue protective nucleic acid sequences and stresscopin like peptides for the treatment of acute heart failure.

BACKGROUND INFORMATION

[004] Heart failure is a common cardiovascular condition and has reached epidemic proportions in the United States and Europe. The number of hospital admissions for acute heart failure is approaching 1 million each year in the United States alone. Currently, readmission rates and mortality have reached 30% to 40% within 60 days following discharge. In acute heart failure, worsening of hemodynamic function, in particular with very high left ventricular end-diastolic pressure is common.

[005] The current treatment for acute heart failure is multifactorial and often differs among patients. While diuretics, vasodilators, and positive inotropes remain the mainstay in the treatment of patients with acute heart failure, these treatments are associated with mortality and high readmission rates.

[006] Furthermore, existing inotropic therapies (eg, dobutamine) result in improved cardiac output, but with increased heart rate and increased myocardial oxygen consumption. These inotropic agents also carry with them a proarrhythmic potential in patients with heart failure. This cardiac liability is believed to be associated with the energy expense and calcium drive associated with these agents' direct positive inotropic actions. [007] In an effort to meet this growing unmet medical need, many new approaches have been studied with limited success in safely improving the hemodynamic status and outcome of patients with this syndrome. Described herein is a novel method of treatment for heart failure, specifically decompressed acute heart failure, comprising the administration of a cardiac protective nucleic acid and a stresscopin-like peptide.

SUMMARY OF THE INVENTION

[008] The present invention is based on methods for treating acute heart failure comprising administering a cardiac tissue protective nucleic acid sequence and a stresscopin like peptide. The present invention also provides vectors and kits comprising a cardiac tissue protective nucleic acid and stresscopin like peptides for use in the methods of the invention.

[009] In one embodiment, the present invention provides a method of treating a heart condition comprising administering a cardiac tissue protective nucleic acid sequence and a stresscopin-like peptide. In one aspect, the cardiac condition is heart failure, congenital heart disease or arrhythmias. In another aspect, the heart failure is systolic heart failure, diastolic heart failure, reduced ejection fraction heart failure, preserved ejection fraction heart failure or acute heart failure. In a specific aspect, the heart failure is acute heart failure. In one 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 another 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, AAVl l or AAV12. In a specific aspect, the vector is AAV6. In another aspect, the heart condition is precipitated by surgery, pulmonary embolism repair, a toxic substance, stroke, advanced age, severe metabolic disturbance, anemia, liver dysfunction, kidney dysfunction, paraneoplastic syndrome and/or chemotherapy.

[0010] In another aspect, the transgene is adenylyl cyclase 6 (AC6), urocortin or stresscopin. In a further aspect, the transgene is urocortin 1, urocortin 2, urocortin 3 or stresscopin. In a further aspect, the stresscopin like peptide comprises SEQ ID NO:2. In a specific aspect, the stresscopin like peptide comprises the amino acid sequence TKFTLSLDVPTNIMNLLFNIAKAKNLRAQAAANAHLMAQI. In one aspect, following administration of the nucleic acid sequence and stresscopin-like peptide diastolic function is improved, Ca2+ uptake is increased, LV dilation is decreased, left ventricular ejection fraction is increased, stroke volume is increased, cardiac output is increased, left ventricular end diastolic pressure is decreased and left ventricular end systolic volume is decreased. [0011] In an additional aspect, an additional therapeutic agent is administered. In a further aspect, the therapeutic agent is 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, Urea, dobutamine, dopamine, milrinone, enoximone, levosimendan, norepinephrine, epinephrine, nitroglycerin, isosorbide dinitrate, nitroprusside and nesiritide. In another aspect, renal replacement therapy is administered.

[0012] In one aspect, the nucleic acid sequence and the stresscopin-like peptide are administered at simultaneously or are co-administered. In an alternative aspect, the nucleic acid sequence is administered after the stresscopin like-peptide is administered. In an additional aspect, the nucleic acid sequence is administered within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30 or 36 hours after the stresscopin like peptide is administered.

[0013] In another embodiment, the present invention provides for a kit comprising a cardiac tissue protective nucleic acid sequence and a stresscopin like peptide. 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). In a further aspect, the vector is selected from the group consisting of: Ad5, AAV1, AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12. In a specific aspect, the vector is AAV6. In one aspect, the transgene is adenylyl cyclase 6 (AC6), urocortin or stresscopin. In another aspect, the transgene is selected from the group consisting of urocortin 1, urocortin 2, urocortin 3 and stresscopin. In a further aspect, the stresscopin-like peptide comprises SEQ ID NO:2. In an alternative aspect, the stresscopin-like peptide comprises the amino acid sequence TKFTLSLDVPTNIMNLLFNIAKAKNLRAQAAANAHLMAQI.

DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention is based on methods for treating acute heart failure comprising administering a cardiac tissue protective nucleic acid sequence and a stresscopin like peptide. The present invention also provides vectors and kits comprising a cardiac tissue protective nucleic acid and stresscopin like peptides for use in the methods of the invention. [0015] 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.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] A specific type of heart failure is acute heart failure (ADHF). ADHF is defined as a sudden worsening of HF symptoms and is usually caused by cardiogenic pulmonary edema with rapid fluid accumulation in the lungs, although it can occur without pulmonary edema. Hypertension, ischemia, and/or ventricular dysfunction causes a decrease in cardiac output, which leads to an activation of the neurohormonal pathway. The sympathetic system increases norepinephrine to improve peripheral perfusion via vasoconstriction and activates the renin-angiotensin-aldosterone system to increase renal perfusion through water retention. An acute increase in left ventricular filling pressure causes protein-poor fluid to leak into the lung alveoli and interstitium, but no compromise of pulmonary membrane integrity occurs. Compensatory mechanisms increase heart rate and systemic vascular resistance in an attempt to improve cardiac output, and a vicious cycle ensues.

[0021] Common causes of ADHF include left ventricular or diastolic dysfunction with or without coronary artery disease (CAD) or valvular abnormalities. Although most patients hospitalized with ADHF have a worsening of preexisting HF, up to 20% of patients have no prior diagnosis of HF. ADHF can also occur in patients without any preexisting cardiac disease, including conditions such as severe hypertension, fluid overload, severe renal disease, or renal artery stenosis. Factors precipitating an event involve a change in the flow of blood through the heart; ADHF can be induced by hypertensive crisis, MI or ischemia, atrial obstruction, acute mitral regurgitation, fluid overload, or nonadherence to HF medications. Certain medications, such as beta-blockers, nondihydropyridine calcium channel blockers, and nonsteroidal anti-inflammatory drugs (NSAIDs), can also precipitate ADHF.

[0022] Treatment consists of reducing the fluid level with diuretics and improving heart function with nitrates, or levosimendan; other treatments such as aquapheresis ultra-filtration may also be required. Examples of diuretics include, but are not limited to, chlorothiazide, chlorthalidone, indapamide, hydrochlorothiazide, methyclothiazide, metolazone, bumetanide, furosemide, ethacrynate, torsemide, Amiloride hydrochloride, spironolactone, triamterene, Amiloride hydrochloride, spironolactone, triamterene, Acetazolamide Injection, Acetazol amide, Methazol amide, glycerin, Isosorbide, mannitol and urea. Nitrates include, but are not limited to, isosorbide dinitrate, isosorbide mononitrate and nitroglycerin.

[0023] 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.

[0024] 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.

[0025] 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

[0026] 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.

[0027] 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 (NCX1), protein phosphatase 1 (PP1), 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.

[0028] 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). Include figures-all public domain sequences (in Kurt's patents too) 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.

[0029] 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.

[0030] Table 1

[0031] 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; 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.

[0032] 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.

[0033] 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.

[0034] Urocortin 2 has been studied in healthy subjects and patients with heart failure. This peptide was shown to increase left ventricular ejection fraction (LVEF) and cardiac output (CO) in a model of heart failure in sheep. In subsequent intravenous infusion studies in 8 healthy subjects and in 8 subjects with heart failure, the increases in LVEF and CO were accompanied by an increase in heart rate and decrease in blood pressure at both doses examined in each of the two studies.

[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 "Stresscopin-like peptide" refers to a peptide that comprises an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) or 100%) identical to the SEQ ID NO:2. The stresscopin-like peptide may include up to, but not more than, four amino acid deletions and/or one or more conservative amino acid substitution to SEQ ID NO:2. Conservative substitutions may be made, for example, according to the following: aliphatic non-polar, polar-uncharged, and polar charged amino acids can be substituted for another aliphatic amino acid that is non-polar, polar-uncharged, or polar-charged amino acid, respectively. Preferably, aliphatic non-polar substitutions occur between amino acids in the group consisting of G, A, and P or between amino acids in the group consisting of I, L, and V. Preferably, aliphatic polar-uncharged substitutions occur between amino acids in the group consisting of C, S, T, and M or between amino acids in the group consisting of N and Q. Preferably, aliphatic polar-charged substitutions occur between amino acids in the group consisting of D and E or between amino acids in the group consisting of K and R. Conservative amino acid substitutions can also be made between aromatic amino acids that include H, F, W and Y. Preferably, at least a portion of the homologous stresscopin-like peptide comprises an amino acid sequence with a 90% sequence identity to h-SCP concerning amino acid deletions and/or non- conservative substitutions.

[0050] Generally, a stresscopin-like peptide refers to a peptide that displays an agonistic activity towards human corticotrophin releasing hormone receptor type 1 (CRHR1 ) and type 2 (CRHR2) closely resembling the CRHR1 and CRHR2 activity of stresscopin. A stresscopin-like peptide is a selective CRHR2 agonist with less activity towards CRHR1 . Selectivity towards a receptor hereby refers to the potency of a peptide to induce an activity response in the receptor that the peptide is selective towards in comparison to other receptors, in which the peptide might also induce activity, but with less potency. The definition of stresscopin-like peptides is not limited to agonist, but can also include partial agonists. The CRHR1 and CRHR2 activity of a stresscopin-like peptide can for instance be assessed in an adenosine 3',5'-cyclic monophosphate (cAMP) assay.

[0051] As used herein, the term "Pharmaceutically acceptable carrier" refers to a substance that is non-toxic, biologically tolerable, and otherwise biologically suitable for administration to a subject, such as an inert substance, added to a pharmacological composition or otherwise used as a vehicle, carrier, or diluent to facilitate administration of an agent and that is compatible therewith. Examples of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

[0052] As used herein, the term "Pharmaceutically acceptable salt" means an acid or base salt of the compounds of the invention that is of sufficient purity and quality for use in the formulation of a composition or medicament of the present invention and are tolerated and sufficiently non-toxic to be used in a pharmaceutical preparation. Suitable pharmaceutically acceptable salts include acid addition salts which may, for example, be formed by reacting the drug compound with a suitable pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid.

[0053] 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. 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) and a stresscopin-like peptide. The administration of the stresscopin-like peptide can be simultaneous or c-administration with, prior to or following administration of the cardiac protective protein. 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 (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, Urea, dobutamine, dopamine, milrinone, enoximone, levosimendan, norepinephrine, epinephrine, nitroglycerin, isosorbide dinitrate, nitroprusside and nesiritide. In another aspect, renal replacement therapy is administered. 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.

[0054] 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.

[0055] 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.

[0056] The cardiac tissue protective nucleic acid mat be administered by oral, subcutaneous, topical, rectal, nasal, intraarterial, intravenous, intramuscular, intracardiac, or transdermal routes.

[0057] 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.

[0058] Treatment with a stresscopin-like peptide in a subject having heart failure might be evidence by increased left ventricular ejection fraction (LVEF), stroke volume (SV), cardiac output (CO) and/or cardiac index (CI) and a decrease in left ventricular end diastolic pressure (LVEDP), left ventricular pressure during isovolumetric relaxation (LV-dP/dt), systemic vascular resistance (SVR) and/or left ventricular end-systolic volume (LVESV). [0059] In one embodiment, the present invention provides a method of treating a heart condition comprising administering a cardiac tissue protective nucleic acid sequence and a stresscopin-like peptide. In one aspect, the cardiac condition is heart failure, congenital heart disease or arrhythmias. In another aspect, the heart failure is systolic heart failure, diastolic heart failure, reduced ejection fraction heart failure, preserved ejection fraction heart failure or acute heart failure. In a specific aspect, the heart failure is acute heart failure. In one 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 another 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, AAVl l or AAV12. In a specific aspect, the vector is AAV6.

[0060] In another aspect, the transgene is adenylyl cyclase 6 (AC6), urocortin or stresscopin. In a further aspect, the transgene is urocortin 1, urocortin 2, urocortin 3 or stresscopin. In a further aspect, the stresscopin like peptide comprises SEQ ID NO:2. In a specific aspect, the stresscopin like peptide comprises the amino acid sequence TKFTLSLDVPTNIMNLLFNIAKAKNLRAQAAANAHLMAQI. In one aspect, following administration of the nucleic acid sequence and stresscopin-like peptide diastolic function is improved, Ca2+ uptake is increased, LV dilation is decreased, left ventricular ejection fraction is increased, stroke volume is increased, cardiac output is increased, left ventricular end diastolic pressure is decreased and left ventricular end systolic volume is decreased. In an additional aspect, an additional therapeutic agent is administered.

[0061] In a further aspect, the therapeutic agent is 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, Urea, dobutamine, dopamine, milrinone, enoximone, levosimendan, norepinephrine, epinephrine, nitroglycerin, isosorbide dinitrate, nitroprusside and nesiritide. In another aspect, renal replacement therapy is administered.

[0062] In one aspect, the nucleic acid sequence and the stresscopin-like peptide are administered at simultaneously or are co-administered. In an alternative aspect, the nucleic acid sequence is administered after the stresscopin like-peptide is administered. In an additional aspect, the nucleic acid sequence is administered within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30 or 36 hours after the stresscopin like peptide is administered.

[0063] In another embodiment, the present invention provides for a kit comprising a cardiac tissue protective nucleic acid sequence and a stresscopin like peptide. 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). In a further aspect, the vector is selected from the group consisting of: Ad5, AAV1, AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12. In a specific aspect, the vector is AAV6. In one aspect, the transgene is adenylyl cyclase 6 (AC6), urocortin or stresscopin. In another aspect, the transgene is selected from the group consisting of urocortin 1, urocortin 2, urocortin 3 and stresscopin. In a further aspect, the stresscopin-like peptide comprises SEQ ID NO:2. In an alternative aspect, the stresscopin-like peptide comprises the amino acid sequence TKFTLSLDVPTNIMNLLFNIAKAK LRAQAAANAHLMAQI.

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 NO.: 1

CKFTL SLDVP TNIMN LLFNI AKAKN LRAQA AA AH LMAQI-NH₂ SEQ ID NO.: 2

TCFTL SLDVP TNIMN LLFNI AKAKN LRAQA AANAH LMAQI-NH₂ SEQ ID NO.: 3

TKCTL SLDVP TNIMN LLFNI AKAKN LRAQA AANAH LMAQI-NH₂ SEQ ID NO.: 4

TKFCL SLDVP TNIMN LLFNI AKAKN LRAQA AANAH LMAQI-NH₂ SEQ ID NO.: 5

TKFTC SLDVP TNIMN LLFNI AKAKN LRAQA AANAH LMAQI-NH₂ SEQ ID NO.: 6

TKFTL CLDVP TNIMN LLFNI AKAKN LRAQA AANAH LMAQI-NH₂ SEQ ID NO.: 7

TKFTL SCDVP TNIMN LLFNI AKAKN LRAQA AANAH LMAQI-NHj SEQ ID NO.: 8

TKFTL SLCVP TNIMN LLFNI AKAKN LRAQA AANAH LMAQI-NHj SEQ ID NO.: 9

TKFTL SLDCP TNIMN LLFNI AKAKN LRAQA AANAH LMAQI-NHj SEQ ID NO.: 10

TKFTL SLDVC TNIMN LLFNI AKAKN LRAQA AANAH LMAQI-NHj SEQ ID NO.: 11

TKFTL SLDVP CNIMN LLFNI AKAKN LRAQA AANAH LMAQI-NHj SEQ ID NO.: 12

TKFTL SLDVP TCIMN LLFNI AKAKN LRAQA AANAH LMAQI-NHj SEQ ID NO.: 13

TKFTL SLDVP TNCMN LLFNI AKAKN LRAQA AANAH LMAQI-NHj SEQ ID NO.: 14

TKFTL SLDVP TNICN LLFNI AKAKN LRAQA AANAH LMAQI-NHj SEQ ID NO.: 15

TKFTL SLDVP TNIMC LLFNI AKAKN LRAQA AANAH LMAQI-NHj SEQ ID NO.: 16

TKFTL SLDVP TNIMN CLFNI AKAKN LRAQA AANAH LMAQI-NHj SEQ ID NO.: 17

TKFTL SLDVP TNIMN LCFNI AKAKN LRAQA AANAH LMAQI-NHj SEQ ID NO.: 18

TKFTL SLDVP TNIMN LLCNI AKAKN LRAQA AANAH LMAQI-NHj SEQ ID NO.: 19

TKFTL SLDVP TNIMN LLFCI AKAKN LRAQA AANAH LMAQI-NHj SEQ ID NO.: 20 TABLE 2

Human stresscopin with amidated C-terminus and

Cys-variant stresscopin-like peptides

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)-NH₂

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 LLFNI AKAC (-NES) N LRAQA SEQ ID

AANAH LMAQI-NH;

NO.: 50

TKFTL SLDVP TNIMN LLFNC (-NES) AKAKN LRAQA SEQ ID

AANAH LMAQI-NH;

NO.: 5 !

TKFTL SLDVP TNIMN LLFC (-NES) I AKAKN SEQ ID

LRAQA AANAH LMAQI-NH;

NO.: 52

TKFTL SLDVP TNIMN LC (-NES) FNI AKAKN LRAQA SEQ ID

AANAH LMAQI-NH;

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

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 TNIMC (-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 TABLE 4

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

TKFTL SLDVP TNIMN LLFNI AKC (-NES-PEG) KN LRAQA AANAH LMAQI-NH₂ SEQIDNO.: 77

TKFTL SLDVP TNIMN LLFNI AKAC (-NES-PEG) N LRAQA AANAH LMAQI-NH₂ SEQIDNO.: 78

TKFTL SLDVP TNIMN LLFNI AKAKC (-NES-PEG) LRAQA AANAH LMAQI-NH₂ SEQIDNO.: 79

TKFTL SLDVP TNIMN LLFNI AKAKN C (-NES-PEG) RAQA AANAH LMAQI-NH₂ SEQIDNO.: 80

TKFTL SLDVP TNIMN LLFNI AKAKN LC (-NES-PEG) AQA AANAH LMAQI-NH₂ SEQIDNO.: 81

TKFTL SLDVP TNIMN LLFNI AKAKN LRC (-NES-PEG) QA AANAH LMAQI-NH₂ SEQIDNO.: 82

TKFTL SLDVP TNIMN LLFNI AKAKN LRAC (-NES-PEG) A AANAH LMAQI-NH₂ SEQIDNO.: 83

TKFTL SLDVP TNIMN LLFNI AKAKN LRAQC (-NES-PEG) AANAH LMAQI-NH₂ SEQIDNO.: 84

TKFTL SLDVP TNIMN LLFNI AKAKN LRAQA C (-NES-PEG) ANAH LMAQI-NH₂ SEQIDNO.: 85

TKFTL SLDVP TNIMN LLFNI AKAKN LRAQA AC (-NES-PEG) NAH LMAQI-NH₂ SEQIDNO.: 86

TKFTL SLDVP TNIMN LLFNI AKAKN LRAQA AAC (-NES-PEG) AH LMAQI-NH₂ SEQIDNO.: 87

TKFTL SLDVP TNIMN LLFNI AKAKN LRAQA AANC (-NES-PEG) H LMAQI-NH₂ SEQIDNO.: 88

TKFTL SLDVP TNIMN LLFNI AKAKN LRAQA AANAC (-NES-PEG) LMAQI-NH₂ SEQIDNO.: 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) -NH₂ 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

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 (5)

L16S- MeO-PEG (20,000)-IA-A28C- STR (6)

L16N- MeO-PEG (20,000)-IA-A28C- STR (7)

L5A- MeO-PEG (20,000)-IA-A28C- STR (8)

L5S- MeO-PEG (20,000)-IA-A28C-STR (9)

L5N- MeO-PEG (20,000)-IA-A28C-STR (10)

MeO-PEG (20,000)-IA-K2N-A28C-STR (11)

MeO-PEG (20,000)-IA-K24N-A28C-STR (12)

MeO-PEG (20,000)-IA-R27N-A28C-STR (13)

MeO-PEG (20,000)-IA-H35N-A28C-STR (14)

MeO-PEG (20,000)-IA-N12K-A28C-STR (15)

MeO-PEG (20,000)-IA-N12R-A28C-STR (16)

MeO-PEG (20,000)-IA-N25K-A28C-STR (17)

MeO-PEG (20,000)-IA-N25R-A28C-STR (18)

2MeO-PEG (20,000)-IA-A21C-A28C- STR (19)

2MeO-PEG (20,000)-IA-A23C-A28C- STR (20)

2MeO-PEG (20,000)-IA-A28C-A31C- STR (21)

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 (27)

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 (31)

[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 treating a heart condition comprising administering a cardiac tissue protective nucleic acid sequence and a stresscopin-like peptide.

2. The method of claim 1, wherein the cardiac condition is selected from the group consisting of: heart failure, congenital heart disease and arrhythmias.

3. The method of claim 2, wherein the heart failure is selected from the group consisting of systolic heart failure, diastolic heart failure, reduced ejection fraction heart failure, preserved ejection fraction heart failure and acute heart failure.

4. The method of claim 3, wherein the heart failure is acute heart failure.

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

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

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

8. The method of claim 7, 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.

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

10. The method of claim 5, wherein the transgene is adenylyl cyclase 6 (AC6), urocortin or stresscopin.

11. The method of claim 10, wherein the transgene is selected from the group consisting of urocortin 1, urocortin 2, urocortin 3 and stresscopin.

12. The method of claim 1, wherein the stresscopin like peptide comprises SEQ ID NO:2.

13. The method of claim 1, wherein the stresscopin like peptide comprises the amino acid sequence TKFTLSLDVPTNIMNLLFNIAKAK LRAQAAANAHLMAQI.

14. The method of any of claims 1 - 3, wherein following administration of the nucleic acid sequence and the stresscopin-like peptide diastolic function is improved, Ca2+ uptake is increased, LV dilation is decreased, left ventricular ejection fraction is increased, stroke volume is increased, cardiac output is increased, left ventricular end diastolic pressure is decreased and left ventricular end systolic volume is decreased.

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

16. The method of claim 15, wherein 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), Amilonde hydrochloride, spironolactone (Aldactone), triamterene (Dyrenium), Acetazolamide, Methazol amide, glycerin (Glycerol), Isosorbide, Mannitol, Urea, dobutamine, dopamine, milrinone, enoximone, levosimendan, norepinephrine, epinephrine, nitroglycerin, isosorbide dinitrate, nitroprusside and nesiritide.

17. The method of claim 1, wherein the nucleic acid sequence and the stresscopin-like peptide are administered at simultaneously or are co-administered.

18. The method of claim 1, wherein the nucleic acid sequence is administered after the stresscopin like peptide is administered.

19. The method of claim 22, wherein the nucleic acid sequence is administered within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30 or 36 hours after the stresscopin like peptide is administered.

20. A kit comprising a cardiac tissue protective nucleic acid sequence and a stresscopin like peptide.

21. The kit of claim 20, wherein the nucleic acid sequence comprises a transgene and an operably linked promoter.

22. The kit of claim 20, wherein the nucleic acid sequence is in a vector.

23. The kit of claim 22, wherein the vector is an adenovirus or an adeno-associated virus (AAV).

24. The kit of claim 23, 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.

25. The kit of claim 24, wherein the vector is AAV6.

26. The kit of claim 20, wherein the transgene is adenylyl cyclase 6 (AC6), urocortin or stresscopin.

27. The kit of claim 26, wherein the transgene is selected from the group consisting of urocortin 1, urocortin 2, urocortin 3 and stresscopin.

28. The kit of claim 27, wherein the stresscopin like peptide comprises SEQ ID NO:2.

29. The kit of claim 28, wherein the stresscopin like peptide comprises the amino acid sequence TKFTLSLDVPTNIMNLLFNIAKAK LRAQAAANAHLMAQI.

30. The method of claim 10 or the kit of claim 26 wherein the stresscopin is cysteine- modified and/or pegylated.

31. The method or kit of claim 30, wherein the stresscopin is selected from peptides of Tables 2-5.