RU2788124C2 - Pharmaceutical composition for prevention or treatment of heart failure - Google Patents

Abstract

FIELD: biotechnology; pharmaceutical industry.

SUBSTANCE: genetic structure is presented, containing polynucleotide encoding SERCA2a protein and polynucleotide encoding CCN5 protein; a recombinant expression vector, a method for the prevention or treatment of heart failure, a pharmaceutical composition, the use of a genetic structure for the prevention or treatment of heart failure, and the use of a recombinant expression vector are also presented.

EFFECT: pharmaceutical composition allows for obtainment of a synergetic therapeutic effect provided by a function of SERCA2a protein, which consists in prevention of the loss of cardiomyocytes and increase in the activity of cardiomyocytes, and a function of CCN5 protein, which consists in suppression of fibrosis of heart cells and tissues.

33 cl, 32 dwg, 4 ex

Description

FIELD OF TECHNOLOGY

The present invention relates to a pharmaceutical composition for the prevention or treatment of heart failure. In particular, the present invention relates to a gene construct that contains a nucleotide sequence encoding a SERCA2a protein or a fragment thereof, and a nucleotide sequence encoding a CCN5 protein or a fragment thereof, as well as a pharmaceutical composition for the prevention or treatment of heart failure, which contains the gene construct in as an active ingredient.

BACKGROUND OF THE INVENTION

Heart failure (HF) is a disease in which complex symptoms occur due to structural and functional dysfunction of the ventricular pump that draws in or ejects blood. Heart failure is a disease with a higher mortality rate than cancer, with a mortality rate of 50% or more within 5 years of diagnosis. The number of patients with heart failure worldwide is 38 million, and the prevalence of this disease also increases with age. However, there is no fundamental cure for heart failure, and current treatments can only slow the progression of the disease. Therefore, medical care for this disease is associated with high treatment costs, which is a heavy burden for patients (Braunwald E. Lancet, 2015; 385: 812-824).

Heart failure can be caused by a wide range of heart conditions that result from heart disorders, genetic defects, and systemic diseases. Diseases associated with the onset of heart failure generally include ischemic diseases, hypertension and valvular heart disease, in addition, they include primary cardiomyopathy, secondary cardiomyopathy including amyloidosis, congenital heart disease, pericardial disease and the like, which develop due to genetic or acquired causes (Maron BJ. et al., Circulation., 2006; 113: 1807-1816). Heart failure, which develops due to various heart diseases, leads to pathological remodeling of the heart, which causes structural changes and dysfunction of the heart. In particular, with regard to remodeling: there is remodeling at the cellular level caused by changes in the size, shape and function of cardiomyocytes, and remodeling at the tissue level caused by fibrosis of cardiac tissue as a result of excessive accumulation of extracellular matrix (ECM). Such pathological remodeling of the heart includes disease-related transcriptional, signaling, structural, electrophysiological and functional changes in cardiomyocytes.

The myocardial extracellular matrix (ECM) is a complex structure that maintains mechanical function, allowing for efficient contraction and relaxation of cardiomyocytes, and plays a role in ensuring adequate force transmission, electrical signal transmission, cell-to-cell communication, metabolite exchange, and the like. in the microenvironment of the heart muscle. Increased stress, damage, and disease of the heart wall lead to the development of fibrosis in the extracellular matrix, thereby causing impairment of motor function, such as contraction and relaxation, of cardiomyocytes and myofibrils (Li AH. et al., Circ Res. , 2014; 114(5) : 916-27).

In addition, pathological remodeling leads to dysfunction of contraction and relaxation of the heart muscle and the disappearance of cardiomyocytes. Hypertrophy and death of cardiomyocytes at the cellular level affect the myocardial excitation-contraction relationship and also lead to pathological changes in the molecular mechanism that regulates cardiomyocyte contraction, cell survival, mitochondrial function associated with energy metabolism, and oxidative stress (Koitabashi N., et al . ., Nat. rev. Cardiol. , 2012; 9:147-157).

Cardiac tissues undergo pathological structural changes such as myofibroblast formation and fibrosis, increased vascular smooth muscle, dysfunction of vascular endothelial cells, and inflammatory action of immune cells. This disease progression at the cellular level leads to remodeling at the tissue level through integrated processes such as cardiomyocyte hypertrophy and death, loss of blood vessels, fibrosis, inflammation, metabolic dysfunction, and electrophysiological remodeling, thereby causing heart failure (Burchfield JS et al., Circulation 2013; 128: 388-400) .

Neurohormonal blockers have been used for the past 40 years to treat patients with heart failure. However, despite the effectiveness of the treatment of heart failure, which consists in relieving symptoms and reducing stress on the heart, the prognosis in patients with heart failure is very poor, their mortality reaches 50% at 5 years after the onset of the disease and 90% at 10 years after the onset of the disease. In addition, if the heart failure is severely advanced, there is no other treatment than the placement of a heart assist device and a heart transplant. Therefore, there is an urgent need to develop a fundamental cure for heart failure and new therapies that ensure the recovery of the heart.

In terms of new therapies, there are active developments including drugs, cell therapies, miRNAs, and gene therapies that regulate disease-related signaling systems. Among the above treatments, some drugs have been reported to be effective in animal models of heart failure or in small phase II clinical trials (Braunwald E. Lancet, 2015; 385: 812-824, VonLueder TG. et al., Nat. Rev. Cardiol. , 2015; 12: 730-740).

In particular, various preclinical trials for the treatment of heart disease in animals have been carried out due to gene-level understanding of the mechanism of heart disease, the discovery of therapeutic genes, methods for constructing and packaging gene carriers, the rapid development of delivery methods, and the like. (Gorski PA, et al. Cell Metabol. 2015; 21: 183-194).

In recent years, numerous studies have shown that delivery of the SERCA2a gene to animal models of heart failure leads to an increase in survival as well as an increase in cardiac contractility. However, in phase IIb clinical trials with a recombinant adeno-associated virus expressing SERCA2a, AAV-SERCA2a, involving a large number of clinical patients, up to 250, gene therapy did not show real clinical benefits for the treatment of heart failure, in contrast to experimental results obtained in pigs. , sheep, dogs and even primates (Rincon M Y. et al., Cardiovas. Res., 2015; 108: 4-20).

In the development of drugs for the treatment of heart failure, cases have been reported where new drugs that demonstrate a therapeutic effect in phase II clinical trials were less than expected, or were not effective in phase III clinical trials (Vaduganathan M, et al., Nat. Rev. Cardiol., 2013; 10: 85-97).

Modern methods of treating heart failure are aimed at relieving symptoms and reducing overload stress in the heart. However, despite the effectiveness of treatment, the prognosis for patients with heart failure is very pessimistic: their mortality reaches 50% after 5 years from the onset of the disease and 90% after 10 years from the onset of the disease. Among the mechanisms of disease development that have been recently uncovered in the development of treatments for heart failure, cardiac remodeling, including fibrosis, which directly affects the function of the heart pump, and damage to cardiomyocytes interact with each other, they are closely related to each other. affecting the development, progression and prognosis of the disease, and form a vicious circle.

Therefore, since treatment directed at a single drug target or disease mechanism may lead to insufficient results in the treatment of heart failure, there is a need to develop a new complex treatment that can achieve functional recovery at the cellular level and provide treatment for histological remodeling of the heart.

DESCRIPTION OF THE INVENTION

TECHNICAL PROBLEM

The present inventors conducted research to develop an effective method for treating heart failure, and as a result found that a gene construct, an expression vector, and a recombinant virus each containing a nucleotide sequence encoding a SERCA2a protein or a fragment thereof, and a nucleotide sequence encoding a CCN5 protein or its fragment, have a synergistic therapeutic effect on dysfunction caused by heart failure in a mouse model of heart failure caused by multiple etiologies, thereby completing the present invention.

SOLUTION

In one aspect, the present invention provides a gene construct comprising (i) a nucleotide sequence encoding a SERCA2a protein or fragment thereof; and (ii) a nucleotide sequence encoding a CCN5 protein or a fragment thereof.

In another aspect, the present invention provides a recombinant expression vector loaded with a gene construct.

In a further aspect, the present invention provides a recombinant virus containing a gene construct.

In a further aspect, the present invention provides a pharmaceutical composition for the prevention or treatment of heart failure, comprising, as an active ingredient, a gene construct, a recombinant expression vector, or a recombinant virus.

In a further aspect, the present invention provides a method for the prevention or treatment of heart failure, comprising the step of administering the pharmaceutical composition to an individual.

In a further aspect, the present invention provides a pharmaceutical composition for the prevention or treatment of heart failure, comprising an expression vector loaded with a nucleotide sequence encoding a SERCA2a protein or a fragment thereof; and an expression vector loaded with a nucleotide sequence encoding a CCN5 protein or a fragment thereof.

In a further aspect, the present invention provides a pharmaceutical composition for the prevention or treatment of heart failure, comprising a recombinant virus that contains a nucleotide sequence encoding a SERCA2a protein or a fragment thereof; and a recombinant virus that contains a nucleotide sequence encoding a CCN5 protein or a fragment thereof.

In a further aspect, the present invention provides a method for the prevention or treatment of heart failure, comprising the step of administering to an individual (i) an expression vector loaded with a nucleotide sequence encoding a SERCA2a protein or a fragment thereof; and (ii) an expression vector loaded with a nucleotide sequence encoding a CCN5 protein or a fragment thereof.

In a further aspect, the present invention provides a method for the prevention or treatment of heart failure, comprising the step of administering to an individual (i) a recombinant virus that contains a nucleotide sequence encoding a SERCA2a protein or a fragment thereof; and (ii) a recombinant virus that contains a nucleotide sequence encoding a CCN5 protein or a fragment thereof.

BENEFICIAL EFFECTS OF THE INVENTION

A common symptom of heart failure is the disappearance of cardiomyocytes and the development of fibrosis of the heart tissue, and this structural remodeling of the heart causes dysfunction of the heart pump. However, the disappearance of cardiomyocytes and fibrosis of the heart tissue may vary depending on the stage of development of the disease and may interact with each other, forming a vicious circle. Therefore, the simultaneous treatment of the disappearance of cardiomyocytes and fibrosis of cardiac tissue may be most effective.

The pharmaceutical composition of the present invention for the prevention and treatment of heart failure provides simultaneous expression of the SERCA2a protein and the CCN5 protein. The pharmaceutical composition can have a synergistic therapeutic effect by preventing the disappearance of cardiomyocytes and increasing their activity under the action of the SERCA2a protein, as well as by preventing fibrosis of heart cells and tissues under the action of the CCN5 protein, and therefore, this composition can be effectively used for the prevention or treatment of heart failure, complex disease with different causes.

BRIEF DESCRIPTION OF THE DRAWINGS

In FIG. 1a shows the structure of the pTR-CMV-SERCA2a vector.

In FIG. 1b shows the structure of the pTR-CMV-CCN5 vector.

In FIG. 1c shows the structure of the pTR-CMV-SERCA2a-P2A-CCN5 vector.

In FIG. 1d shows the structure of the pTR-CMV-CCN5-P2A-SERCA2a vector.

In FIG. 2a shows the results of intracellular expression and extracellular expression of pTR-CMV-SERCA2a, pTR-CMV-CCN5 and pTR-CMV-SERCA2a-P2A-CCN5.

In FIG. 2b shows the results of a comparison of Ca ²⁺ uptake-related activity of the SERCA2a protein expressed in cells transformed with pTR-CMV-SERCA2a-P2A-CCN5 or pTR-CMV-CCN5-P2A-SERCA2a (n=5, **<0.01) .

In FIG. 2c shows the results of measuring the expression levels of SERCA2a protein and CCN5 protein in cells transformed with pTR-CMV-SERCA2a-P2A-CCN5 or pTR-CMV-CCN5-P2A-SERCA2a.

In FIG. 3 shows a conceptual diagram of the results of animal experiments, namely mice with ischemia-reperfusion injury-induced heart failure.

In FIG. 4a shows the results of identification of the expression of SERCA2a protein and CCN5 protein in heart tissues, achieved by injecting mice with ischemia-reperfusion injury-induced heart failure with recombinant viruses AAV9-Control, AAV9-SERCA2a, AAV9-CCN5, AAV9-SERCA2a+AAV9-CCN5.

In FIG. 4b shows the results of comparing the levels of SERCA2a protein expression in heart tissues (n=5, *<0.05, **<0.01) achieved by injecting mice with ischemia-reperfusion injury-induced heart failure with recombinant AAV9-Control viruses, AAV9-SERCA2a, AAV9-CCN5, AAV9-SERCA2a+AAV9-CCN5.

In FIG. 4c shows the results of comparison of CCN5 protein expression levels in heart tissues (n=5, **<0.01) achieved by injecting mice with ischemia-reperfusion injury-induced heart failure with recombinant viruses AAV9-Control, AAV9-SERCA2a, AAV9- CCN5, AAV9-SERCA2a+AAV9-CCN5.

In FIG. 5 shows photographs of mouse hearts after injection of recombinant viruses AAV9-Control, AAV9-SERCA2a, AAV9-CCN5, AAV9-SERCA2a+AAV9-CCN5 into mice with ischemia-reperfusion injury-induced heart failure: the area of heart tissue infarction is marked with a red line.

In FIG. 6 shows photographs of mouse hearts after administration of recombinant viruses AAV9-Control, AAV9-SERCA2a, AAV9-CCN5, AAV9-SERCA2a+AAV9-CCN5 to mice with ischemic-reperfusion injury-induced heart failure, and the results of staining of a cross section of heart tissue using picrosirius. red: the red line marks the area of transmural infarction of the heart tissue.

In FIG. 7 shows the results of quantitative determination of the proportion of the infarcted area of the extracted hearts (n=5, * <0.05, ** <0.01) after administration of recombinant viruses AAV9-Control, AAV9- SERCA2a, AAV9-CCN5, AAV9-SERCA2a+AAV9-CCN5.

In FIG. 8 shows results showing the contraction fraction (n=5, *<0.05, **<0.01, ***<0.001) after administration of recombinant AAV9-Control viruses to mice with ischemia-reperfusion injury-induced heart failure, AAV9-SERCA2a, AAV9-CCN5, AAV9-SERCA2a+AAV9-CCN5 followed by echocardiography.

In FIG. 9a shows results showing end-systolic volume-pressure ratio (ESPVR) (n=5, *<0.05, **<0.01, ***<0.001) after administration to mice with ischemia-reperfusion-induced heart failure. damage, recombinant viruses AAV9-Control, AAV9-SERCA2a, AAV9-CCN5, AAV9-SERCA2a+AAV9-CCN5, followed by measurement of hemodynamic parameters.

In FIG. 9b shows results showing the end-diastolic volume pressure ratio (EDPVR) (n=5, *<0.05, **<0.01) after administration of recombinant AAV9- viruses to mice with ischemia-reperfusion injury-induced heart failure. Control, AAV9-SERCA2a, AAV9-CCN5, AAV9-SERCA2a+AAV9-CCN5, followed by measurement of hemodynamic parameters.

In FIG. 10 shows a conceptual diagram of the results of animal experiments, namely mice with heart failure induced by transverse aortic contraction.

In FIG. 11a shows the results of identification of the expression of SERCA2a protein and CCN5 protein in heart tissues after administration of recombinant viruses AAV9-Control, AAV9-SERCA2a, AAV9-CCN5, AAV9-SERCA2a-P2A-CCN5 to mice with heart failure induced by transverse aortic contraction.

In FIG. 11b shows the results of comparison of SERCA2a protein expression levels in heart tissues (n=5, * <0.05, ** <0.01) after administration of recombinant viruses AAV9-Control, AAV9-SERCA2a to mice with heart failure induced by transverse aortic contraction. , AAV9-CCN5, AAV9-SERCA2a-P2A-CCN5.

In FIG. 11c shows the results of comparison of CCN5 protein expression levels in heart tissues (n=5, **<0.01) after administration of AAV9-Control, AAV9-SERCA2a, AAV9-CCN5, AAV9 recombinant viruses to mice with heart failure induced by transverse aortic contraction. -SERCA2a-P2A-CCN5.

In FIG. 12 shows photographs of mouse hearts after administration of recombinant viruses AAV9-Control, AAV9-SERCA2a, AAV9-CCN5, AAV9-SERCA2a-P2A-CCN5 to mice with aortic transverse contraction-induced heart failure, and results of staining of a cross section of heart tissue using trichrome according to Masson.

In FIG. 13 shows results showing contraction fraction (n=5, *<0.05, **<0.01) after administration of recombinant viruses AAV9-Control, AAV9-SERCA2a, AAV9-CCN5 to mice with heart failure induced by transverse aortic contraction , AAV9-SERCA2a-P2A-CCN5, followed by echocardiography.

In FIG. 14a shows results showing end-systolic volume-pressure ratio (ESPVR) (n=5, *<0.05, **<0.01) after administration of recombinant AAV9-Control viruses to mice with aortic transverse contraction-induced heart failure. , AAV9-SERCA2a, AAV9-CCN5, AAV9-SERCA2a-P2A-CCN5, followed by measurement of hemodynamic parameters.

In FIG. 14b shows results showing end-diastolic pressure-volume ratio (EDPVR) (n=5, *<0.05, **<0.01) after administration of recombinant AAV9-control viruses to mice with aortic transverse contraction-induced heart failure. , AAV9-SERCA2a, AAV9-CCN5, AAV9-SERCA2a-P2A-CCN5, followed by measurement of hemodynamic parameters.

In FIG. 15 shows a conceptual diagram of the results of animal experiments, namely mice with heart failure induced by angiotensin II (AngII) infusion.

In FIG. 16a shows the results of identification of the expression of SERCA2a protein and CCN5 protein in heart tissues after administration of recombinant viruses AAV9-Control, AAV9-SERCA2a, AAV9-CCN5, AAV9-SERCA2a-P2A-CCN5 to mice with angiotensin II infusion-induced heart failure.

In FIG. 16b shows the results of comparison of SERCA2a protein expression levels in heart tissues (n=5, **<0.01) after administration of AAV9-Control, AAV9-SERCA2a, AAV9-CCN5 recombinant viruses to mice with angiotensin II infusion-induced heart failure, AAV9-SERCA2a-P2A-CCN5.

In FIG. 16c shows results of comparison of CCN5 protein expression levels in heart tissues (n=5, *<0.05, **<0.01) after administration of recombinant viruses AAV9-Control, AAV9- to mice with angiotensin II infusion-induced heart failure. SERCA2a, AAV9-CCN5, AAV9-SERCA2a-P2A-CCN5.

In FIG. 17 shows photographs of mouse hearts after administration of recombinant viruses AAV9-Control, AAV9-SERCA2a, AAV9-CCN5, AAV9-SERCA2a-P2A-CCN5 to mice with angiotensin II infusion-induced heart failure, and results of staining of a cross section of heart tissue using trichrome. according to Masson.

In FIG. 18 shows results showing left ventricular wall thickening (n=5, *<0.05, **<0.01) following administration of recombinant AAV9-Control, AAV9-SERCA2a viruses to mice with angiotensin II infusion-induced heart failure. , AAV9-CCN5, AAV9-SERCA2a-P2A-CCN5, followed by echocardiography.

In FIG. 19 shows results showing the reduction fraction (n=5, *<0.05, **<0.01) after administration of recombinant viruses AAV9-Control, AAV9-SERCA2a, AAV9 to mice with angiotensin II infusion-induced heart failure -CCN5, AAV9-SERCA2a-P2A-CCN5, followed by echocardiography.

BEST MODE FOR CARRYING OUT THE INVENTION

In one aspect, the present invention provides a gene construct comprising (i) a nucleotide sequence encoding a SERCA2a protein or fragment thereof; and (ii) a nucleotide sequence encoding a CCN5 protein or a fragment thereof. In accordance with this document, the nucleotide sequence may be in the form of mRNA.

As used herein, the term "SERCA2a protein", which is short for sarcoplasmic Ca ²⁺ -ATPase 2a, refers to a protein that supplies calcium to the sarcoplasmic reticulum using ATP energy. The SERCA2a protein may have the amino acid sequence described in SEQ ID NO: 1. And the nucleotide sequence encoding the SERCA2a protein may be the sequence described in SEQ ID NO: 2 or SEQ ID NO: 13.

A fragment of the SERCA2a protein can be obtained by cutting off the N-terminal and/or C-terminal region of wild-type SERCA2a, provided that the fragment retains the activity of the SERCA2a protein. Namely, a fragment of the SERCA2a protein can be obtained by cutting off 1-100, 1-50, 1-20 or 1-10 amino acids from the N-terminus or C-terminus.

As used herein, the term "CCN5 protein" refers to an extracellular matrix protein belonging to the CCN family that plays various roles in the regulation of cellular functions, for example, it may be involved in the induction of vascular disease, angiogenesis, oncogenesis, the induction of fibrotic diseases, cell differentiation and survival. . The CCN5 protein, unlike other proteins of the CCN family, does not have a C-terminal domain and may also be referred to as WISP-2, HICP, Cop1, CTGF-L, and the like. In addition, the CCN5 protein consists of a single polypeptide chain containing 250 amino acids. Due to the secretory leader sequence, consisting of 22 amino acids and located at the N-terminus, the CCN5 protein is secreted from the cell and functions as a signaling protein.

The CCN5 protein may have the amino acid sequence described in SEQ ID NO: 3. And the nucleotide sequence encoding the CCN5 protein may be the sequence described in SEQ ID NO: 4 or SEQ ID NO: 14.

A CCN5 protein fragment can be obtained by truncating the N-terminal and/or C-terminal portion of wild-type CCN5, provided that the fragment retains the activity of the CCN5 protein. Namely, a CCN5 protein fragment can be obtained by cutting off 1-30, 1-20, 1-10, or 1-5 amino acids from the N-terminus or C-terminus.

The gene construct may further comprise a self-cleaving sequence located between the nucleotide sequence (i) and the nucleotide sequence (ii). The self-cleaving sequence may be the 2A peptide sequence derived from positive strand RNA viruses such as Picornaviridae, Iflaviruses, Tetraviridae and Discistroviridae or the 2A peptide sequence derived from double stranded RNA viruses such as Rotaviruses, Cypoviruses and Totiviridae (Garry A Luke, et al Journal of General Virology, 2008; 89: 1036-1042).

A nucleotide sequence encoding a 2A peptide derived from porcine teshovirus-1, Thosea asigna virus, equine rhinitis virus A, or foot-and-mouth disease virus, which is commonly used in research, may be present between the nucleotide sequence encoding the SERCA2a protein or a fragment thereof and the nucleotide sequence encoding the CCN5 protein or a fragment thereof. Namely, the self-cleaving sequence may be, without limitation, the nucleotide sequence encoding the 2A peptide derived from porcine teshovirus-1. In addition, the self-cleaving sequence may be the nucleotide sequence described in SEQ ID NO: 6.

The nucleotide sequence encoding the 2A peptide derived from porcine teshovirus-1 may be the nucleotide sequence encoding the amino acid sequence described in SEQ ID NO: 5. In addition, the nucleotide sequence encoding the amino acid sequence described in SEQ ID NO: 5 may be the nucleotide sequence described in SEQ ID NO: 6.

The nucleotide sequence encoding the 2A peptide obtained from Thosea asigna virus may be the nucleotide sequence encoding the amino acid sequence described in SEQ ID NO: 7. In addition, the nucleotide sequence encoding the amino acid sequence described in SEQ ID NO: 7 may be the nucleotide sequence described in SEQ ID NO: 8.

The nucleotide sequence encoding the 2A peptide derived from equine rhinitis virus A may be the nucleotide sequence encoding the amino acid sequence described in SEQ ID NO: 9. In addition, the nucleotide sequence encoding the amino acid sequence described in SEQ ID NO: 9, may be the nucleotide sequence described in SEQ ID NO: 10.

The nucleotide sequence encoding the 2A peptide derived from FMDV may be the nucleotide sequence encoding the amino acid sequence described in SEQ ID NO: 11. In addition, the nucleotide sequence encoding the amino acid sequence described in SEQ ID NO: 11 may be is the nucleotide sequence described in SEQ ID NO: 12.

In addition to the 2A peptide sequence, which is an example of a self-cleaving sequence, there are other self-cleaving sequences including picornavirus 2A sequences (e.g., derived from encephalomyocarditis virus, Tayler murine encephalomyelitis virus, Tayler virus-like virus, Saffold virus, equine rhinitis B virus, bovine rhinovirus, Ljungan virus, Seneca Valley virus, Duck hepatitis virus) present in mammals, and 2A iflaviral sequences (e.g. derived from infectious flacheria virus, Ectropisobliqua picorn-like virus, Perina nuda picorn-like virus), Tetraviruses (e.g. Euprosterna elaeasa, Providence virus) or Dicistroviridae (e.g., from cricket paralysis virus, Drosophila C virus, acute bee paralysis virus, Kashmir bee virus, Israeli acute bee paralysis virus) present in insects, among positive-strand RNA viruses. In addition, examples of such sequences derived from double-stranded RNA viruses may include 2A rotavirus sequences (e.g., derived from porcine rotavirus A, bovine rotavirus C, human rotavirus C, adult diarrhea virus) present in mammals, 2A cipovirus sequences (e.g. , derived from silkworm cytoplasmic polyhedrosis virus, gypsy moth cypovirus, Dendrolimus punctatus cypovirus, moth cypovirus) present in insects, and 2A totivirus sequences (eg, derived from infectious myonecrosis virus) present in Penaeid Shrimp. As such, there may be different sequences 2A, which are not limited to the above.

In addition, the gene construct may contain nucleotide sequences (i) and (ii), which in the direction from the 5'end to the 3'end are arranged in the order (i)-(ii). If SERCA2a-P2A-CCN5, an embodiment of the gene construct, is expressed in a cell, the SERCA2a protein can be inserted into the sarcoplasmic reticulum membrane and the CCN5 protein can be secreted from the cell.

In addition, the gene construct (i) or (ii) may contain a promoter sequence operably linked to it.

As used herein, the term "operably linked" refers to the operability of a nucleotide regulatory sequence (such as a promoter, signal sequence, or collection of transcription factor binding sites) to other nucleotide sequences. A regulatory sequence regulates transcription and/or translation of other nucleotide sequences.

Namely, a promoter linked to a nucleotide sequence encoding a SERCA2a protein or a CCN5 protein can function in animal cells, preferably mammalian cells, to regulate the transcription of the SERCA2a gene or the CCN5 gene. The promoter may include promoters derived from mammalian viruses and promoters derived from mammalian cell genomes. In addition, the promoter includes a synthetic promoter (synthetic muscle and heart restricted promoter, SP _C5-12 ) obtained by combining genomic sequences of mammalian cells and designed to increase specific expression in muscle and heart. The promoter can function specifically in heart cells and can also function in any cell.

In one embodiment, the promoter may be inserted in one of the following orders: i) promoter-SERCA2a-P2A-promoter-CCN5, ii) promoter-CCN5-P2A-SERCA2a, iii) promoter-SERCA2a-P2A-CCN5, or iv) promoter -CCN5-P2A-SERCA2a.

The promoter may be selected from the group consisting of the cytomegalovirus (CMV) promoter, adenovirus late promoter, vaccinia 7.5K promoter, SV40 promoter, HSV tk promoter, RSV promoter, EF1 alpha promoter, metallothionein promoter, beta-actin promoter, human the IL-2 gene, the human IFN gene promoter, the human IL-4 gene promoter, the human lymphotoxin gene promoter, and the human GM-CSF gene promoter, as well as the synthetic muscle- and heart-restricted promoter (SP _C5-12 ). However, the promoter is not limited to the examples. Specifically, the promoter may be the CMV promoter.

In addition, the gene constructs of the present invention can be delivered into the cell using liposomes. Liposomes are formed spontaneously from phospholipids dispersed in an aqueous medium, and liposomes containing the SERCA2a gene and the CCN5 gene can interact with cells through a mechanism such as endocytosis, adsorption on the cell surface, or fusion with the plasma membrane, thus delivering the SERCA2a gene and the CCN5 gene in a cell.

In the present invention, if the pharmaceutical composition of the present invention is based on a viral vector containing a gene construct, the method of administering the pharmaceutical composition may include virus infection methods known in the art. In addition, in the present invention, if the gene construct is contained in a naked recombinant DNA molecule or a plasmid, a microinjection method, a liposome-mediated transfection method, a DEAE-dextran treatment method, and a gene bombardment method can be used to introduce the gene into cells.

In another aspect, the present invention provides a recombinant expression vector loaded with a gene construct.

As used herein, the term "expression vector" refers to a recombinant vector capable of expressing a protein of interest in a target host cell, wherein the recombinant vector is a gene construct that contains the necessary regulatory elements operably linked to the gene insert and permits its expression.

In addition, the expression vector may contain a signal sequence that provides secretion of the protein. Efficient translation of the inserted nucleotide sequence may also require specific initiation signals. Such signals contain the ATG start codon and adjacent sequences. Expression efficiency can be increased by introducing an appropriate transcriptional or translational enhancer.

The expression vector may be a vector selected from the group consisting of plasmid vectors and cosmid vectors.

The plasmid vector may include, without limitation, commercially available plasmids such as pUC18, pBAD, and pIDTSAMRT-AMP.

In a further aspect, the present invention provides a recombinant virus containing a gene construct.

The virus may be any virus selected from the group consisting of adenovirus, adeno-associated virus (AAV), retrovirus, lentivirus, herpes simplex virus, vaccinia virus, and the like. Specifically, the virus may be, without limitation, adeno-associated virus.

Adenovirus is widely used as a gene transfer vector due to its medium genome size, ease of manipulation, high titer, wide target cell range, and excellent infectivity. Its genome can be flanked by 100-200 bp. inverted terminal repeat (ITR), which is an essential cis element for DNA replication and packaging. Adenovirus may additionally contain regions of the E1 genome (E1A and E1B) that encode proteins involved in viral DNA replication.

Among adenoviral vectors, replication-incapable adenoviruses lacking E1 regions can be used. On the other hand, the E3 region is removed from conventional adenoviral vectors to provide a site for the insertion of a foreign gene.

Thus, the gene construct of the present invention containing the SERCA2a gene and the CCN5 gene can be inserted into regions from which E1 regions (E1A region and/or E1B region, preferably E1B region) or E3 region have been deleted. In one embodiment, a gene construct containing the SERCA2a gene and the CCN5 gene can be inserted into the E3 region.

In addition, since up to about 105% of the wild-type genome can be packaged in an adenovirus, about 2 kb. can be further packaged in adenovirus. Thus, the foreign sequence to be inserted into the adenovirus can be further linked to the adenovirus genome.

Adenovirus includes 42 different serotypes and subgroups A to F. In one embodiment, the adenoviral vector of the present invention can be derived from adenovirus type 5 belonging to subgroup C. Biochemical and genetic information about adenovirus type 5 is well known.

Foreign genes delivered by adenovirus replicate as episomes and therefore have very low genotoxicity to host cells.

The retrovirus is widely used as a gene transfer vector because the retrovirus is able to insert its gene into the host genome and deliver a large amount of foreign genetic material, and in addition, it can infect a wide range of cells.

To construct a retroviral vector, a gene construct containing the SERCA2a gene and the CCN5 gene can be inserted into the retrovirus genome in place of the retroviral sequence, resulting in a replication-incapable virus. To obtain virions, a packaging cell line can be constructed and used that expresses the gag, pol and env genes and does not express the long terminal repeat (LTR) and the Ψ sequence.

Adeno-associated virus (AAV) can be used as the gene delivery system of the present invention because it is able to infect non-dividing cells and has the ability to infect different types of cells. Details of the construction and use of AAV vectors are disclosed in US Pat. CCN5 flanked by two AAV terminal repeats and an expression plasmid containing the wild-type AVV coding sequence lacking terminal repeats.

Vectors derived from vaccinia, lentivirus, or herpes simplex virus can also be used to deliver the CCN5 gene and target nucleotide sequence to be delivered into a cell.

In a further aspect, the present invention provides a pharmaceutical composition for the prevention or treatment of heart failure, comprising, as an active ingredient, a gene construct, a recombinant expression vector, or a recombinant virus of the present invention.

When the pharmaceutical composition of the present invention is formulated, it may contain pharmaceutically acceptable carriers including, but not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The dosage form of the pharmaceutical composition may vary depending on the route of administration, and may be in the form of injections.

The dose of the pharmaceutical composition of the present invention is preferably determined taking into account the age, sex, condition of the patient, the degree of absorption of the active ingredients in the body, the rate of inactivation and drugs used in combination; and if the pharmaceutical composition is a virus, the pharmaceutical composition can be administered to an adult in an amount of from 1.0×10 ³ to 1.0×10 ²⁰ viral genomes per day. Specifically, the pharmaceutical composition of the present invention can be administered to an adult individual in an amount of from 1.0×10 ³ to 1.0×10 ²⁰ , 1.0×10 ⁸ to 1.0×10 ¹⁶ , 1.0×10 ¹² to 1.0 ×10 ¹⁵ , or 1.0×101 ³ to 1.0×10 ¹⁴ viral genomes per day.

If the pharmaceutical composition is a plasmid vector, the pharmaceutical composition may be administered to an adult subject at a concentration of 0.1 μg/1 μl to 1 mg/1 μl per day. In addition, if the pharmaceutical composition is a plasmid vector, the dose may be 0.1 ml, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml or higher , and can include all values and ranges in between.

In a further aspect, the present invention provides a pharmaceutical composition for the prevention or treatment of heart failure, comprising, as an active ingredient, a SERCA2a protein and a CCN5 protein.

The pharmaceutical composition of the present invention is administered parenterally, wherein parenteral administration includes intravenous administration, subcutaneous administration, intramuscular administration, intraperitoneal administration, transdermal administration, direct tissue administration, and the like.

In this description, the term "acceptable carrier" refers to some or all of the following substances and includes substances suitable for a particular form: solvents, diluents, liquid media, dispersing agents, suspension aids, surfactants, isotonic agents, thickeners, emulsifiers, preservatives, solid binders, lubricants, etc. Alfanso R. Gennaro, Remington's Pharmaceutical Sciences, 19th edition, 1995, ^Macna Publishing Co. Easton, PA describes various carriers for use in pharmaceutical compositions using known methods and formulations. Examples of pharmaceutically acceptable carriers for the pharmaceutical composition include, without limitation, those listed below. The pharmaceutical composition may include glucose, sucrose, starch such as corn starch and potato starch, cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; tragacanth powder; malt; gelatin; talc; excipients such as cocoa butter, suppository wax, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free distilled water; isotonic saline; Ringer's solution; water based on ethyl alcohol and phosphate, sodium lauryl sulfate and magnesium stearate, colorants, release agents, coating agents, sweeteners, flavors and flavors, antioxidants, etc., at the discretion of the manufacturer.

In a further aspect, the present invention provides a method for the prevention or treatment of heart failure, comprising the step of administering the pharmaceutical composition to an individual.

As used herein, the individual may be a mammal, preferably a human. Namely, the individual may be a human or a non-human mammal, where the individual may be suffering from heart failure, or at risk of heart failure.

In a further aspect, the present invention provides a pharmaceutical composition for the prevention or treatment of heart failure, comprising an expression vector loaded with a gene construct that contains a nucleotide sequence encoding a SERCA2a protein or a fragment thereof; and an expression vector loaded with a gene construct that contains a nucleotide sequence encoding a CCN5 protein or a fragment thereof.

The SERCA2a protein or fragment thereof described above for the gene construct is used. The CCN5 protein or fragment thereof described above for the gene construct is used.

In addition, the gene construct may contain an operably linked promoter sequence.

Namely, a promoter linked to a nucleotide sequence encoding a CCN5 protein or a fragment thereof can function, preferably in animal cells, more preferably in mammalian cells, to regulate the transcription of the CCN5 gene. The promoter includes promoters derived from mammalian viruses and promoters derived from mammalian cell genomes. The promoter may function specifically in heart cells, or it may function in any cell.

The promoter is the promoter described above, namely, it may be the CMV promoter.

In a further aspect, the present invention provides a pharmaceutical composition for the prevention or treatment of heart failure, comprising a recombinant virus comprising a gene construct that contains a nucleotide sequence encoding a SERCA2a protein or a fragment thereof; and a recombinant virus comprising a gene construct that contains a nucleotide sequence encoding a CCN5 protein or a fragment thereof.

A gene construct that contains a nucleotide sequence encoding a SERCA2a protein or a fragment thereof and a gene construct that contains a nucleotide sequence encoding a CCN5 protein or a fragment thereof are described for "pharmaceutical composition for the prevention or treatment of heart failure comprising an expression vector loaded with the gene construct , which contains a nucleotide sequence encoding the SERCA2a protein or a fragment thereof; and an expression vector loaded with a gene construct that contains a nucleotide sequence encoding the CCN5 protein or a fragment thereof".

In a further aspect, the present invention provides a method for the prevention or treatment of heart failure, comprising (i) the step of administering to an individual an expression vector loaded with a nucleotide sequence encoding a SERCA2a protein or a fragment thereof; and (ii) the step of administering to the individual an expression vector loaded with a nucleotide sequence encoding a CCN5 protein or a fragment thereof.

As used herein, the individual may be a mammal, preferably a human. Namely, the individual may be a human or a non-human mammal, where the individual may be suffering from heart failure, or at risk of heart failure.

The introduction steps (i) and (ii) can be carried out simultaneously. Or, after step (i) or (ii) has been carried out, another step of administration can be carried out after a certain time interval.

In a further aspect, the present invention provides a method for the prevention or treatment of heart failure, comprising (i) the step of administering a recombinant virus that contains a nucleotide sequence encoding a SERCA2a protein; and (ii) the step of introducing a recombinant virus that contains a nucleotide sequence encoding a CCN5 protein.

As used herein, the individual may be a mammal, preferably a human. Namely, the individual may be a human or a non-human mammal, where the individual may be suffering from heart failure, or at risk of heart failure.

The introduction steps (i) and (ii) can be carried out simultaneously. Or, after step (i) or (ii) has been carried out, another step of administration can be carried out after a certain time interval.

In a further aspect, the present invention provides the use of the gene construct of the present invention for the prevention or treatment of heart failure.

In a further aspect, the present invention provides the use of a recombinant expression vector of the present invention for the prevention or treatment of heart failure.

In a further aspect, the present invention provides the use of a recombinant virus of the present invention for the prevention or treatment of heart failure.

In a further aspect, the present invention provides the use of the gene construct of the present invention for the preparation of a pharmaceutical composition for the prevention or treatment of heart failure.

In a further aspect, the present invention provides the use of a recombinant expression vector of the present invention for the preparation of a pharmaceutical composition for the prevention or treatment of heart failure.

In a further aspect, the present invention provides the use of a recombinant virus of the present invention for the preparation of a pharmaceutical composition for the prevention or treatment of heart failure.

METHOD FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail using experimental examples and examples. However, the following Experimental Examples and Examples are only intended to illustrate the present invention, and the present invention is not limited to the following Production Examples and Examples.

Production method example 1. Construction of gene constructs and AAV9-SERCA2a-P2A-CCN5

Gene constructs PTR-CMV-SERCA2a, pTR-CMV-CCN5, pTR-CMV-SERC2a-P2A-CCN5 and pTR-CMV-CCN5-P2A-SERC2a are constructed to express CCN5 protein and SERCA2a protein separately or simultaneously (Fig. 1a -1d).

The SERCA2a fragment consists of the full length cDNA sequence of the human SERCA2a protein. The next linked P2A fragment is a self-cleaving region derived from porcine teshovirus-1 and consisting of a nucleotide sequence encoding 22 amino acids. Finally, the CCN5 fragment consists of the full length cDNA sequence of the human CCN5 protein.

The recombinant plasmid pTR-CMV-SERCA2a-P2A-CCN5 is obtained by removing the luciferase fragment from the pTR-CMV-luciferase vector and inserting the SERCA2a-P2A-CCN5 gene construct in its place. The protein produced by the recombinant plasmid separates into the SERCA2a protein and the CCN5 protein by self-cleavage between the 21st amino acid, glycine, and ^{the 22nd} amino acid, proline, at the P2A region. The SERCA2a protein can remain in the endoplasmic reticulum membrane and perform its inherent function. The CCN5 protein can migrate to the endoplasmic reticulum and then be secreted from the cell in a form that the signal peptide cleaves, thereby performing its intended function. The recombinant plasmid pTR-CMV-CCN5-P2A-SERCA2a is also constructed by removing the luciferase fragment from the pTR-CMV-luciferase vector and inserting the CCN5-P2A-SERCA2a gene construct in its place. Again, the resulting protein splits into a SERCA2a protein and a CCN5 protein due to the same function of the P2A sequence.

To obtain a self-complementary adeno-associated virus (AAV, serotype 9), the human CCN5 gene and the SERCA2a gene are cloned in the pds-AAV2-EGFP vector. To improve virus packaging and virus delivery efficiency, the eGFP sequence is removed during construction of the AAV vector. Recombinant AAV is constructed using 293T cells. AAV particles in cell culture are harvested and precipitated with ammonium sulfate. The resulting product is purified by ultracentrifugation using an iodixanol gradient. The AAV particles are enriched by several dilution and enrichment processes such that iodixanol is exchanged with the lactate-containing Ringer's solution using centrifugation. AAV concentration is determined by quantitative RT-PCR and SDS-PAGE.

Experimental method 1. Calcium uptake analysis

The 293T cell line containing the expressed gene is homogenized in a pH 7.0 solution containing 40 mM imidazole, 10 mM NaF, 1 mM EDTA, 300 mM sucrose and 0.5 mM DTT, after which 500 μg of the lysate is added to the absorbance measurement buffer pH 7.0 containing 100 mM KCl, 5 mM MgCl ₂ , 5 mM NaN ₃ , 0.5 M EGTA and 40 mM imidazole. Absorbance is measured using pCa 6 (0.0185 µmol) calcium-containing radioisotopes. Treat with 1 μM ruthenium red (Sigma Aldrich) and leave to stand at 37° C. for 3 minutes. The reaction mixture was then allowed to stand until treated with 5 mM K oxalate and Mg-ATP (Sigma Aldrich). 500 μl of the reaction product is filtered through a 0.45 μm filter (Millipore) for up to 4 minutes at 1 minute intervals and the number of pulses per minute (cpm) is measured using a scintillation counter (Beckman).

Experimental method 2. Western blotting

Heart cells or tissues are homogenized in a minimum volume of 50 mM Tris-HCl, pH 7.4, to which a mixture of broad spectrum protease inhibitors (Calbiochem) is added. Proteins are separated by SDS-PAGE and transferred to a polyvinylidene fluoride membrane (Schleicher & Schuell). After blocking with 5% (w/v) skim milk for 1 hour and washing on a TBST membrane, the membrane is allowed to react with an anti- ^SERCA2a antibody (21st Century Biochemical), an anti-CCN5 antibody (Sigma Aldrich), and an anti-GAPDH antibody (Sigma-Aldrich) ). The membrane is then reacted with a horseradish peroxidase conjugated secondary antibody (Jackson ImmunoResearch, WestGrove, PA, USA) and stained using a chemiluminescent substrate (Dogen). The result is photographed and quantified using the LAS software.

Experimental Method 3: Fabric Dyeing

Heart tissues are taken from animal models and then fixed with 10% (w/v) formalin at room temperature for 5 days. Then washed with PBS. Each sample is embedded in paraffin and the tissue block is cut to produce 7 µm sections. To check the degree of fibrosis and myocardial infarction, the result is stained with picrosirius red (Sigma Aldrich) and Masson's trichrome (Sigma Aldrich). Then observed with an optical microscope.

Experimental method 4. Measurement of myocardial function by echocardiography

Mice are anesthetized by intraperitoneal injection of ketamine (95 mg/kg) and xylazine (5 mg/kg) and echocardiography is performed. Recording was performed using 2D imaging and M-mode tracking, contraction fraction and ventricular size ratio were determined (GE Vivid Vision).

Experimental method 5. Measurement of myocardial function using hemodynamic parameters

In vivo hemodynamic measurements are performed using a 1.2Fr pressure-volume catheter (Scisense Inc., Ontario, Canada). Mice are anesthetized by intraperitoneal injection of ketamine (95 mg/kg) and xylazine (5 mg/kg), intubated by tracheostomy, and ventilated by adjusting mechanical airflow to a tidal volume of 7 µl/g and 120 breaths per minute. A pressure-volume (PV) catheter is placed in the left ventricle, and volume-pressure measurements are analyzed using IOX2 software (emka TECHNOLOGIES).

Example 1 Protein Expression Identification

In order to observe the expression of SERCA2a protein and CCN5 protein by the recombinant plasmid, pTR-CMV-SERA2a, pTR-CMV-CCN5 or pTR-CMV-SERCA2a-P2A-CCN5 obtained in Experimental Example 1 was delivered to cultured cells using Lipofectamine. The obtained cells and cultures were subjected to Western blotting according to the method described in Experimental Method 2 to confirm the expression of SERCA2a, CCN5 and GAPDH proteins.

As a result, it was found that in the case of expression under the control of one promoter, the SERCA2a protein is retained in the cytoplasm, while the CCN5 protein is secreted from the cell (Fig. 2a).

In addition, to compare the level of expression and activity of proteins, pTR-CMV-SERC2a-P2A-CCN5 and pTR-CMV-CCN5-P2A-SERC2a obtained in Experimental Example 1 were delivered to cultured cells using Lipofectamine. The resulting cells and cultures were subjected to calcium uptake assay as described in Experimental Method 1.

As a result, no significant differences in expression levels of SERCA2a protein and CCN5 protein are observed, and cells expressing pTR-CMV-CCN5-P2A-SERC2a are characterized by low levels of calcium uptake (FIGS. 2b and 2c).

This is because the CCN5 protein is a secreted protein, suggesting that when SERCA2a protein and CCN5 protein are expressed in the order SERCA2a-CCN5, both proteins are expressed in their normal structure, while if SERCA2a protein and CCN5 protein are expressed in the order CCN5- SERCA2a, a SERCA2a protein that is translated downstream of the CCN5 protein, can be generated as an abnormal protein with a topology opposite to its original topology.

I. Identification of therapeutic effect in relation to heart failure

In order to identify a therapeutic effect on heart failure, a recombinant virus of the present invention containing a gene construct that contains a nucleotide sequence encoding a SERCA2a protein and a nucleotide sequence encoding a CCN5 protein is administered to mice with multiple etiology induced heart failure.

Induced heart failure mice include an ischemia-reperfusion injury model of heart failure, an aortic transverse contraction-induced heart failure model, and an angiotensin II-induced heart failure model. Mice with induced heart failure are subdivided into AAV9 control group, AAV9-SERCA2a receiving group, AAV9-CCN5 receiving group and AAV9-SERCA2a-P2A-CCN5 receiving group, after which the therapeutic effects on heart failure in different groups are compared. .

Experimental Example 1 Identification of a therapeutic effect on heart failure in mice with ischemia-reperfusion injury-induced heart failure.

Experimental example 1.1. Generation of mice with ischemia-reperfusion injury-induced heart failure and administration of recombinant virus

B6C3F1 mice, 8-10 weeks old, are anesthetized by intraperitoneal injection of ketamine (95 mg/kg) and xylazine (5 mg/kg) and undergo surgery. To induce ischemia, a 1 mm polyethylene 10 (PE10) tube is placed on the left anterior descending coronary artery and tied. After 30 minutes, reperfusion is induced by disconnecting and removing the PE10 tubing. Simultaneously with the induction of reperfusion, each mouse is injected via the tail vein with 1×10 ¹¹ viral genomes (vg) AAV9-control, AAV9-CCN5, AAV9-SERCA2a or AAV9-SERCA2a+AAV9-CCN5. After 4 weeks, myocardial function is measured and histological analysis is performed (Fig. 3).

Experimental example 1.2. Identification of CCN5 protein and/or SERCA2a protein expression

First, to determine whether effective delivery of the viral genome has occurred, the expression of the SERCA2a protein and CCN5 protein is checked by Western blotting according to the method described in Experimental Method 2.

As a result, compared with the AAV9 control group, CCN5 protein expression was significantly increased in the AAV9-CCN5 group, and SERCA2a protein expression was largely normalized in the AAV9-SERCA2a group. In addition, it was found that in the group receiving the joint introduction of AAV9-CCN5 and AAV9-SERCA2a, the expression of CCN5 protein and SERCA2a protein was significantly increased (Fig. 4a-4c).

Experimental example 1.3. Identification of the effect of reducing the infarct area of the heart

Hearts are removed from mice in which heart failure has been induced by ischemia-reperfusion injury and photographed. Tissue staining is carried out according to the method described in experimental method 3.

The results demonstrate that in the AAV9-CCN5 group, in the AAV9-SERCA2a group, and in the AAV9-CCN5 and AAV9-SERCA2a combination group, there is a significantly smaller infarct area compared to the infarct area in the AAV9 group -control. In particular, in the group receiving the combination of AAV9-CCN5 and AAV9-SERCA2a, a significantly smaller infarct area was observed (FIGS. 5 and 6). In addition, from the graph obtained by quantifying the proportion of infarct area to total heart area, it can be seen that in the group receiving the combination of AAV9-CCN5 and AAV9-SERCA2a, there is a significant decrease in the proportion of infarct area (Fig. 7).

Experimental example 1.4. Identification of the effect of increased cardiac contractility

Echocardiography is performed and hemodynamic parameters are determined to determine if morphological changes in the heart tissue can affect cardiac contractility.

To measure the contraction fraction (FS), a parameter of cardiac contractility, echocardiography is performed according to the method described in experimental method 4. The results obtained show that the contraction fraction, which was reduced due to ischemia-reperfusion injury, is largely restored in the AAV9- CCN5, in the group receiving AAV9-SERCA2a, and in the group receiving the combination of AAV9-CCN5 and AAV9-SERCA2a. In particular, in the group receiving the combination of AAV9-CCN5 and AAV9-SERCA2a, an increase in the contraction fraction was noticeable (Fig. 8).

In addition, hemodynamic measurement is carried out according to the method described in Experimental Method 5 to accurately analyze cardiac contractility. As a result, the end systolic pressure to volume ratio (ESPVR), a parameter of myocardial systolic strength, decreases due to ischemia-reperfusion injury. The AAV9-SERCA2a group has a much higher ESPVR, while the AAV9-CCN5 group does not show a significant increase in ESPVR. The same phenomenon is observed in the CCN5 study, confirming the fact that CCN5 does not affect heart failure. In addition, a synergistically increased ESPVR was observed in the group receiving the combination of AAV9-CCN5 and AAV9-SERCA2a (Figure 9a).

In addition, end-diastolic volume pressure ratio (EDPVR), a parameter of diastolic myocardial strength, has been found to increase with ischemia-reperfusion injury. In the AAV9-CCN5 group, there is a significant reduction in EDPVR, and in the AAV9-SERCA2a group, there is no significant reduction in EDPVR. This result, as in previous studies, confirms the fact that the SERCA2a protein does not affect diastolic heart failure. In addition, the group receiving the combination of AAV9-CCN5 and AAV9-SERCA2a showed a significant decrease in EDPVR (Fig. 9b).

Based on the data obtained, it was found that the combination of the introduction of AAV9-CCN5 and AAV9-SERCA2a allows achieving a synergistic therapeutic effect.

Experimental Example 2 Identification of a Therapeutic Effect on Heart Failure in Mice with Aortic Transverse Contraction-Induced Heart Failure

Experimental example 2.1. Production of mice with heart failure induced by transverse aortic contraction and administration of recombinant virus

B6C3F1 mice, 8 to 10 weeks old, are anesthetized by intraperitoneal injection of ketamine (95 mg/kg) and xylazine (5 mg/kg) and undergo surgery. To provide a field of view, the aortic arch is longitudinally dissected from 2 to 3 mm of the proximal part of the sternum. After that, using a 27-gauge needle, a connection is established between the innominate artery and the carotid artery. The tying is then carried out and the needle is removed, which causes narrowing of the transverse aorta. 8 weeks after induction of heart failure by constriction, each mouse is injected via the tail vein with 1×10 ¹¹ μg of AAV9-control, AAV9-CCN5, AAV9-SERCA2a, or AAV9-SERCA2a-P2A-CCN5. After 8 weeks, measurements of myocardial function and histological analysis are performed (Fig. 10).

Experimental example 2.2. Identification of CCN5 protein and/or SERCA2a protein expression

First, to determine whether the effective delivery of the viral genome was carried out, the expression of the SERCA2a protein and the CCN5 protein was examined by Western blotting according to the method described in Experimental Method 2. The results show that, compared with the AAV9 control group, the expression of the CCN5 protein significantly increased in the AAV9-CCN5 group, and SERCA2a protein expression was significantly increased in the AAV9-SERCA2a group. In addition, it was found that in the group receiving AAV9-SERCA2a-P2A-CCN5, CCN5 protein expression and SERCA2a protein expression were significantly increased (Fig. 11a-11c).

Experimental example 2.3. Identification of the effect of a decrease in the internal dimensions of the heart

Hearts are harvested from mice with ischemia-reperfusion injury-induced heart failure, heart tissues are stained with Masson's trichrome as described in Experimental Method 3. The results show that the AAV9 control group showed an increase in internal heart size; and in the group receiving AAV9-CCN5, in the group receiving AAV9-SERCA2a, and in the group receiving AAV9-SERCA2a-P2A-CCN5, there is a decrease in the internal dimensions of the heart. In particular, it was found that in the group receiving AAV9-SERCA2a-P2A-CCN5, there is a significant reduction in internal dimensions. In addition, the AAV9-CCN5 group, the AAV9-SERCA2a group, and the AAV9-SERCA2a-P2A-CCN5 group were found to have a reduction in cardiac fibrosis caused by heart failure (FIG. 12).

Experimental example 2.4. Identification of the effect of increased cardiac contractility

Echocardiography is performed and hemodynamic parameters are determined to determine if morphological changes in the heart tissue can affect cardiac contractility.

To measure the contraction fraction (FS), a parameter of cardiac contractility, echocardiography is performed according to the method described in experimental method 4. The results obtained show that the contraction fraction, which was reduced due to aortic contraction, is largely restored in the AAV9-CCN5 group, in the group receiving AAV9-SERCA2a and in the group receiving the combination of AAV9-CCN5 and AAV9-SERCA2a. In particular, in the group receiving AAV9-SERCA2a-P2A-CCN5, an increase in the contraction fraction was noticeable (Fig. 13).

In addition, hemodynamic measurement is carried out according to the method described in Experimental Method 5 to accurately analyze cardiac contractility. As a result, the end systolic pressure to volume ratio (ESPVR), a parameter of systolic myocardial strength, decreases due to aortic contraction. In the group receiving AAV9-SERCA2a, there is a significant recovery of ESPVR, and in the group receiving AAV9-CCN5, there is no significant recovery of ESPVR. In addition, a significant increase in ESPVR was observed in the AAV9-SERCA2a-P2A-CCN5 group (Fig. 14a).

In addition, end-diastolic volume pressure ratio (EDPVR), a parameter of diastolic myocardial strength, has been found to increase with ischemia-reperfusion injury. In the AAV9-CCN5 group, there is a significant reduction in EDPVR, and in the AAV9-SERCA2a group, there is no significant reduction in EDPVR. In addition, a significant reduction in EDPVR was observed in the AAV9-SERCA2a-P2A-CCN5 group (Fig. 14b).

Based on the data obtained, it was found that the introduction of AAV9-SERCA2a-P2A-CCN5 allows achieving a synergistic therapeutic effect.

Experimental Example 3 Identification of a Therapeutic Effect on Heart Failure in Angiotensin II (Ang II) Induced Heart Failure Mice

Experimental example 3.1. Obtaining mice with heart failure induced by the introduction of angiotensin II, and the introduction of recombinant virus

8 to 10 week old B6C3F1 mice are anesthetized by intraperitoneal injection of ketamine (95 mg/kg) and xylazine (5 mg/kg) and heart failure is induced by subcutaneous infusion of angiotensin II (Ang II). Angiotensin II is administered subcutaneously for 2 weeks at a concentration of 3 mg/kg per day using a small osmotic pump (Alzet 1002, Alzet). 2 weeks after induction of heart failure with Ang II, each mouse was injected with 1 x 10 ¹¹ viral genomes (vgs) of AAV9-control, AAV9-CCN5, AAV9-SERCA2a, or AAV9-SERCA2a-P2A-CCN5 via the tail vein. After 4 weeks, myocardial function is measured and histological analysis is performed (FIG. 15).

Experimental example 3.2. Identification of CCN5 protein and/or SERCA2a protein expression

First, to determine whether the viral genome has been efficiently delivered, the expression of SERCA2a protein and CCN5 protein is checked by Western blotting according to the method described in Experimental Method 2.

The results show that, compared with the AAV9 control group, CCN5 protein expression is significantly increased in the AAV9-CCN5 group, and SERCA2a protein expression is significantly increased in the AAV9-SERCA2a group. In addition, it was found that in the group receiving AAV9-SERCA2a-P2A-CCN5, CCN5 protein expression and SERCA2a protein expression were significantly increased (Fig. 16a-16c).

Experimental example 3.3. Identification of the effect of a decrease in the internal dimensions of the heart and a change in the thickness of the internal wall of the heart

Hearts are harvested from mice with angiotensin II-induced heart failure, heart tissues are stained with Masson's trichrome as described in Experimental Method 3.

The results show that in the AAV9 control group, there is an increase in the internal dimensions of the heart; and in the group receiving AAV9-CCN5, in the group receiving AAV9-SERCA2a, and in the group receiving AAV9-SERCA2a-P2A-CCN5, there is a decrease in the internal dimensions of the heart. In particular, it was found that in the group receiving AAV9-SERCA2a-P2A-CCN5, there is a significant decrease in the size of the lumen (Fig. 17).

In addition, echocardiography was carried out according to the method described in Example 6 to measure the thickness of the inner wall of the heart. The change in the thickness of the inner wall of the heart is determined quantitatively and graphically.

The results obtained show that the thickness of the inner wall of the heart decreases after angiotensin II infusion, and in the group receiving AAV9-CCN5, in the group receiving AAV9-SERCA2a, and in the group receiving AAV9-SERCA2a-P2A-CCN5, there is a significant restoration of the thickness of the internal walls of the heart (Fig. 18).

Experimental example 3.4. Identification of the effect of increased cardiac contractility

Echocardiography is performed and hemodynamic parameters are determined to determine if morphological changes in the heart tissue can affect cardiac contractility.

To measure the contraction fraction (FS), a parameter of cardiac contractility, echocardiography is performed according to the method described in experimental method 4. The results obtained show that the contraction fraction, which was reduced due to aortic contraction, is largely restored in the AAV9-CCN5 group, in the group receiving AAV9-SERCA2a and in the group receiving the combination of AAV9-CCN5 and AAV9-SERCA2a. In particular, in the group receiving AAV9-SERCA2a-P2A-CCN5, the reduction fraction was markedly restored (Fig. 19). Based on the data obtained, it was found that the introduction of AAV9-SERCA2a-P2A-CCN5 allows achieving a synergistic therapeutic effect.

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Met Glu Asn Ala His Thr Lys Thr Val Glu Glu Val Leu Gly His Phe

1 5 10 15

Gly Val Asn Glu Ser Thr Gly Leu Ser Leu Glu Gln Val Lys Lys Leu

20 25 30

Lys Glu Arg Trp Gly Ser Asn Glu Leu Pro Ala Glu Glu Gly Lys Thr

35 40 45

Leu Leu Glu Leu Val Ile Glu Gln Phe Glu Asp Leu Leu Val Arg Ile

50 55 60

Leu Leu Leu Ala Ala Cys Ile Ser Phe Val Leu Ala Trp Phe Glu Glu

65 70 75 80

Gly Glu Glu Thr Ile Thr Ala Phe Val Glu Pro Phe Val Ile Leu Leu

85 90 95

Ile Leu Val Ala Asn Ala Ile Val Gly Val Trp Gln Glu Arg Asn Ala

100 105 110

Glu Asn Ala Ile Glu Ala Leu Lys Glu Tyr Glu Pro Glu Met Gly Lys

115 120 125

Val Tyr Arg Gln Asp Arg Lys Ser Val Gln Arg Ile Lys Ala Lys Asp

130 135 140

Ile Val Pro Gly Asp Ile Val Glu Ile Ala Val Gly Asp Lys Val Pro

145 150 155 160

Ala Asp Ile Arg Leu Thr Ser Ile Lys Ser Thr Thr Leu Arg Val Asp

165 170 175

Gln Ser Ile Leu Thr Gly Glu Ser Val Ser Val Ile Lys His Thr Asp

180 185 190

Pro Val Pro Asp Pro Arg Ala Val Asn Gln Asp Lys Lys Asn Met Leu

195 200 205

Phe Ser Gly Thr Asn Ile Ala Ala Gly Lys Ala Met Gly Val Val Val

210 215 220

Ala Thr Gly Val Asn Thr Glu Ile Gly Lys Ile Arg Asp Glu Met Val

225 230 235 240

Ala Thr Glu Gln Glu Arg Thr Pro Leu Gln Gln Lys Leu Asp Glu Phe

245 250 255

Gly Glu Gln Leu Ser Lys Val Ile Ser Leu Ile Cys Ile Ala Val Trp

260 265 270

Ile Ile Asn Ile Gly His Phe Asn Asp Pro Val His Gly Gly Ser Trp

275 280 285

Ile Arg Gly Ala Ile Tyr Tyr Phe Lys Ile Ala Val Ala Leu Ala Val

290 295 300

Ala Ala Ile Pro Glu Gly Leu Pro Ala Val Ile Thr Thr Cys Leu Ala

305 310 315 320

Leu Gly Thr Arg Arg Met Ala Lys Lys Asn Ala Ile Val Arg Ser Leu

325 330 335

Pro Ser Val Glu Thr Leu Gly Cys Thr Ser Val Ile Cys Ser Asp Lys

340 345 350

Thr Gly Thr Leu Thr Asn Gln Met Ser Val Cys Arg Met Phe Ile

355 360 365

Leu Asp Arg Val Glu Gly Asp Thr Cys Ser Leu Asn Glu Phe Thr Ile

370 375 380

Thr Gly Ser Thr Tyr Ala Pro Ile Gly Glu Val His Lys Asp Asp Lys

385 390 395 400

Pro Val Asn Cys His Gln Tyr Asp Gly Leu Val Glu Leu Ala Thr Ile

405 410 415

Cys Ala Leu Cys Asn Asp Ser Ala Leu Asp Tyr Asn Glu Ala Lys Gly

420 425 430

Val Tyr Glu Lys Val Gly Glu Ala Thr Glu Thr Ala Leu Thr Cys Leu

435 440 445

Val Glu Lys Met Asn Val Phe Asp Thr Glu Leu Lys Gly Leu Ser Lys

450 455 460

Ile Glu Arg Ala Asn Ala Cys Asn Ser Val Ile Lys Gln Leu Met Lys

465 470 475 480

Lys Glu Phe Thr Leu Glu Phe Ser Arg Asp Arg Lys Ser Met Ser Val

485 490 495

Tyr Cys Thr Pro Asn Lys Pro Ser Arg Thr Ser Met Ser Lys Met Phe

500 505 510

Val Lys Gly Ala Pro Glu Gly Val Ile Asp Arg Cys Thr His Ile Arg

515 520 525

Val Gly Ser Thr Lys Val Pro Met Thr Ser Gly Val Lys Gln Lys Ile

530 535 540

Met Ser Val Ile Arg Glu Trp Gly Ser Gly Ser Asp Thr Leu Arg Cys

545 550 555 560

Leu Ala Leu Ala Thr His Asp Asn Pro Leu Arg Arg Glu Glu Met His

565 570 575

Leu Glu Asp Ser Ala Asn Phe Ile Lys Tyr Glu Thr Asn Leu Thr Phe

580 585 590

Val Gly Cys Val Gly Met Leu Asp Pro Pro Arg Ile Glu Val Ala Ser

595 600 605

Ser Val Lys Leu Cys Arg Gln Ala Gly Ile Arg Val Ile Met Ile Thr

610 615 620

Gly Asp Asn Lys Gly Thr Ala Val Ala Ile Cys Arg Arg Ile Gly Ile

625 630 635 640

Phe Gly Gln Asp Glu Asp Val Thr Ser Lys Ala Phe Thr Gly Arg Glu

645 650 655

Phe Asp Glu Leu Asn Pro Ser Ala Gln Arg Asp Ala Cys Leu Asn Ala

660 665 670

Arg Cys Phe Ala Arg Val Glu Pro Ser His Lys Ser Lys Ile Val Glu

675 680 685

Phe Leu Gln Ser Phe Asp Glu Ile Thr Ala Met Thr Gly Asp Gly Val

690 695 700

Asn Asp Ala Pro Ala Leu Lys Lys Ala Glu Ile Gly Ile Ala Met Gly

705 710 715 720

Ser Gly Thr Ala Val Ala Lys Thr Ala Ser Glu Met Val Leu Ala Asp

725 730 735

Asp Asn Phe Ser Thr Ile Val Ala Ala Val Glu Glu Gly Arg Ala Ile

740 745 750

Tyr Asn Asn Met Lys Gln Phe Ile Arg Tyr Leu Ile Ser Ser Asn Val

755 760 765

Gly Glu Val Val Cys Ile Phe Leu Thr Ala Ala Leu Gly Phe Pro Glu

770 775 780

Ala Leu Ile Pro Val Gln Leu Leu Trp Val Asn Leu Val Thr Asp Gly

785 790 795 800

Leu Pro Ala Thr Ala Leu Gly Phe Asn Pro Asp Leu Asp Ile Met

805 810 815

Asn Lys Pro Pro Arg Asn Pro Lys Glu Pro Leu Ile Ser Gly Trp Leu

820 825 830

Phe Phe Arg Tyr Leu Ala Ile Gly Cys Tyr Val Gly Ala Ala Thr Val

835 840 845

Gly Ala Ala Ala Trp Trp Phe Ile Ala Ala Asp Gly Gly Pro Arg Val

850 855 860

Ser Phe Tyr Gln Leu Ser His Phe Leu Gln Cys Lys Glu Asp Asn Pro

865 870 875 880

Asp Phe Glu Gly Val Asp Cys Ala Ile Phe Glu Ser Pro Tyr Pro Met

885 890 895

Thr Met Ala Leu Ser Val Leu Val Thr Ile Glu Met Cys Asn Ala Leu

900 905 910

Asn Ser Leu Ser Glu Asn Gln Ser Leu Leu Arg Met Pro Pro Trp Glu

915 920 925

Asn Ile Trp Leu Val Gly Ser Ile Cys Leu Ser Met Ser Leu His Phe

930 935 940

Leu Ile Leu Tyr Val Glu Pro Leu Pro Leu Ile Phe Gln Ile Thr Pro

945 950 955 960

Leu Asn Val Thr Gln Trp Leu Met Val Leu Lys Ile Ser Leu Pro Val

965 970 975

Ile Leu Met Asp Glu Thr Leu Lys Phe Val Ala Arg Asn Tyr Leu Glu

980 985 990

Pro Ala Ile Leu Glu

995

<210> 2

<211> 2991

<212> DNA

<213> Homo sapiens

<400> 2

atggagaacg cgcacaccaa gacggtggag gaggtgctgg gccacttcgg cgtcaacgag 60

agtacggggc tgagcctgga acaggtcaag aagcttaagg agagatgggg ctccaacgag 120

ttaccggctg aagaaggaaa aaccttgctg gaacttgtga ttgagcagtt tgaagacttg 180

ctagttagga ttttattact ggcagcatgt atatcttttg ttttggcttg gtttgaagaa 240

ggtgaagaaa caattacagc ctttgtagaa ccttttgtaa ttttactcat attagtagcc 300

aatgcaattg tgggtgtatg gcaggaaaga aatgctgaaa atgccatcga agcccttaag 360

gaatatgagc ctgaaatggg caaagtgtat cgacaggaca gaaagagtgt gcagcggatt 420

aaagctaaag acatagttcc tggtgatatt gtagaaattg ctgttggtga caaagttcct 480

gctgatataa ggttaacttc catcaaatct accacactaa gagttgacca gtcaattctc 540

acaggtgaat ctgtctctgt catcaagcac actgatcccg tccctgaccc acgagctgtc 600

aaccaagata aaaagaacat gctgttttct ggtacaaaca ttgctgctgg gaaagctatg 660

ggagtggtgg tagcaactgg agttaacacc gaaattggca agatccggga tgaaatggtg 720

gcaacagaac aggagagaac accccttcag caaaaactag atgaatttgg ggaacagctt 780

tccaaagtca tctcccttat ttgcattgca gtctggatca taaatattgg gcacttcaat 840

gacccggttc atggagggtc ctggatcaga ggtgctattt actactttaa aattgcagtg 900

gccctggctg tagcagccat tcctgaaggt ctgcctgcag tcatcaccac ctgcctggct 960

cttggaactc gcagaatggc aaagaaaaat gccattgttc gaagcctccc gtctgtggaa 1020

acccttggtt gtacttctgt tatctgctca gacaagactg gtacacttac aacaaaccag 1080

atgtcagtct gcaggatgtt cattctggac agagtggaag gtgatacttg ttcccttaat 1140

gagtttacca taactggatc aacttatgca cctattggag aagtgcataa agatgataaa 1200

ccagtgaatt gtcaccagta tgatggtctg gtagaattag caacaatttg tgctctttgt 1260

aatgactctg ctttggatta caatgaggca aagggtgtgt atgaaaaagt tggagaagct 1320

acagagactg ctctcacttg cctagtagag aagatgaatg tatttgatac cgaattgaag 1380

ggtctttcta aaatagaacg tgcaaatgcc tgcaactcag tcattaaaca gctgatgaaa 1440

aaggaattca ctctagagtt ttcacgtgac agaaagtcaa tgtcggttta ctgtacacca 1500

aataaaccaa gcaggacatc aatgagcaag atgtttgtga agggtgctcc tgaaggtgtc 1560

attgacaggt gcacccacat tcgagttgga agtactaagg ttcctatgac ctctggagtc 1620

aaacagaaga tcatgtctgt cattcgagag tggggtagtg gcagcgacac actgcgatgc 1680

ctggccctgg ccactcatga caacccactg agaagagaag aaatgcacct tgaggactct 1740

gccaacttta ttaaatatga gaccaatctg accttcgttg gctgcgtggg catgctggat 1800

cctccgagaa tcgaggtggc ctcctccgtg aagctgtgcc ggcaagcagg catccgggtc 1860

atcatgatca ctggggaca caagggcact gctgtggcca tctgtcgccg catcggcatc 1920

ttcgggcagg atgaggacgt gacgtcaaaa gctttcacag gccgggagtt tgatgaactc 1980

aacccctccg cccagcgaga cgcctgcctg aacgcccgct gttttgctcg agttgaaccc 2040

tcccacaagt ctaaaatcgt agaatttctt cagtcttttg atgagattac agctatgact 2100

ggcgatggcg tgaacgatgc tcctgctctg aagaaagccg agattggcat tgctatgggc 2160

tctggcactg cggtggctaa aaccgcctct gagatggtcc tggcggatga caacttctcc 2220

accattgtgg ctgccgttga ggaggggcgg gcaatctaca acaacatgaa acagttcatc 2280

cgctacctca tctcgtccaa cgtcggggaa gttgtctgta ttttcctgac agcagccctt 2340

ggatttcccg aggctttgat tcctgttcag ctgctctggg tcaatctggt gacagatggc 2400

ctgcctgcca ctgcactgggg gttcaaccct cctgatctgg acatcatgaa taaacctccc 2460

cggaacccaa aggaaccatt gatcagcggg tggctctttt tccgttactt ggctattggc 2520

tgttacgtcg gcgctgctac cgtgggtgct gctgcatggt ggttcattgc tgctgacggt 2580

ggtccaagag tgtccttcta ccagctgagt catttcctac agtgtaaaga ggacaacccg 2640

gactttgaag gcgtggattg tgcaatcttt gaatccccat acccgatgac aatggcgctc 2700

tctgttctag taactataga aatgtgtaac gccctcaaca gcttgtccga aaaccagtcc 2760

ttgctgagga tgcccccctg ggagaacatc tggctcgtgg gctccatctg cctgtccatg 2820

tcactccact tcctgatcct ctatgtcgaa cccttgccac tcatcttcca gatcacaccg 2880

ctgaacgtga cccagtggct gatggtgctg aaaatctcct tgcccgtgat tctcatggat 2940

gagacgctca agtttgtggc ccgcaactac ctggaacctg caatactgga g 2991

<210> 3

<211> 250

<212> Protein

<213> Homo sapiens

<400> 3

Met Arg Gly Thr Pro Lys Thr His Leu Leu Ala Phe Ser Leu Leu Cys

1 5 10 15

Leu Leu Ser Lys Val Arg Thr Gln Leu Cys Pro Thr Pro Cys Thr Cys

20 25 30

Pro Trp Pro Pro Pro Arg Cys Pro Leu Gly Val Pro Leu Val Leu Asp

35 40 45

Gly Cys Gly Cys Cys Arg Val Cys Ala Arg Arg Leu Gly Glu Pro Cys

50 55 60

Asp Gln Leu His Val Cys Asp Ala Ser Gln Gly Leu Val Cys Gln Pro

65 70 75 80

Gly Ala Gly Pro Gly Gly Arg Gly Ala Leu Cys Leu Leu Ala Glu Asp

85 90 95

Asp Ser Ser Cys Glu Val Asn Gly Arg Leu Tyr Arg Glu Gly Glu Thr

100 105 110

Phe Gln Pro His Cys Ser Ile Arg Cys Arg Cys Glu Asp Gly Gly Phe

115 120 125

Thr Cys Val Pro Leu Cys Ser Glu Asp Val Arg Leu Pro Ser Trp Asp

130 135 140

Cys Pro His Pro Arg Arg Val Glu Val Leu Gly Lys Cys Cys Pro Glu

145 150 155 160

Trp Val Cys Gly Gln Gly Gly Gly Leu Gly Thr Gln Pro Leu Pro Ala

165 170 175

Gln Gly Pro Gln Phe Ser Gly Leu Val Ser Ser Leu Pro Pro Gly Val

180 185 190

Pro Cys Pro Glu Trp Ser Thr Ala Trp Gly Pro Cys Ser Thr Thr Cys

195 200 205

Gly Leu Gly Met Ala Thr Arg Val Ser Asn Gln Asn Arg Phe Cys Arg

210 215 220

Leu Glu Thr Gln Arg Arg Leu Cys Leu Ser Arg Pro Cys Pro Pro Ser

225 230 235 240

Arg Gly Arg Ser Pro Gln Asn Ser Ala Phe

245 250

<210> 4

<211> 750

<212> DNA

<213> Homo sapiens

<400> 4

atgagaggca caccgaagac ccacctcctg gccttctccc tcctctgcct cctctcaaag 60

gtgcgtaccc agctgtgccc gaccacatgt acctgcccct ggccacctcc ccgatgcccg 120

ctgggagtac ccctggtgct ggatggctgt ggctgctgcc gggtatgtgc acggcggctg 180

ggggagccct gcgaccaact ccacgtctgc gacgccagcc agggcctggt ctgccagccc 240

ggggcaggac ccggtggccg gggggccctg tgcctcttgg cagaggacga cagcagctgt 300

gaggtgaacg gccgcctgta tcgggaaggg gagaccttcc agccccactg cagcatccgc 360

tgccgctgcg aggacggcgg cttcacctgc gtgccgctgt gcagcgagga tgtgcggctg 420

cccagctggg actgccccca cccgggagg gtcgaggtcc tgggcaagtg ctgccctgag 480

tgggtgtgcg gccaaggagg gggactgggg acccagcccc ttccagccca aggaccccag 540

ttttctggcc ttgtctcttc cctgccccct ggtgtcccct gcccagaatg gagcacggcc 600

tggggaccct gctcgaccac ctgtgggctg ggcatggcca cccgggtgtc caaccagaac 660

cgcttctgcc gactggagac ccagcgccgc ctgtgcctgt ccaggccctg cccaccctcc 720

aggggtcgca gtccacaaaa cagtgccttc 750

<210> 5

<211> 22

<212> Protein

<213> Artificial sequence

<220>

<223> self-cleaving sequence derived from porcine teshovirus-1

<400> 5

Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val

1 5 10 15

Glu Glu Asn Pro Gly Pro

20

<210> 6

<211> 66

<212> DNA

<213> Artificial sequence

<220>

<223> self-cleaving sequence derived from porcine teshovirus-1

<400> 6

ggaagcggag ctactaactt cagcctgctg aagcaggctg gagacgtgga ggagaaccct 60

ggacct 66

<210> 7

<211> 21

<212> Protein

<213> Artificial sequence

<220>

<223> self-cleaving sequence derived from Thoseaasigna virus

<400> 7

Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu

1 5 10 15

Glu Asn Pro Gly Pro

20

<210> 8

<211> 63

<212> DNA

<213> Artificial sequence

<220>

<223> self-cleaving sequence derived from Thoseaasigna virus

<400> 8

ggaagcggag agggcagagg aagtctgcta acatgcggtg acgtcgagga gaatcctgga 60

cct 63

<210> 9

<211> 23

<212> Protein

<213> Artificial sequence

<220>

<223> self-cleaving sequence derived from equine rhinitis virus A

(ERAV)

<400> 9

Gly Ser Gly Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp

1 5 10 15

Val Glu Ser Asn Pro Gly Pro

20

<210> 10

<211> 69

<212> DNA

<213> Artificial sequence

<220>

<223> self-cleaving sequence derived from equine rhinitis virus A

(ERAV)

<400> 10

ggaagcggac agtgtactaa ttatgctctc ttgaaattgg ctggagatgt tgagagcaac 60

cctggacct 69

<210> 11

<211> 25

<212> Protein

<213> Artificial sequence

<220>

<223> self-cleaving sequence derived from FMDV 2A

<400> 11

Gly Ser Gly Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala

1 5 10 15

Gly Asp Val Glu Ser Asn Pro Gly Pro

20 25

<210> 12

<211> 75

<212> DNA

<213> Artificial sequence

<220>

<223> self-cleaving sequence derived from FMDV 2A

<400> 12

60

tccaaccctg gacct 75

<210> 13

<211> 2994

<212> DNA

<213> Homo sapiens

<400> 13

atggagaacg cgcacaccaa gacggtggag gaggtgctgg gccacttcgg cgtcaacgag 60

agtacggggc tgagcctgga acaggtcaag aagcttaagg agagatgggg ctccaacgag 120

ttaccggctg aagaaggaaa aaccttgctg gaacttgtga ttgagcagtt tgaagacttg 180

ctagttagga ttttattact ggcagcatgt atatcttttg ttttggcttg gtttgaagaa 240

ggtgaagaaa caattacagc ctttgtagaa ccttttgtaa ttttactcat attagtagcc 300

aatgcaattg tgggtgtatg gcaggaaaga aatgctgaaa atgccatcga agcccttaag 360

gaatatgagc ctgaaatggg caaagtgtat cgacaggaca gaaagagtgt gcagcggatt 420

aaagctaaag acatagttcc tggtgatatt gtagaaattg ctgttggtga caaagttcct 480

gctgatataa ggttaacttc catcaaatct accacactaa gagttgacca gtcaattctc 540

acaggtgaat ctgtctctgt catcaagcac actgatcccg tccctgaccc acgagctgtc 600

aaccaagata aaaagaacat gctgttttct ggtacaaaca ttgctgctgg gaaagctatg 660

ggagtggtgg tagcaactgg agttaacacc gaaattggca agatccggga tgaaatggtg 720

gcaacagaac aggagagaac accccttcag caaaaactag atgaatttgg ggaacagctt 780

tccaaagtca tctcccttat ttgcattgca gtctggatca taaatattgg gcacttcaat 840

gacccggttc atggagggtc ctggatcaga ggtgctattt actactttaa aattgcagtg 900

gccctggctg tagcagccat tcctgaaggt ctgcctgcag tcatcaccac ctgcctggct 960

cttggaactc gcagaatggc aaagaaaaat gccattgttc gaagcctccc gtctgtggaa 1020

acccttggtt gtacttctgt tatctgctca gacaagactg gtacacttac aacaaaccag 1080

atgtcagtct gcaggatgtt cattctggac agagtggaag gtgatacttg ttcccttaat 1140

gagtttacca taactggatc aacttatgca cctattggag aagtgcataa agatgataaa 1200

ccagtgaatt gtcaccagta tgatggtctg gtagaattag caacaatttg tgctctttgt 1260

aatgactctg ctttggatta caatgaggca aagggtgtgt atgaaaaagt tggagaagct 1320

acagagactg ctctcacttg cctagtagag aagatgaatg tatttgatac cgaattgaag 1380

ggtctttcta aaatagaacg tgcaaatgcc tgcaactcag tcattaaaca gctgatgaaa 1440

aaggaattca ctctagagtt ttcacgtgac agaaagtcaa tgtcggttta ctgtacacca 1500

aataaaccaa gcaggacatc aatgagcaag atgtttgtga agggtgctcc tgaaggtgtc 1560

attgacaggt gcacccacat tcgagttgga agtactaagg ttcctatgac ctctggagtc 1620

aaacagaaga tcatgtctgt cattcgagag tggggtagtg gcagcgacac actgcgatgc 1680

ctggccctgg ccactcatga caacccactg agaagagaag aaatgcacct tgaggactct 1740

gccaacttta ttaaatatga gaccaatctg accttcgttg gctgcgtggg catgctggat 1800

cctccgagaa tcgaggtggc ctcctccgtg aagctgtgcc ggcaagcagg catccgggtc 1860

atcatgatca ctggggaca caagggcact gctgtggcca tctgtcgccg catcggcatc 1920

ttcgggcagg atgaggacgt gacgtcaaaa gctttcacag gccgggagtt tgatgaactc 1980

aacccctccg cccagcgaga cgcctgcctg aacgcccgct gttttgctcg agttgaaccc 2040

tcccacaagt ctaaaatcgt agaatttctt cagtcttttg atgagattac agctatgact 2100

ggcgatggcg tgaacgatgc tcctgctctg aagaaagccg agattggcat tgctatgggc 2160

tctggcactg cggtggctaa aaccgcctct gagatggtcc tggcggatga caacttctcc 2220

accattgtgg ctgccgttga ggaggggcgg gcaatctaca acaacatgaa acagttcatc 2280

cgctacctca tctcgtccaa cgtcggggaa gttgtctgta ttttcctgac agcagccctt 2340

ggatttcccg aggctttgat tcctgttcag ctgctctggg tcaatctggt gacagatggc 2400

ctgcctgcca ctgcactgggg gttcaaccct cctgatctgg acatcatgaa taaacctccc 2460

cggaacccaa aggaaccatt gatcagcggg tggctctttt tccgttactt ggctattggc 2520

tgttacgtcg gcgctgctac cgtgggtgct gctgcatggt ggttcattgc tgctgacggt 2580

ggtccaagag tgtccttcta ccagctgagt catttcctac agtgtaaaga ggacaacccg 2640

gactttgaag gcgtggattg tgcaatcttt gaatccccat acccgatgac aatggcgctc 2700

tctgttctag taactataga aatgtgtaac gccctcaaca gcttgtccga aaaccagtcc 2760

ttgctgagga tgcccccctg ggagaacatc tggctcgtgg gctccatctg cctgtccatg 2820

tcactccact tcctgatcct ctatgtcgaa cccttgccac tcatcttcca gatcacaccg 2880

ctgaacgtga cccagtggct gatggtgctg aaaatctcct tgcccgtgat tctcatggat 2940

gagacgctca agtttgtggc ccgcaactac ctggaacctg caatactgga gtag 2994

<210> 14

<211> 753

<212> DNA

<213> Homo sapiens

<400> 14

atgagaggca caccgaagac ccacctcctg gccttctccc tcctctgcct cctctcaaag 60

gtgcgtaccc agctgtgccc gaccacatgt acctgcccct ggccacctcc ccgatgcccg 120

ctgggagtac ccctggtgct ggatggctgt ggctgctgcc gggtatgtgc acggcggctg 180

ggggagccct gcgaccaact ccacgtctgc gacgccagcc agggcctggt ctgccagccc 240

ggggcaggac ccggtggccg gggggccctg tgcctcttgg cagaggacga cagcagctgt 300

gaggtgaacg gccgcctgta tcgggaaggg gagaccttcc agccccactg cagcatccgc 360

tgccgctgcg aggacggcgg cttcacctgc gtgccgctgt gcagcgagga tgtgcggctg 420

cccagctggg actgccccca cccgggagg gtcgaggtcc tgggcaagtg ctgccctgag 480

tgggtgtgcg gccaaggagg gggactgggg acccagcccc ttccagccca aggaccccag 540

ttttctggcc ttgtctcttc cctgccccct ggtgtcccct gcccagaatg gagcacggcc 600

tggggaccct gctcgaccac ctgtgggctg ggcatggcca cccgggtgtc caaccagaac 660

cgcttctgcc gactggagac ccagcgccgc ctgtgcctgt ccaggccctg cccaccctcc 720

aggggtcgca gtccacaaaa cagtgccttc tag 753

Claims (35)

1. A gene construct for the prevention or treatment of heart failure, comprising: (i) a nucleotide sequence encoding a SERCA2a protein; and (ii) a nucleotide sequence encoding a CCN5 protein. 2. The gene construct according to claim 1, wherein the SERCA2a protein is the amino acid sequence described in SEQ ID NO: 1. 3. The gene construct according to claim 2, where the nucleotide sequence encoding the SERCA2a protein is the sequence described in SEQ ID NO: 2. 4. The gene construct according to claim 1, wherein the CCN5 protein is the amino acid sequence described in SEQ ID NO: 3. 5. The gene construct according to claim 4, wherein the nucleotide sequence encoding the CCN5 protein is the sequence described in SEQ ID NO: 4. 6. The gene construct according to claim 1, wherein the gene construct contains a self-cleaving sequence located between the nucleotide sequences (i) and (ii). 7. The gene construct according to claim 6, wherein the self-cleaving sequence is a nucleotide sequence encoding peptide 2A derived from porcine teshovirus-1, Thosea asigna virus, equine rhinitis A virus, or foot-and-mouth disease virus. 8. The gene construct according to claim 6, wherein the self-cleaving sequence is a nucleotide sequence encoding peptide 2A derived from porcine teshovirus-1. 9. The gene construct according to claim 6, wherein the self-cleaving sequence is the nucleotide sequence described in SEQ ID NO: 6. 10. The gene construct according to claim 1, wherein the gene construct contains the nucleotide sequences (i) and (ii) in the 5' to 3' direction in the order (i)-(ii). 11. The gene construct of claim 1, wherein the gene construct further comprises an operably linked promoter sequence. 12. The gene construct of claim 11, wherein the promoter is any promoter selected from the group consisting of cytomegalovirus promoter, adenovirus late promoter, vaccinia 7.5K promoter, SV40 promoter, HSV tk promoter, RSV promoter, EF1 alpha promoter, a metallothionein promoter, a beta-actin promoter, a human IL-2 gene promoter, a human IFN gene promoter, a human IL-4 gene promoter, a human lymphotoxin gene promoter, a human GM-CSF gene promoter, and a synthetic muscle- and heart-restricted promoter. 13. A recombinant expression vector loaded with a gene construct according to any one of claims 1-12. 14. The recombinant expression vector of claim 13, wherein the expression vector is any vector selected from the group consisting of plasmid vectors and cosmid vectors. 15. A recombinant viral vector containing a gene construct according to any one of claims 1-12. 16. The recombinant viral vector of claim 15, wherein the virus is any virus selected from the group consisting of adenovirus, adeno-associated virus (AAV), retrovirus, lentivirus, herpes simplex virus, and vaccinia virus. 17. The recombinant viral vector of claim 15, wherein the virus is an adeno-associated virus. 18. A pharmaceutical composition for the prevention or treatment of heart failure, containing as an active ingredient a gene construct according to claim 1, a recombinant expression vector according to claim 13, or a recombinant viral vector according to claim 15, and a pharmaceutically acceptable carrier. 19. A method for preventing or treating heart failure, comprising the step of administering to an individual a pharmaceutical composition according to claim 18. 20. Pharmaceutical composition for the prevention or treatment of heart failure, containing an expression vector loaded with a nucleotide sequence encoding the SERCA2a protein; and an expression vector loaded with a nucleotide sequence encoding a CCN5 protein and a pharmaceutically acceptable carrier. 21. Pharmaceutical composition for the prevention or treatment of heart failure, containing a recombinant virus that contains a nucleotide sequence encoding the SERCA2a protein; and a recombinant virus that contains a nucleotide sequence encoding a CCN5 protein and a pharmaceutically acceptable carrier. 22. A method for preventing or treating heart failure, comprising the steps of administering to an individual (i) an expression vector loaded with a nucleotide sequence encoding a SERCA2a protein; and (ii) an expression vector loaded with a nucleotide sequence encoding a CCN5 protein. 23. The method of claim 22, wherein the administration steps (i) and (ii) are carried out simultaneously. 24. The method of claim 22, wherein, after step (i) or (ii), another step of administration is carried out after a certain time interval. 25. A method for the prevention or treatment of heart failure, comprising the steps of administering (i) a recombinant virus that contains a nucleotide sequence encoding a SERCA2a protein; and (ii) a recombinant virus that contains a nucleotide sequence encoding a CCN5 protein. 26. The method of claim 25, wherein the administration steps (i) and (ii) are carried out simultaneously. 27. The method of claim 25, wherein, after step (i) or (ii), another step of administration is carried out after a certain time interval. 28. The use of a gene construct according to claim 1 for the prevention or treatment of heart failure. 29. The use of a recombinant expression vector according to claim 13 for the prevention or treatment of heart failure. 30. The use of a recombinant viral vector according to claim 15 for the prevention or treatment of heart failure. 31. The use of a gene construct according to claim 1 to obtain a pharmaceutical composition for the prevention or treatment of heart failure. 32. The use of a recombinant expression vector according to claim 13 for the preparation of a pharmaceutical composition for the prevention or treatment of heart failure. 33. The use of a recombinant viral vector according to claim 15 for the preparation of a pharmaceutical composition for the prevention or treatment of heart failure.

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