RU2778806C2 - Pharmaceutical composition for prevention or treatment of cardiac arrhythmia - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: use of a pharmaceutical composition including as an active ingredient a genetic structure, which contains a nucleotide sequence encoding protein CCN5, and a pharmaceutically acceptable carrier is proposed for the prevention or treatment of cardiac arrhythmia. The use of a pharmaceutical composition including as an active ingredient an expression vector carrying a nucleotide sequence encoding protein CCN5 and a pharmaceutically acceptable carrier is proposed for the prevention or treatment of cardiac arrhythmia. The use of a pharmaceutical composition including as an active ingredient a recombinant virus, which contains a nucleotide sequence encoding protein CCN5, and a pharmaceutically acceptable carrier is proposed for the prevention or treatment of cardiac arrhythmia. The use of a pharmaceutical composition including protein CCN5 and a pharmaceutically acceptable carrier is proposed for the prevention or treatment of cardiac arrhythmia. The use of a pharmaceutical composition containing as an active ingredient a genetic structure, which contains a nucleotide sequence encoding protein CCN5, or an expression vector carrying a nucleotide sequence encoding protein CCN5, or a recombinant virus, which contains a nucleotide sequence encoding protein CCN5, and a pharmaceutically acceptable carrier is also proposed for the manufacture of a drug for the prevention or treatment of cardiac arrhythmia. A method for the prevention or treatment of cardiac arrhythmia is proposed, including the administration to a subject of protein CCN5, as well as a method for the prevention or treatment of cardiac arrhythmia, including the administration of a pharmaceutical composition containing as an active ingredient a genetic structure, which contains a nucleotide sequence encoding protein CCN5, or an expression vector carrying a nucleotide sequence encoding protein CCN5, or a recombinant virus, which contains a nucleotide sequence encoding protein CCN5, and a pharmaceutically acceptable carrier.

EFFECT: invention can be effectively used for the prevention or treatment of cardiac arrhythmia.

26 cl, 1 tbl, 2 ex, 77 dwg

Description

Technical field

The present invention relates to a pharmaceutical composition for the prevention or treatment of cardiac arrhythmia. Specifically, the present invention relates to a pharmaceutical composition comprising, as an active ingredient, the CCN5 protein or a nucleotide sequence encoding the same.

Background of the invention

Cardiac arrhythmia is a heart disease caused by irregularities in the heart's rhythm and effective contraction of the heart due to problems with the electrical impulses in the heart. By definition, cardiac arrhythmia is classified into atrial arrhythmia and ventricular arrhythmia, depending on where it occurs. Specifically, atrial arrhythmia is classified into atrial fibrillation, atrial tachycardia, and sinus node dysfunction; and ventricular arrhythmias are classified into ventricular tachycardia and ventricular fibrillation (Swarminathan PD, et al. Circ Res 2012; 110:1661-1677).

Cardiac arrhythmia causes significant morbidity and mortality, especially in developed countries, and cardiac arrest is one of the leading causes of death in developed countries (Mozaffarian D, et al. Circulation 2015; 131: e29-e322). In particular, ventricular arrhythmia is the leading cause of sudden cardiac death, and other risk factors for heart disease precipitate and exacerbate ventricular arrhythmia (Roberts-Thomson KC, et al. Nat Rev Cardiol 2011; 8: 311-321). In addition, in the classification of cardiac arrhythmias, atrial fibrillation is the most common arrhythmia with an increasing incidence and significantly increases the incidence of diseases such as cardiac arrhythmia and stroke (Andrada D, et al. Circ Res 2014; 114; 1453-1458).

Therapies for the treatment of cardiac arrhythmia include antiarrhythmic drugs, catheter ablation, an implantable cardioverter defibrillator for the treatment of ventricular arrhythmias. However, these therapies have limited therapeutic effects. Blockade of ion channels, which is the main mechanism in the treatment of arrhythmias, has limitations in the long-term treatment and prevention of arrhythmias. In clinical studies related to the suppression of cardiac arrhythmias, in the treatment of extrasystole, antiarrhythmic drugs have been shown to increase mortality from cardiovascular diseases in patients with myocardial infarction. A common side effect of currently used antiarrhythmic drugs is the risk of arrhythmia (Camm J, Int Cardiol 2012; 155: 363-371).

Meanwhile, studies have shown that CaMKII (Ca ²⁺ /calmodulin-dependent protein kinase II) plays a central role in the electrical aspects of cardiac arrhythmia. Increased activity of CaMKII leads to hyperactivation of ion channels, defects in intracellular Ca ²⁺ homeostasis, and tissue damage, thereby contributing to arrhythmia. Thus, inhibition of CaMKII activity can be used as an effective way to treat arrhythmias.

In order to create an effective agent for the treatment of cardiac arrhythmia, there is a need to develop such an agent for treatment by understanding its pathological mechanism of action and discovering new therapeutic targets.

Accordingly, the present invention is intended to solve the problem of developing a treatment agent that simultaneously targets CaMKII, which is a key target of electrical dysfunction, and fibrosis, which is the main cause of structural dysfunction, as a treatment agent for the onset and course of arrhythmia and for prolonged worsening symptoms of arrhythmia.

Description of the invention

Technical task

In this regard, the present inventors conducted a study to develop an effective agent for treating cardiac arrhythmia, and as a result, found that a pharmaceutical composition that contains a gene encoding a CCN5 protein or a fragment thereof inhibits the pathological activity of CaMKII and inhibits the activity of myofibroblasts, thereby developing the present invention. In addition, the present inventors have found that a pharmaceutical composition that contains a gene encoding a CCN5 protein and a SERCA2a protein exhibits a synergistic therapeutic effect on electrical dysfunction in an animal model of cardiac arrhythmia, thereby developing the present invention.

The solution of the problem

In one aspect, the present invention provides a pharmaceutical composition for the prevention or treatment of cardiac arrhythmia, comprising as an active ingredient a genetic construct that contains a nucleotide sequence encoding a CCN5 protein or a fragment thereof.

In another aspect, the present invention provides a pharmaceutical composition for the prevention or treatment of cardiac arrhythmias, comprising as an active ingredient an expression vector carrying a nucleotide sequence encoding a CCN5 protein or a fragment thereof.

In yet another aspect, the present invention provides a pharmaceutical composition for the prevention or treatment of cardiac arrhythmia, comprising as an active ingredient a recombinant virus that contains a nucleotide sequence encoding a CCN5 protein or a fragment thereof.

In yet another aspect of the present invention, there is provided a method for preventing or treating cardiac arrhythmias, comprising the step of administering to a subject a pharmaceutical composition of the present invention.

In another aspect, the present invention provides a pharmaceutical composition for the prevention or treatment of cardiac arrhythmias, comprising the CCN5 protein as an active ingredient.

In another aspect, the present invention provides a method for preventing or treating a cardiac arrhythmia, comprising the step of administering a CCN5 protein to a subject.

In yet another aspect of the present invention, there is provided a method for preventing or treating cardiac arrhythmias, comprising the step of administering to a subject a genetic construct comprising a nucleotide sequence encoding a CCN5 protein.

In yet another aspect, the present invention provides a method for preventing or treating cardiac arrhythmias, comprising the step of administering to a subject an expression vector carrying a nucleotide sequence encoding a CCN5 protein.

In another aspect, the present invention provides a method for the prevention or treatment of cardiac arrhythmias, comprising the step of administering to a subject a recombinant virus containing a nucleotide sequence encoding a CCN5 protein.

Positive effects of the invention

The pharmaceutical composition for the prevention or treatment of cardiac arrhythmia of the present invention suppresses the pathological activity of CaMKII, which causes cardiac electrical dysfunction, the main cause of atrial arrhythmia and ventricular arrhythmia, so that the electrical functions of the heart are restored, and inhibits the activity of myofibroblasts, which cause structural dysfunction. Therefore, the pharmaceutical composition of the present invention can be effectively used to prevent or treat cardiac arrhythmia.

Brief description of the figures

In FIG. 1 shows a schematic representation of the structure of the pTR-CMV-CCN5 vector.

In FIG. 2 shows a schematic representation of the structure of the pTR-CMV-SERCA2a-P2A-CCN5 vector.

In FIG. 3a shows a graphical representation of a conceptual model of animal experiments using wild-type or CCN5 TG mice to determine the inhibitory effect of CCN5 protein on cardiac fibrosis.

In FIG. 3b shows photographs obtained by administering angiotensin II to wild-type or CCN5 TG mice for 14 days, extracting atrial tissue from it, and staining the atrial tissue with Masson's trichrome.

In FIG. 3c shows the results obtained by administering angiotensin II to wild-type or CCN5 TG mice for 14 days, extracting atrial tissues from them, staining the atrial tissues with Masson's trichrome, and quantifying the degree of fibrosis of the stained atrial tissues (*: p<0.05; **: p<0.01).

In FIG. 3d shows the results obtained by administering angiotensin II to wild-type or CCN5 TG mice for 14 days, extracting atrial tissues from them, and determining the level of α-SMA mRNA expression in atrial tissues by qRT-PCR (**: p<0.01 ).

In FIG. 3e shows the results obtained by administering angiotensin II to wild-type or CCN5 TG mice for 14 days, extracting atrial tissues from them, and determining the expression level of Collagen I mRNA in atrial tissues by qRT-PCR (*: p<0.05; * *: p<0.01).

In FIG. 3f shows the results obtained by administering angiotensin II to wild-type or CCN5 TG mice for 14 days, extracting atrial tissues from them, and determining the level of TGF-β1 mRNA expression in atrial tissues by qRT-PCR (*: p<0.05; **: p<0.01).

In FIG. 3g shows results obtained by administering angiotensin II to wild-type or CCN5 TG mice for 14 days, extracting atrial tissues from them, and determining the level of IL-1β mRNA expression in atrial tissues by qRT-PCR (*: p<0.05) .

In FIG. 3h shows the results obtained by administering angiotensin II to wild-type or CCN5 TG mice for 14 days, extracting atrial tissues from them, and determining the level of RANTES mRNA expression in atrial tissues by qRT-PCR (*: p<0.05; ** : p<0.01).

In FIG. 3i shows results obtained by administering angiotensin II to wild-type or CCN5 TG mice for 14 days, extracting atrial tissues from them, and determining by qRT-PCR the level of expression of F4/80 mRNA in atrial tissues (**: p<0.01 ).

In FIG. 3j shows results obtained by administering angiotensin II to wild-type or CCN5 TG mice for 14 days, extracting atrial tissues from them, and determining by qRT-PCR the level of MCP-1 mRNA expression in atrial tissues (**: p<0.01 ).

In FIG. 4a is a graphical representation of a conceptual model of animal experiments using wild-type or CCN5 TG mice to determine the inhibitory effect of CCN5 protein on atrial fibrillation.

In FIG. 4b shows the results obtained by administering angiotensin II to wild-type or CCN5 TG mice for 14 days, extracting the heart, applying electrical stimulation to it to induce atrial fibrillation, and obtaining an electrocardiogram.

In FIG. 4c shows the results obtained by administering angiotensin II to wild-type or CCN5 TG mice for 14 days, extracting the heart from them, and determining the rate of atrial fibrillation induced when the heart is exposed to electrical stimulation to induce atrial fibrillation.

In FIG. 5a shows a graphical representation of a conceptual model of experiments using HL-1 cells to determine the inhibitory effect of the CCN5 protein on atrial fibrillation.

In FIG. 5b shows results obtained by treating HL-1 cells with angiotensin II and simultaneously with CM-Con or CM-CCN5, cultured for 48 hours, and then determined by Western blotting for protein expression, p-CaMKII (Thr286), CaMKII, pRyR2 ( Ser2808), pRyR2 (Ser2814), RyR2, calsequestrin 2, Na ⁺ /Ca ⁺ exchanger 2 (NCX2) and GAPDH in HL-1 cells.

In FIG. 5c shows the results obtained by treating HL-1 cells with angiotensin II and simultaneously with CM-Con or CM-CCN5, cultured for 48 hours, and then calculated p-CaMKII (Thr286)/CaMKII values in HL-1 cells (** : p<0.01).

In FIG. 5d shows the results obtained by treating HL-1 cells with angiotensin II and simultaneously with CM-Con or CM-CCN5, cultured for 48 hours, and then calculated pRyR2 (Ser2808)/RyR2 values in HL-1 cells (**: p < 0.01).

In FIG. 5e shows the results obtained by treating HL-1 cells with angiotensin II and simultaneously with CM-Con or CM-CCN5, cultured for 48 hours, and then calculated pRyR2 (Ser2814)/RyR2 values in HL-1 cells (*: p<0 .05, **: p<0.01).

In FIG. 5f shows the results obtained by treating HL-1 cells with angiotensin II and simultaneously with CM-Con or CM-CCN5, cultured for 48 hours, and then calculated calsequestrin2/GAPDH values in HL-1 cells (*: p<0.05; **: p<0.01).

In FIG. 5g shows the results obtained by treating HL-1 cells with angiotensin II and simultaneously with CM-Con or CM-CCN5, cultured for 48 hours, and then calculated NCX2/GAPDH values in HL-1 cells (**: p<0.01 ).

In FIG. 6a shows a graphical representation of a conceptual model of experiments using rat atrial fibroblasts to determine the inhibitory effect of the CCN5 protein on atrial fibrillation.

In FIG. 6b shows photographs taken by treating rat atrial fibroblasts with angiotensin II and simultaneously with CM-Con or CM-CCN5, cultured for 48 hours, and then staining the cultured rat atrial fibroblasts with fluorescent immunochemistry.

In FIG. 6c shows results obtained by treating rat atrial fibroblasts with angiotensin II and simultaneously with CM-Con or CM-CCN5, cultured for 48 hours, and then determined by Western blotting for expression of proteins, α-SMA, Collagen I, TGF-β1 and α tubulin in atrial fibroblasts.

In FIG. 6d shows the results obtained by treating rat atrial fibroblasts with angiotensin II and simultaneously with CM-Con or CM-CCN5, performing culture for 48 hours, and then calculating α-SMA/α-tubulin values in atrial fibroblasts (*: p<0.05 **: p<0.01).

In FIG. 6e shows the results obtained by treating rat atrial fibroblasts with angiotensin II and simultaneously with CM-Con or CM-CCN5, cultured for 48 hours, and then calculated Collagen I/α-tubulin values in atrial fibroblasts (*: p<0.05; **: p<0.01).

In FIG. 6f shows results obtained by treating rat atrial fibroblasts with angiotensin II and concurrently with CM-Con or CM-CCN5, cultured for 48 hours, and then calculated TGF-β1/α-tubulin values in atrial fibroblasts (*: p<0.05 **: p<0.01).

In FIG. 6g shows the results obtained by treating rat atrial fibroblasts with angiotensin II and simultaneously with CM-Con or CM-CCN5, cultured for 48 hours, and then determined by qRT-PCR the level of expression of α-SMA mRNA in atrial tissues (*: p<0 .05).

In FIG. 6h shows the results obtained by treating rat atrial fibroblasts with angiotensin II and simultaneously with CM-Con or CM-CCN5, cultured for 48 hours, and then determined by qRT-PCR the expression level of Collagen I mRNA in atrial tissues (**: p<0 .01).

In FIG. 6i shows the results obtained by treating rat atrial fibroblasts with angiotensin II and simultaneously with CM-Con or CM-CCN5, cultured for 48 hours, and then determined by qRT-PCR the level of TGF-β1 mRNA expression in the atrial tissues (*: p<0 .05, **: p<0.01).

In FIG. 7a is a graphical representation of a conceptual model of animal experiments using mice with induced atrial fibrosis to determine the therapeutic effect of the AAV-CCN5 protein on atrial fibrosis.

In FIG. 7b shows the results obtained by administering angiotensin II to wild-type mice for 14 days, injecting them with AAV-Control or AAV-CCN5, extracting atrial tissue from them 4 weeks after injection, and determining CCN5 protein expression by Western blotting.

In FIG. 7c shows the results obtained by administering angiotensin II to wild-type mice for 14 days, injecting them with AAV-Control or AAV-CCN5, extracting atrial tissue from them 4 weeks after injection, and determining levels of CCN5 protein and mRNA expression.

In FIG. 7d shows photographs obtained by administering angiotensin II to wild-type mice for 14 days, injecting them with AAV-Control or AAV-CCN5, extracting atrial tissue from them 4 weeks after injection, and staining the atrial tissue with Masson's trichrome.

In FIG. 7e shows the results obtained by administering angiotensin II to wild-type mice for 14 days, injecting them with AAV-Control or AAV-CCN5, extracting atrial tissues from them 4 weeks after injection, staining the atrial tissues with Masson's trichrome, and quantifying fibrosis in stained atrial tissues (*: p<0.05).

In FIG. 7f shows results obtained by administering angiotensin II to wild-type mice for 14 days, injecting them with AAV-Control or AAV-CCN5, extracting atrial tissue from them 4 weeks after injection, and determining α-SMA/18s rRNA values by qRT-PCR in atrial tissues (*: p<0.05).

In FIG. 7g shows the results obtained by administering angiotensin II to wild-type mice for 14 days, injecting them with AAV-Control or AAV-CCN5, extracting atrial tissue from them 4 weeks after injection, and determining Collagen I/18s rRNA values by qRT-PCR in atrial tissues (*: p<0.05; **: p<0.01).

In FIG. 7h shows the results obtained by administering angiotensin II to wild-type mice for 14 days, injecting them with AAV-Control or AAV-CCN5, extracting atrial tissue from them 4 weeks after injection, and determining TGF-β1/18s rRNA values by qRT-PCR in atrial tissues (**: p<0.01).

In FIG. 7i shows the results obtained by administering angiotensin II to wild-type mice for 14 days, injecting them with AAV-Control or AAV-CCN5, extracting atrial tissue from them 4 weeks after injection, and determining IL-1β/18s rRNA values by qRT-PCR in atrial tissues (*: p<0.05).

In FIG. 7j shows the results obtained by administering angiotensin II to wild-type mice for 14 days, injecting them with AAV-Control or AAV-CCN5, extracting atrial tissues from them 4 weeks after injection, and determining RANTES/18s rRNA values in tissues by qRT-PCR atrial (**: p<0.01).

In FIG. 7k shows the results obtained by administering angiotensin II to wild-type mice for 14 days, injecting them with AAV-Control or AAV-CCN5, extracting atrial tissue from them 4 weeks after injection, and determining F4/80/18s rRNA values by qRT-PCR in atrial tissues (*: p<0.05; **: p<0.01).

In FIG. 7l shows the results obtained by administering angiotensin II to wild-type mice for 14 days, injecting them with AAV-Control or AAV-CCN5, extracting atrial tissue from them 4 weeks after injection, and determining MCP-1/18s rRNA values by qRT-PCR in atrial tissues (**: p<0.01).

In FIG. 8a shows a graphical representation of a conceptual model of animal experiments using mice with induced atrial fibrosis to determine the atrial fibrosis-suppressing effect of the AAV-CCN5 protein.

In FIG. 8b shows electrocardiogram data obtained by administering angiotensin II to wild-type mice for 14 days, injecting them with AAV-Control or AAV-CCN5, and obtaining an electrocardiogram 4 weeks after injection.

In FIG. 8c shows the results obtained by administering angiotensin II to wild-type mice for 14 days, injecting them with AAV-Control or AAV-CCN5, and observing the incidence of arrhythmia with electrical stimulation 4 weeks after injection.

In FIG. 8d shows the results obtained by administering angiotensin II to wild-type mice for 14 days, injecting them with AAV-Control or AAV-CCN5, and observing the intensity of electrical stimulation required to induce arrhythmia 4 weeks after injection.

In FIG. 8e shows the results obtained by administering angiotensin II to wild-type mice for 14 days, injecting them with AAV-Control or AAV-CCN5, and determining the Ca ²⁺ action potential 4 weeks after injection.

In FIG. 8f shows results obtained by administering angiotensin II to wild-type mice for 14 days, injecting them with AAV-Control or AAV-CCN5 and determining action potential duration 50 (APD ₅₀ ) and action potential duration 75 (APD ₇₅ ) 4 weeks after injection. .

In FIG. 8g shows the results obtained by administering angiotensin II to wild-type mice for 14 days, injecting them with AAV-Control or AAV-CCN5, and determining the rate of depolarization 4 weeks after injection (*: p<0.05).

In FIG. 9a shows the shortening fraction and change in body weight over 6 weeks in wild-type and ventricular arrhythmia-induced mice.

In FIG. 9b shows optical mapping obtained by applying 10 Hz electrical stimulation to the right ventricle (RV) of wild-type and ventricular-induced mice and photographing.

In FIG. 9c shows optical mapping obtained by applying 20 Hz electrical stimulation to the right ventricle (RV) of wild-type and ventricular-induced mice and photographing.

In FIG. 9d shows the results obtained by determining the action potential of Ca ²⁺ in wild-type mice and mice with induced ventricular arrhythmia.

In FIG. 9e shows the results obtained by determining action potential duration 50 (APD ₅₀ ) and action potential duration 75 (APD ₇₅ ) in wild-type mice and mice with induced ventricular arrhythmia (*: p<0.05).

In FIG. 9f shows the results obtained by determining the variance of action potential duration in wild-type mice and mice with induced ventricular arrhythmia (*: p<0.05).

In FIG. 9g shows conduction velocity and depolarization velocity in wild type and ventricular arrhythmia mice (***: p<0.001).

In FIG. 10a shows changes in Ca ²⁺ action potential with ISO treatment in wild-type and ventricular-induced mice.

In FIG. 10b shows changes in Ca ²⁺ repolarization pattern with ISO treatment in the right ventricle of wild-type and ventricular-induced mice.

In FIG. 10c shows changes in 75 action potential duration (APD ₇₅ ) with ISO treatment in wild-type and ventricular-induced mice.

In FIG. 10d shows Ca ²⁺ optical mapping obtained after ISO treatment of wild-type mice and mice with induced ventricular arrhythmia.

In FIG. 10e shows the results obtained after ISO treatment of wild-type mice and mice with induced ventricular arrhythmia and then determination of action potential duration 75 (APD ₇₅ ) (*: p<0.05).

In FIG. 10f shows changes in depolarization rate with ISO treatment in WT and ventricular arrhythmia-induced mice (***: p<0.001).

In FIG. 11a shows the shortening fraction over 6 weeks in WT mice, ventricular arrhythmia-induced mice injected with AAV9-Control, ventricular arrhythmia-induced mice injected with AAV9-CCN5, and ventricular arrhythmia-induced mice injected with AAV9-SERCA2a-P2A-CCN5 .

In FIG. 11b shows changes in left ventricular wall thickness over 6 weeks in WT mice, ventricular arrhythmia-induced mice injected with AAV9-Control, ventricular arrhythmia-induced mice injected with AAV9-CCN5, and ventricular arrhythmia-induced mice injected with AAV9-SERCA2a- P2A-CCN5.

In FIG. 11c shows Ca ²⁺ optical mapping for ventricular arrhythmia induced mice injected with AAV9-CCN5 and ventricular arrhythmia induced mice injected with ISO and AAV9-CCN5.

In FIG. 11d shows Ca ²⁺ optical mapping for ventricular arrhythmia-induced mice injected with AAV9-SERCA2a-P2A-CCN5 and ventricular arrhythmia-induced mice injected with ISO and AAV9-SERCA2a-P2A-CCN5.

In FIG. 11e shows changes in Ca ²⁺ action potential as a function of ISO treatment in ventricular arrhythmia induced mice injected with AAV9-CCN5 and ventricular arrhythmia induced mice injected with AAV9-SERCA2a-P2A-CCN5.

In FIG. 11f shows the pattern of Ca ²⁺ repolarization in the left ventricle of wild-type mice, ventricular arrhythmia-induced mice injected with AAV9-Control, ventricular arrhythmia-induced mice injected with AAV9-CCN5, and ventricular arrhythmia-induced mice injected with AAV9-SERCA2a-P2A- CCN5.

In FIG. 11g shows changes in action potential duration 75 (APD ₇₅ ) as a function of ISO treatment in wild-type mice, ventricular arrhythmia-induced mice injected with AAV9-Control, ventricular arrhythmia-induced mice injected with AAV9-CCN5, and ventricular arrhythmia-induced mice. injected with AAV9-SERCA2a-P2A-CCN5 (*: p<0.05; **: p<0.01; ***: p<0.001).

In FIG. 11h shows conduction velocity versus ISO treatment in wild-type mice, ventricular arrhythmia-induced mice injected with AAV9-Control, ventricular arrhythmia-induced mice injected with AAV9-CCN5, and ventricular arrhythmia-induced mice injected with AAV9-SERCA2a-P2A- CCN5 (*: p<0.05; ***: p<0.001).

In FIG. 11i shows depolarization rate versus ISO treatment in wild-type mice, ventricular arrhythmia-induced mice injected with AAV9-Control, ventricular arrhythmia-induced mice injected with AAV9-CCN5, and ventricular arrhythmia-induced mice injected with AAV9-SERCA2a-P2A- CCN5 (**: p<0.01; ***: p<0.001).

In FIG. 12a shows changes in the electrocardiogram observed when electrical stimulation was applied to wild-type mice, ventricular arrhythmia-induced mice injected with AAV9-Control, ventricular arrhythmia-induced mice injected with AAV9-CCN5, and ventricular arrhythmia-induced mice injected with AAV9. -SERCA2a-P2A-CCN5.

In FIG. 12b shows the intensity of electrical stimulation required to induce arrhythmia in wild-type mice, ventricular arrhythmia-induced mice injected with AAV9-Control, ventricular arrhythmia-induced mice injected with AAV9-CCN5, and ventricular arrhythmia-induced mice injected with AAV9-SERCA2a-P2A. -CCN5.

In FIG. 12c shows the frequency of electrically stimulated arrhythmias in wild-type mice, ventricular arrhythmia-induced mice injected with AAV9-Control, ventricular arrhythmia-induced mice injected with AAV9-CCN5, and ventricular arrhythmia-induced mice injected with AAV9-SERCA2a-P2A-CCN5.

In FIG. 13a shows photographs obtained by extracting hearts from wild-type mice, ventricular arrhythmia-induced mice injected with AAV9-Control, ventricular arrhythmia-induced mice injected with AAV9-CCN5, and ventricular arrhythmia-induced mice injected with AAV9-SERCA2a-P2A-CCN5 , and staining of the heart with Masson's trichrome.

In FIG. 13b shows results obtained by Western blot determination of protein expression, Nav 1.5, FAP, Connexin 43, MsrA, Kir2.1, tubulin, SERCA2a, Smad7, and CCN5 in heart tissue from wild-type mice, ventricular arrhythmia-induced mice injected with AAV9 -Control, ventricular arrhythmia-induced mice injected with AAV9-CCN5, and ventricular arrhythmia-induced mice injected with AAV9-SERCA2a-P2A-CCN5.

The best way to carry out the invention

Hereinafter, the present invention is described in detail.

In one aspect, the present invention provides a pharmaceutical composition for the prevention or treatment of cardiac arrhythmia, comprising as an active ingredient a genetic construct that contains a nucleotide sequence encoding a CCN5 protein or a fragment thereof.

As used herein, the term "CCN5 protein" refers to a matrix cellular protein belonging to the CCN family that plays various roles in the regulation of cellular functions such as vascular disease induction, angiogenesis, tumorigenesis, fibrosis induction, cell differentiation, and survival. The CCN5 protein, unlike other proteins of the CCN family, does not have a C-terminal domain and is also referred to as WISP-2, HICP, Cop1, CTGF-L, or the like. In addition, the CCN5 protein consists of a single polypeptide chain with a 250 amino acid sequence. Through a secretory leader sequence of 22 amino acids at the N-terminus, the CCN5 protein is secreted from the cell and functions as a signaling protein. Thus, when a nucleotide sequence is expressed in a cell, the CCN5 protein can be secreted from the cell. According to this document, the nucleotide sequence may be in the form of mRNA.

In particular, the CCN5 protein may have the amino acid sequence shown by SEQ ID NO: 1. In addition, the nucleotide sequence encoding the CCN5 protein can be the sequence shown by SEQ ID NO: 2 or SEQ ID NO: 41.

In addition, the fragment of the CCN5 protein may be a fragment obtained by truncating a portion of the N-terminus and/or C-terminus of wild-type CCN5, to the extent that this fragment maintains the activity of the CCN5 protein. Specifically, the CCN5 protein fragment may be a fragment obtained by shortening 1 to 30, 1 to 20, 1 to 10, or 1 to 5 amino acids from the N-terminus or C-terminus.

In addition, the genetic construct may contain a promoter sequence operably linked to it.

As used herein, the term "operably linked" refers to a functional relationship between an expression control nucleotide sequence (such as a promoter, a signal sequence, or a set of transcription factor binding sites) and other nucleotide sequences. A regulatory sequence regulates transcription and/or translation of other nucleotide sequences.

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

The promoter may be any promoter selected from the group consisting of the cytomegalovirus (CMV) promoter, adenovirus late promoter, vaccinia 7.5K promoter, SV40 promoter, HSV tk promoter, RSV promoter, EF1alpha promoter, metallothionein promoter, β-actin promoter, a human IL-2 gene promoter, a human IFN gene promoter, a human IL-4 gene promoter, a human lymphotoxin gene promoter, and a human GM-CSF gene promoter. However, the list of promoters is not limited to this. In particular, the promoter may be a CMV promoter.

The genetic construct may further comprise a nucleotide sequence encoding a SERCA2a protein or a fragment thereof. As used herein, the nucleotide sequence may be in the form of mRNA.

As used herein, in a genetic construct, the nucleotide sequence encoding the SERCA2a protein or fragment thereof may be in the 5' to 3' direction in the order "nucleotide sequence encoding the SERCA2a protein or fragment thereof-nucleotide sequence encoding the CCN5 protein or fragment thereof" . As used herein, the nucleotide sequence encoding the CCN5 protein or fragment thereof may contain a stop codon.

In addition, in a genetic construct, the nucleotide sequence encoding the SERCA2a protein or fragment thereof may be in the 5' to 3' direction in the order "nucleotide sequence encoding the CCN5 protein or fragment thereof-nucleotide sequence encoding the SERCA2a protein or fragment thereof" . As used herein, the nucleotide sequence encoding the SERCA2a protein or fragment thereof may contain a stop codon.

In addition, the genetic construct may further comprise a self-cleaving sequence between the nucleotide sequence encoding the SERCA2a protein or fragment thereof and the nucleotide sequence encoding the CCN5 protein or fragment thereof.

As used herein, the term "SERCA2a protein" refers to a protein that functions to cause calcium reuptake into the sarcoplasmic reticulum using the energy of ATP. It has been reported that patients suffering from heart failure with reduced ejection fraction (HFrEF) have a markedly reduced level of expression of the SERCA2a protein. Decreased reuptake of calcium into the sarcoplasmic reticulum, which is a consequence of a decrease in the expression of the SERCA2a protein, abnormally increases the calcium concentration in the cytoplasm, weakens the contraction-relaxation function of cardiomyocytes, and acts as a direct cause of death of cardiomyocytes, causing the formation of harmful oxygen, impaired energy metabolism, and the like from for the influx of calcium into the mitochondria.

In particular, the SERCA2a protein may have the amino acid sequence shown by SEQ ID NO: 3. In addition, the nucleotide sequence encoding the SERCA2a protein can be the sequence shown by SEQ ID NO: 4 or SEQ ID NO: 42.

In addition, a fragment of the SERCA2a protein may be a fragment obtained by truncating a portion of the N-terminus and/or C-terminus of wild-type SERCA2a, to the extent that this fragment maintains the activity of the SERCA2a protein. In particular, the SERCA2a protein fragment may be a fragment obtained by truncating 1 to 100, 1 to 50, 1 to 20, or 1 to 10 amino acids from the N-terminus or C-terminus.

The self-cleaving sequence may be 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. In particular, the self-cleaving sequence may be a nucleotide sequence encoding a 2A peptide derived from porcine teshovirus 1. In addition, the self-cleaving sequence may be the nucleotide sequence represented by 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 shown by SEQ ID NO: 5. In addition, the nucleotide sequence encoding the amino acid sequence shown by SEQ ID NO: 5 may be the nucleotide sequence shown in SEQ ID NO: 6.

The nucleotide sequence encoding the 2A peptide derived from Thosea asigna virus may be the nucleotide sequence encoding the amino acid sequence shown by SEQ ID NO: 7. In addition, the nucleotide sequence encoding the amino acid sequence shown by SEQ ID NO: 7 may be the nucleotide sequence represented by SEQ ID NO: 8.

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

The nucleotide sequence encoding the 2A peptide derived from FMDV may be the nucleotide sequence encoding the amino acid sequence shown by SEQ ID NO: 11. In addition, the nucleotide sequence encoding the amino acid sequence shown by SEQ ID NO: 11 may be the nucleotide sequence the sequence represented by SEQ ID no: 12.

When SERCA2a-P2A-CCN5, a genetic construct variant, is expressed in a cell, the SERCA2a protein can be incorporated into the sarcoplasmic reticulum membrane and the CCN5 protein can be secreted from the cell. In addition, when CCN5-P2a-SERCA2a, a variant of the genetic construct of the present invention, is expressed in a cell, the SERCA2a protein can be incorporated into the sarcoplasmic reticulum membrane, and the CCN5 protein can be secreted from the cell.

As used herein, the term "cardiac arrhythmia" refers to a disease in which the heart does not continue to beat regularly due to poor generation of electrical stimulation in the heart or poor transmission of stimulation, and the heart beat becomes abnormally fast, slow, or irregular.

Cardiac arrhythmia is classified into atrial arrhythmia and ventricular arrhythmia, depending on where it occurs. Atrial arrhythmias may include atrial fibrillation, atrial tachycardia, or sinus node dysfunction. Ventricular arrhythmia may include ventricular tachycardia or ventricular fibrillation.

In addition, the genetic construct of the present invention can be delivered to the cell using liposomes. Liposomes are formed automatically from phospholipids dispersed in an aqueous phase, and liposomes containing a nucleotide sequence encoding a CCN5 protein and/or a nucleotide sequence encoding a SERCA2a protein enable a nucleotide sequence encoding a CCN5 protein and/or a nucleotide sequence encoding a SERCA2a protein , be delivered to the cell through a mechanism such as endocytosis, adsorption on the cell surface, or fusion with the plasma membrane of the cell.

In another aspect, the present invention provides a pharmaceutical composition for the prevention or treatment of cardiac arrhythmias, comprising as an active ingredient an expression vector carrying a nucleotide sequence encoding a CCN5 protein or a fragment thereof.

The CCN5 protein is as described above in the description of a pharmaceutical composition for the prevention or treatment of cardiac arrhythmia, which contains a genetic construct as an active ingredient.

As used herein, the term "expression vector" refers to a recombinant vector capable of expressing a target protein in a target host cell, wherein the recombinant vector is a genetic construct that contains important regulatory elements operably linked to a gene insert such that the insert the gene is expressed.

In addition, the expression vector may contain a signal sequence for the secretion of the fusion polypeptide, so that the isolation of the protein from the cell culture is facilitated. Specific initiation signals may also be required for efficient translation of the inserted nucleic acid sequence. These signals contain an ATG start codon and adjacent sequences. In some cases, exogenous translational regulatory signals must be provided, which may contain an ATG start codon. These exogenous translational regulatory signals and start codons can come from a variety of natural and synthetic sources. Expression efficiency can be improved by introducing an appropriate transcription or translation enhancing element.

In addition, the expression vector may further contain a nucleotide sequence encoding a SERCA2a protein or a fragment thereof. As used herein, in an expression vector, the nucleotide sequence encoding the SERCA2a protein or fragment thereof may be in the 5' to 3' direction, in the order "nucleotide sequence encoding the SERCA2a protein or fragment thereof-nucleotide sequence encoding the CCN5 protein or fragment thereof". ".

The expression vector may further comprise a self-cleaving sequence between the nucleotide encoding the SERCA2a protein or fragment thereof and the nucleotide encoding the CCN5 protein or fragment thereof. The self-cleaving sequence is as described above in the description of a pharmaceutical composition for the prevention or treatment of cardiac arrhythmias, which contains the genetic construct as an active ingredient.

The expression vector may contain a nucleotide sequence encoding a CCN5 protein or fragment thereof, and/or a nucleotide sequence encoding a SERCA2a protein or fragment thereof, of the present invention. Herein, the vector to be used is not specifically limited as long as it can produce the CCN5 protein and/or the SERCA2a protein of the present invention. The expression vector may be any one selected from the group consisting of plasmid vectors and cosmid vectors.

The plasmid vector may include, but is not limited to, commercially available plasmids such as pUC18, pBAD, and pIDTSAMRT-AMP.

The cardiac arrhythmia is as described above in the description of a pharmaceutical composition for preventing or treating cardiac arrhythmia containing a genetic construct as an active ingredient.

In yet another aspect, the present invention provides a pharmaceutical composition for the prevention or treatment of cardiac arrhythmia, comprising as an active ingredient a recombinant virus that contains a nucleotide sequence encoding a CCN5 protein or a fragment thereof.

The CCN5 protein is as described above in the description of a pharmaceutical composition for the prevention or treatment of cardiac arrhythmias, which contains the genetic construct as an active ingredient.

The virus may be any vector selected from the group consisting of adenovirus, adeno-associated virus (AAV), retrovirus, lentivirus, herpes simplex virus, vaccinia virus, and the like. In particular, the virus may be, but is not limited to, an adeno-associated virus.

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

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

Thus, the CCN5 gene of the present invention can be inserted into remote E1 regions (E1A region and/or E1B region, preferably E1B region) or E3 region. In particular, the CCN5 protein gene can be inserted into the E3 region.

Meanwhile, the target nucleotide sequence to be delivered to the cell may be inserted into remote E1 regions (E1A region and/or E1B region, preferably E1B region) or E3 region, preferably E3 region. In addition, the target nucleotide sequence can also be expressed by a bicistronic expression system linked to an internal ribosome entry site (IRES), such as the promoter-target nucleotide sequence-poly A sequence-IRES-CCN5 protein gene.

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

Adenovirus has 42 different serotypes and subgroups from A to F. Of these, type 5 adenovirus belonging to subgroup C is suitable for obtaining the adenoviral vector of the present invention. Biochemical and genetic information about adenovirus type 5 is well known.

Foreign genes delivered by adenovirus replicate in the same way as episomes and thus 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 has a wide range of cells that it can infect.

To create a retroviral vector, the CCN5 gene and the target nucleotide sequence to be delivered are inserted into the retroviral genome instead of the retroviral sequence to produce a replication-incapable virus. To obtain virions, a packaging cell line is constructed that contains the gag, pol and env genes and lacks the long terminal repeat (LTR) and the ψ sequence. When a recombinant plasmid that contains the CCN5 gene, the target nucleotide sequence to be delivered, the LTR, and the ψ sequence is introduced into a cell line, the ψ sequence produces an RNA transcript of the recombinant plasmid. This transcript is packaged in the virus and the virus is secreted into the medium. The medium containing the recombinant retroviruses is harvested, enriched and used as a gene delivery system.

Adeno-associated virus (AAV) is suitable as a gene delivery system of the present invention because it is able to infect non-dividing cells and has the ability to infect various cell types. Details of the construction and use of AAV vectors are described in US Pat. Nos. 5,139,941 and 4,797,368.

Typically, an AAV virus is produced by co-transformation of a plasmid containing the target gene sequence (the CCN5 gene and the target nucleotide sequence to be delivered), which is flanked by two terminal repeats of AAV, and an expression plasmid containing the wild-type AVV coding sequence that lacks terminal repeats. .

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

In addition, the virus may further comprise a promoter sequence operably linked to the nucleotide sequence. An operably linked promoter sequence is as described above for a pharmaceutical composition for the prevention or treatment of cardiac arrhythmias that contains the genetic construct as an active ingredient.

In addition, the recombinant virus may additionally contain a nucleotide sequence encoding the SERCA2a protein or a fragment thereof. As used herein, in a recombinant virus, the nucleotide sequence encoding the SERCA2a protein or fragment thereof may be in the 5' to 3' direction in the order "nucleotide sequence encoding the SERCA2a protein or fragment thereof-nucleotide sequence encoding the CCN5 protein or fragment thereof" .

In addition, the recombinant virus may contain a self-cleaving sequence between the nucleotide sequence encoding the SERCA2a protein or fragment thereof and the nucleotide sequence encoding the CCN5 protein or fragment thereof. The self-cleaving sequence is as described above in the description of a pharmaceutical composition for the prevention or treatment of cardiac arrhythmias, which contains the genetic construct as an active ingredient.

The cardiac arrhythmia is as described above.

In the present invention, the method of administering a pharmaceutical composition that contains as an active ingredient a virus that contains a genetic construct can be carried out in accordance with methods of viral infection known in the art. In addition, according to the present invention, when the genetic construct as an active ingredient is contained in a recombinant naked DNA molecule or a plasmid, a microinjection method, a liposome-mediated transfection method, a DEAE-dextran treatment method, and a genetic bombardment method can be used to introduce the gene into cells.

The pharmaceutically acceptable carrier to be contained in the pharmaceutical composition of the present invention is one conventionally used to formulate it, and examples thereof include, but are 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 pharmaceutical composition may include, in addition to the ingredients listed above, a lubricant, a wetting agent, a sweetener, a flavor, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are detailed in Remington's Pharmaceutical Sciences (19th ^ed ., 1995).

The dosage form of the pharmaceutical composition may vary depending on the method of application and may be made in the form of injections.

The dose of the pharmaceutical composition of the present invention is desirably determined taking into account the age, sex, condition of the patient, the degree of absorption of the active substances by the body, the degree of inactivation, and the drugs used in combination; and the pharmaceutical composition may be administered in an amount of 0.0001 mg/kg (body weight) to 100 mg/kg (body weight) based on CCN5 protein.

The dose of the pharmaceutical composition of the present invention is desirably determined taking into account the age, sex, condition of the patient, the degree of absorption of the active substances by the body, the degree of inactivation, and the drugs used in combination; and when the pharmaceutical composition is a virus, the pharmaceutical composition may be administered in an amount of 1.0×10 ³ to 1.0×10 ²⁰ viral genomes per day per adult. In particular, the pharmaceutical composition of the present invention may be administered in an amount of 1.0×10 ³ to 1.0×10 ²⁰ , 1.0×10 ⁸ to 1.0×10 ¹⁶ , 1.0×10 ¹² up to 1.0×10 ¹⁵ or from 1.0×10 ¹³ to 1.0×0 ¹⁴ viral genomes per day per adult.

In addition, when the pharmaceutical composition is a plasmid vector, the pharmaceutical composition may be administered at a concentration of 0.1 µg/1 µl to 1 mg/1 µl per day per adult. In addition, when the pharmaceutical composition is a plasmid vector, the dose may include 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 includes all values and ranges in between.

In another aspect, the present invention provides a pharmaceutical composition for the prevention or treatment of cardiac arrhythmias, comprising the CCN5 protein as an active ingredient. The pharmaceutical composition may further include the SERCA2a protein. The CCN5 protein and the SERCA2a protein are as described above.

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

As used herein, the term "acceptable carrier" refers to some or all of the following substances and includes substances suitable for a particular dosage: solvents, diluents, liquid carriers, dispersants, suspension adjuvants, surfactants, isotonic agents, thickeners , emulsifiers, preservatives solid binders, lubricants or the like. In Alfanso R. Gennaro, Remington's Pharmaceutical Sciences, 19th edition, 1995, ^Macna Publishing Co. Easton, PA, provides a variety of carriers for use in pharmaceutical compositions with known methods and compositions. Examples of pharmaceutically acceptable carriers include, but are not limited to, the following: 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; at the discretion of the formulation manufacturer, inert excipients such as cocoa butter, suppository wax, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil and soybean oil may be included; 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; ethyl alcohol and phosphate buffered water, sodium lauryl sulfate and magnesium stearate, colorants, anti-adhesives, coating agents, sweeteners, flavors and fragrances, antioxidants, and the like.

In yet another aspect of the present invention, there is provided a method for preventing or treating cardiac arrhythmias, comprising the step of administering to a subject a pharmaceutical composition of the present invention.

The pharmaceutical composition may be a pharmaceutical composition for the prevention or treatment of cardiac arrhythmia, which contains as an active ingredient a genetic construct containing a nucleotide sequence encoding a CCN5 protein or a fragment thereof. In addition, the pharmaceutical composition may be a pharmaceutical composition for the prevention or treatment of cardiac arrhythmia, which contains, as an active ingredient, an expression vector carrying a nucleotide sequence encoding a CCN5 protein or a fragment thereof. In addition, the pharmaceutical composition may be a pharmaceutical composition for the prevention or treatment of cardiac arrhythmia, which contains, as an active ingredient, a recombinant virus that contains a nucleotide sequence encoding a CCN5 protein or a fragment thereof.

In this case, the subject may be a mammal, preferably a human. Specifically, the subject may be a human or other mammal that suffers from or may be at risk for cardiac arrhythmia.

In yet another aspect of the present invention, there is provided a method for preventing or treating cardiac arrhythmias, comprising administering a CCN5 protein to a subject. In addition, the method may further comprise administering the SERCA2a protein to the subject.

In this case, the subject may be a mammal, preferably a human. Specifically, the subject may be a human or other mammal that suffers from or may be at risk for cardiac arrhythmias.

In yet another aspect, the present invention provides a method for the prevention or treatment of cardiac arrhythmias, comprising administering to a subject a genetic construct that contains a nucleotide sequence encoding a CCN5 protein or a fragment thereof, and a genetic construct that contains a nucleotide sequence encoding a SERCA2a protein or a fragment thereof.

In this case, the subject may be a mammal, preferably a human. Specifically, the subject may be a human or other mammal that suffers from or may be at risk for cardiac arrhythmias.

In yet another aspect, the present invention provides a method for the prevention or treatment of cardiac arrhythmias, comprising administering to a subject an expression vector carrying a nucleotide sequence encoding a SERCA2a protein or a fragment thereof, and an expression vector carrying a nucleotide sequence encoding a CCN5 protein or a fragment thereof.

In this case, the subject may be a mammal, preferably a human. Specifically, the subject may be a human or other mammal that suffers from or may be at risk for cardiac arrhythmias.

In yet another aspect, the present invention provides a method for the prevention or treatment of cardiac arrhythmias, comprising administering to a subject 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 this case, the subject may be a mammal, preferably a human. Specifically, the subject may be a human or other mammal that suffers from or may be at risk for cardiac arrhythmias.

In yet another aspect of the present invention, the use of a pharmaceutical composition of the present invention for the prevention or treatment of cardiac arrhythmias is provided.

In yet another aspect of the present invention, the use of a pharmaceutical composition of the present invention for the manufacture of a medicament for the prevention or treatment of cardiac arrhythmias is provided.

The pharmaceutical composition may be a pharmaceutical composition for the prevention or treatment of cardiac arrhythmia, which contains, as an active ingredient, a genetic construct that contains a nucleotide sequence encoding a CCN5 protein or a fragment thereof. In addition, the pharmaceutical composition may be a pharmaceutical composition for the prevention or treatment of cardiac arrhythmia, which contains, as an active ingredient, an expression vector carrying a nucleotide sequence encoding a CCN5 protein or a fragment thereof. In addition, the pharmaceutical composition may be a pharmaceutical composition for the prevention or treatment of cardiac arrhythmia, which contains, as an active ingredient, a recombinant virus that contains a nucleotide sequence encoding a CCN5 protein or a fragment thereof.

Method for carrying out the invention

Hereinafter, the present invention is described in detail by way of 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 Preparative Examples and Examples.

Preparative Example 1 Construction of AAV9-CCN5 and AAV9-SERCA2a-P2A-CCN5

The pTR-CMV-CCN5 genetic construct was designed to express the CCN5 protein. In addition, the genetic construct pTR-CMV-SERC2a-P2A-CCN5 was designed to simultaneously express the CCN5 protein and the SERCA2a protein (FIGS. 1 and 2).

In the genetic construct, the SERCA2a fragment consists of the cDNA sequence of the human SERCA2a protein. The next linked P2A fragment is a self-cleavage site derived from lead teshovirus 1 and consists of a nucleotide sequence encoding 22 amino acids. Finally, the CCN5 fragment consists of the cDNA sequence of the human CCN5 protein.

The recombinant plasmid pTR-CMV-SERCA2a-P2A-CCN5 was constructed by removing part of the luciferase from the pTR-CMV-luciferase vector and inserting the SERCA2a-P2A-CCN5 genetic construct in its place. The protein produced by the recombinant plasmid was subdivided into a SERCA2a fragment and a CCN5 fragment by self-cleavage between amino acid 21 glycine and amino acid 22 proline at the P2A site. Part of SERCA2a can remain in the endoplasmic reticulum membrane and perform its built-in function. In addition, the CCN5 fragment can migrate to the endoplasmic reticulum and then be secreted from the cell in a form in which the signal peptide is cleaved, thereby performing its insertion function.

The human CCN5 gene was cloned into the pds-AAV2-EGFP vector to construct adeno-associated virus (AAV, serotype 9). To improve virus packaging and virus delivery efficiency, the AAV eGFP sequence was removed during vector construction. Recombinant AAV was constructed using 293T cells. AAV particles in cell culture were collected and precipitated with ammonium sulfate. The resulting product was purified by ultracentrifugation using an iodixanol gradient. The AAV particles were enriched by performing several dilutions and enrichments such that the iodixanol was replaced with Ringer's lactate solution using centrifugation. AAV concentration was quantified using quantitative RT-PCR and SDS-PAGE.

Experimental method 1. Creation of an experimental model and introduction of a gene

Experimental method 1.1. Creation of a mouse model of atrial fibrillation by infusion of angiotensin II into a mouse model by overexpression of CCN5

Male mice C57BL6 WT (wild type, black coat color) and transgenic (TG) mice were used for the experiments, which had heart-specific overexpression of CCN5.

CCN5-TG mice were obtained by subcloning the mouse CCN5 gene into a pNC vector (Clontech, USA) containing the α-MHC promoter, which induces heart-specific gene expression, and introducing the resulting product into fertilized C57BL/6 eggs using a microinjection technique. Additionally, sequencing was commissioned to Macrogen Inc. to acquire and maintain a significant strain of mice. (South Korea). Southern blotting was used to identify the presence of the CCN5 transgene in the mouse genome.

All mice used were 8 to 10 week old mice weighing 20 to 25 g. Mice were anesthetized by intraperitoneal injection of ketamine (95 mg/kg) and xylazine (5 mg/kg) and atrial fibrillation was induced by subcutaneous infusion of angiotensin II. In this case, angiotensin II was administered subcutaneously for 14 days at a concentration of 3.0 mg/kg per day using a small osmotic pump (Alzet 1002, Alzet).

Experimental method 1.2. Creation of a mouse model of atrial fibrillation by angiotensin II infusion and virus injection

8-10 week old B6C3F1 mice (gray coat) were anesthetized by intraperitoneal injection of ketamine (95 mg/kg) and xylazine (5 mg/kg), and atrial fibrillation was induced by subcutaneous infusion of angiotensin II. In this method, angiotensin II was infused subcutaneously for 2 weeks at a concentration of 3 mg/kg per day using a small osmotic pump. 2 weeks after induction of atrial fibrillation with angiotensin II infusion, each mouse was injected with 1×10 ¹¹ viral genomes (vgs) from AAV9-Control or AAV9-CCN5 via the tail vein.

Experimental method 1.3. Creation of a mouse model of ventricular arrhythmia by infusion of angiotensin II and introduction of the virus gene

8-10 week old B6C3F1 mice were anesthetized by intraperitoneal injection of ketamine (95 mg/kg) and xylazine (5 mg/kg), and ventricular arrhythmia was induced by subcutaneous infusion of angiotensin II. Angiotensin II was infused subcutaneously for 2 weeks at a concentration of 3 mg/kg per day using a small osmotic pump. 2 weeks after induction of ventricular arrhythmia by angiotensin II infusion, each mouse was injected with 1×10 ¹¹ vgs from AAV9-Control, AAV9-CCN5, or AAV9-SERCA2a-P2A-CCN5 via the tail vein.

Experimental method 2. Fabric staining

Animals were taken heart tissue and then fixed with 10% (w/v) formalin at room temperature for 5 days. Then washed with PBS. Each sample was embedded in paraffin and the tissue sample block was cut into 7 µm sections.

To measure the degree of fibrosis, Masson trichrome staining was performed. Tissue where fibrosis has progressed stains blue, while normal tissue stains red. The degree of fibrosis was expressed by calculating the fraction in which fibrosis occurred in the entire tissue. This was observed under an optical microscope and analyzed using the Aperio Imagescope software (Leica Biosystems).

Experimental Method 3: Determination of mRNA expression level by real-time PCR

Heart tissues were taken from animals and then mRNA was extracted from them for qRT-PCR. Real time PCR was performed using the QuantiTect SYBR Green Real Time PCR Kit (Qiagen Ltd). Through this, their transcription level was analyzed. RNA was isolated from heart tissue using Trizol (Gibco BRL) and cDNA was synthesized from it. Real time PCR quantitative conditions were as follows: 37 cycles at 94°C for 10 seconds, at 57°C for 15 seconds, at 72°C for 5 seconds. Data on the primers used in the experiment are shown in Table 1.

[Table 1]

primer Sequence Information SEQID No. mouse α-SMA-F 5'-CCCACCCAGAGTGGAGAA-3' SEQ ID NO: 13 mouse α-SMA-R 5'-ACATAGCTGGAGCAGCGTCT-3 SEQ ID NO: 14 Mice Collagen I-F 5'-CATGTTCAGCTTTGTGGACCT-3'' SEQ ID NO: 15 Mouse Collagen I-R 5'-GACGCTGACTTCAGGGATGT-3' SEQ ID NO: 16 mouse TGF-β1-F 5'-TGGAGCAACATGTGGAACTC-3' SEQ ID NO: 17 Mouse TGF-β1-R 5'-CAGCAGCCGGTTACCAAG-3' SEQ ID NO: 18 mouse IL-1β-F 5'-TCCAGGATGAGGACATGATGAGCA-3' SEQ ID NO: 19 mouse IL-1β-R 5'-GAACGTCACACACACCAGCAGGTTA-3' SEQ ID NO: 20 RANTES-F mice 5'-TGCAGAGGACTCTGAGACAGC-3' SEQ ID NO: 21 RANTES-R mice 5'-GAGTGGTGTCCGAGCCATA-3' SEQ ID NO: 22 F4/80-F mice 5'-CCTGGACGAATCCTGTGAAG-3' SEQ ID NO: 23 F4/80-R mice 5'-GGTGGGACCACAGAGAGTTG-3' SEQ ID NO: 24 MCP-1-F mice 5'-CATCCACGTGTTGGCTCA-3' SEQ ID NO: 25 MCP-1-R mice 5'-GATCATCTTGCTGGTGAATGAGT-3' SEQ ID NO: 26 CCN5-F mice 5'-ATACAGGTGCCAGGAAGGTG-3' SEQ ID NO: 27 CCN5-R mice 5'-GTTGGATACTCGGGTGGCTA-3' SEQ ID NO: 28 GAPDH-F mice 5'-CTCATGACCACAGTCCATGC-3' SEQ ID NO: 29 mouse GAPDH-R 5'-TTCAGCTCTGGGATGACCTT-3' SEQ ID no: 30 mouse 18s rRNA-F 5'-GTAACCCGTTGAACCCCATT-3' SEQ ID NO: 31 mouse 18s rRNA-R 5'-CCATCCCAATCGGTAGTAGCG-3' SEQ ID NO: 32 α-SMA-F rat 5'-TCTGTCTCTAGCACACAACTGTGAATG-3' SEQ ID NO: 33 Rat α-SMA-R 5'-TTGACAGGCCAGGGCTAGAAGGG-3' SEQ ID NO: 34 Collagen I-F rat 5'-AATGCACTTTTGGTTTTTGGTCACGT-3' SEQ ID NO: 35 Collagen I-R rat 5'-CAGCCCACTTTGCCCCAACCC-3' SEQ ID NO: 36 Rat TGF-β1-F 5'-TGTTCGCGCTCTCGGCAGTG-3' SEQ ID NO: 37 Rat TGF-β1-R 5'-CGGATGGCCTCGATGCGCTT-3' SEQ ID NO: 38 rat GAPDH-F 5'-ACCCAGCCCAGCAAGGATACTG-3' SEQ ID NO: 39 rat GAPDH-R 5'-ATTCGAGAGAAGGGAGGGCTCCC-3' SEQ ID NO: 40

Experimental method 4. Electrophysiological experimental method

Experimental method 4.1. Electrocardiogram measurement in atrial fibrillation model using CCN5 TG mice

For atrial electrophysiology studies, experiments were performed ex vivo by connecting a remote mouse heart to a Langendorff system. The removed heart was perfused with Krebs-Henseleit buffer (118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO ₄ , 1.25 mM CaCl ₂ , 1.2 mM KH ₂ PO ₄ , 25 mM NaHCO ₃ , 11 mM glucose) and 95% O ₂ /5% gaseous CO ₂ maintained a temperature of 37°C and a pressure of 60 mmHg. Before applying electrical stimulation, the remote heart was stabilized for 10 minutes by connecting to the Langendorff system. Then a Teflon-coated silver bipolar electrode was placed in the right atrium, left atrium, and left ventricle. To induce atrial fibrillation, 2-second pulsed pacing was performed three times using an automatic pacemaker. In the first 2 second pulsed stimulation, a 40 ms cycle duration pacing with a 5 ms pulse duration was applied. After application of the stimulus, stabilization was performed for 3 minutes. A second 2 second burst was applied with a cycle time of 20 ms with a pulse width of 5 ms and stabilization was performed again for 3 minutes. The last 2 second burst was applied with a cycle time of 20 ms with a pulse width of 10 ms. An atrium that showed an irregular RR interval of at least 1 second and showed an irregular, fast rhythm was found to have atrial fibrillation.

Experimental method 4.2. Electrocardiogram measurement in atrial fibrillation mice injected with AAV9-CCN5

Experiments were performed ex vivo by connecting the extracted mouse heart to the Langendorff system as described in Experimental Method 4.1. Specifically, mice were first injected with heparin to prevent blood clotting, and mice were anesthetized using 100% isoflurane (Forane, USP, Baxter Healthcare Corporation). After that, the mouse heart was removed and connected to the Langendorff system. A cannula was placed in the heart through the aorta, perfused with Tyrode's solution (NaCl 130 mM, NaHCO ₃ 24 mM, KCl 4 mM, MgCl ₂ 1 mM, CaCl ₂ 1.8 mM, KH ₂ PO ₄ 1.2 mM, C ₆ H ₁₂ O ₆ 5.6 mM, 1% albumin) at 1.5 to 2.0 min ^-1 flow and 95% O ₂ /5% CO ₂ gas, maintaining temperature 38±1°C, pH 7.3 to pH 7.5 and pressure from 60 mm to 70 mm Hg. (pressure sensor BP-1, World Precision Instruments). To minimize mechanical contraction of the heart, electromechanical decoupling (5 mM blebbistatin, Sigma Aldrich, USA) was used.

A custom-made Ag-AgCl pacemaker was placed in the right atrium and another electrode was attached to both the epicardial surface and the cardiac septum. The anterior surface of the heart was used to continuously display electrical activity in the left atrium in a semi-vertical manner (Mightex BioLED Light, source control module, BLS series).

Volumetric electrocardiography was performed to determine arrhythmias. Amplification and low-pass filtering were performed at 150 Hz using an electronic amplifier to continuously record a cardiac volume electrocardiogram. In addition, digital sampling was performed at 1 kHz (BioPac Systems MP150). To identify the occurrence of a persistent arrhythmia using electrical and optical signals, pacing was started at a fundamental rate of 7 Hz (PCL=140 ms) in the right atrium with a gradual increase in rate while applying a 2 ms pacing duration at a rate of 2 Hz to 3 Hz.

Experimental method 4.3. Experimental optical mapping method in mouse models of atrial fibrillation and ventricular arrhythmia

When the heart rate reached a steady state of 4 Hz to 5 Hz 20-30 minutes after the start of reperfusion of the heart, the heart was stained by injecting 0.3 ml of 15 μM voltage-sensitive dye (Di-4-ANEPPS, Invitrogen, Thermofisher Scientific). Monochromatic light (Mightex, BioLED) was used to excite fluorophores at a wavelength of 530 nm. The emitted wavelengths were filtered through a long band filter (>590 nm) and projected at a spatial resolution of 87.5 µm, a frame rate of 1 kHz, and an 80×80 pixel CCD (SciMeasure, SciMeasure Analytical Systems, USA) at 3× total magnification.

Experimental method 5. Western blotting

The cells used and the heart obtained in the present experiments were prepared using homogenized RIPA buffer (0.1% (w/v) SDS, 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% (w/v) ./vol) NP-40, 0.5% (w/v) sodium deoxycholate) supplemented with a broad-spectrum protease inhibitor cocktail (Calbiochem). Proteins were separated by size using SDS-PAGE gels and transferred to a PVDF membrane (Millipore). After blocking with 5% (w/v) skim milk for 1 hour and washing with TBST, the prepared membrane was allowed to react with p-CaMKII, CaMKII, Na ⁺ /Ca ⁺ exchanger 2 (NCX2), RyR2 (Santa Cruz), pRyR2 ( Ser2808), pRyR2 (Ser2814) (Badrilla), GAPDH (laboratory), α-tubulin, TGF-β1, α-SMA (Sigma), Collagen I (Rockland), NaV 1.5, connexin 43, Kir2.1 (Alomone labs) , fibroblast activation protein (FAP), mitochondrial peptide, methionine sulfoxide reductase (MsrA), tubulin (Abcam), ^SERCA2a (21st Century Biochemicals), Smad7 (Invitrogen), and CCN5 (Genescipt) antibodies. The membrane was then incubated with horseradish peroxidase conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA) and designed using a chemiluminescent substrate (Dogen). The blots were scanned and quantified using LAS software.

Experimental Method 6. Isolation and Culture of Atrial Fibroblasts

For the isolation of fibroblasts from the atria of white rats used the heart of white rats Sprague Dawley (SD). The left atrium was minced into single cells by tissue digestion using a collagenase solution. Cells harvested from the atrium were first centrifuged at 50×g for 3 minutes. The supernatant thus obtained was collected and centrifuged again at 500×g for 10 minutes. The cell layer thus obtained was cultured using a DMEM culture containing 10% fetal bovine serum (FBS) and 1% antibiotics. After 2-3 days, the culture was treated with 100 nM angiotensin II and simultaneously with control conditioned medium (CM-Con) or CCN5-containing conditioned medium (CM-CCN5). The experiment was terminated after 48 hours. For clear experimental results, only left atrial fibroblasts were used as left atrial fibroblasts.

Experimental Method 7 Preparation of CM-CCN5

To obtain CM-CCN5, the pcDNA3.1-CCN5HA plasmid was used. HEK293 cells were distributed 5×10 ⁵ cells in a 60 mm culture dish and stabilized for one day. pcDNA3.1-CCN5HA was then transfected using Lipofectamine (Invitrogen). After 4 hours, the medium was exchanged to remove lipofectamine. Then, cultivation was carried out for 24 hours, and the resulting culture medium was named CM-CCN5.

Experimental method 8. Fluorescent immunochemistry

15,000 cells were spread on a 16 mm coverslip and incubated overnight for stabilization. The cells were then treated with 100 nM angiotensin II and CM-Con or CM-CCN5 and incubated. The resulting cells were fixed with a 4% (w/v) paraformaldehyde solution, permeating the cell membrane using a 0.5% Triton X-100 solution, and then blocked with a 5% (w/v) BSA solution. An anti-α-SMA antibody (Sigma) was then reacted and an Alexa Fluor 488 conjugated antibody (Invitrogen) was used as a secondary antibody. The nuclei were stained using Hoechst stain. For cells that were subjected to immunochemistry, a Fluoview FV 1000 confocal microscope was used.

Experimental method 9. Measurement of myocardial function using echocardiography

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

Experimental Example 1 Detection of the therapeutic effect of CCN5 protein in a mouse model of atrial fibrillation

Experimental example 1.1. Identification of the effect of atrial fibrillation suppression by the CCN5 protein

WT mice and CCN5 TG mice prepared by the method described in Experimental Method 1.1 were infused subcutaneously with angiotensin II for 14 days at a concentration of 3.0 mg/kg per day using a small osmotic pump (Alzet 1002, Alzet). After 2 weeks, the hearts were removed from the mice. Tissue staining was performed in the manner described in Experimental Method 2 to determine the degree of fibrosis (FIG. 3a).

As a result, in the control group of mice injected with angiotensin II, collagen was found to accumulate in approximately 8% of the atrial tissues; on the other hand, in the group of CCN5 TG mice injected with angiotensin II, collagen accumulation was observed in about 4% of the atrial tissues, indicating a significant decrease (Fig. 3b and 3c).

In addition, to identify changes occurring in mRNA expression, mRNA was isolated from the mouse left atrium and qRT-PCR was performed in the manner described in Experimental Method 3.

As a result, cardiac fibrosis marker genes, α-SMA, Collagen I and TGF-β1 and inflammatory response marker genes, IL-1β, RANTES (regulated upon activation, normal T cells are expressed and secreted), F4/80, protein 1 monocyte chemoattractant (MCP-1) expression of their mRNA was increased in the control group of mice that were injected with angiotensin II; on the other hand, the expression of these marker genes was significantly reduced in the group of CCN5 TG mice injected with angiotensin II (Fig. 3d-3j). Based on these results, it was found that CCN5 effectively suppresses angiotensin II-induced atrial fibrosis.

Experimental example 1.2. Identification of the suppression effect of CCN5 atrial fibrillation

For atrial electrophysiology studies, experiments were performed ex vivo by connecting a remote mouse heart to the Langendorff system in the same manner as in experiment 4.1. As a result, in WT mice injected with angiotensin II, atrial fibrillation was induced in 4 out of 6 animals. However, in CCN5 TG mice injected with angiotensin II, all 4 animals showed normal electrocardiogram results even after electrical stimulation (FIGS. 4b and 4c). Based on these results, CCN5 was found to inhibit angiotensin II-induced atrial fibrillation.

Experimental example 1.3. Revealing regulation of intracellular Ca concentration ²⁺ using CCN5

The suggestion that CCN5 regulates Ca ²⁺ in cardiomyocytes was investigated using HL-1 cells (Sigma), which is a mouse atrial cardiomyocyte cell line. In addition, Western blotting was used to reveal its regulatory effect on Ca ²⁺ concentration in cultured HL-1 cells.

In experiments at the cellular level, CM-CCN5 obtained by the method described in Experimental Example 8 was used to detect the effect of CCN5. HL-1 cells were treated with angiotensin II and simultaneously with CM-Con (control) or CM-CCN5 (experimental group). After 48 hours, changes in protein levels were monitored by Western blotting (FIG. 5a).

As a result, it was found that CaMKII (Thr287) phosphorylation increased when HL-1 cells were treated with 400 nM angiotensin II, while CaMKII phosphorylation decreased in cells that were treated simultaneously with CM-CCN5. In HL-1 cells that had been treated with angiotensin II, hyperphosphorylation of the sarcoplasmic reticulum ryanodine receptor 2 (RyR2) at Ser2808 and Ser2814 was induced; however, such hyperphosphorylation was inhibited by CCN5. Increased expression of Na ⁺ /Ca ⁺ exchanger 2 (NCX2), caused by angiotensin II, was also reduced by CCN5. The level of expression of calsequestrin 2, a calcium-binding protein in the sarcoplasmic reticulum, was reduced by angiotensin II, while its expression was increased in cells that were subjected to simultaneous treatment with CM-CCN5 (Fig. 5b-5g). Based on these results, it was found that CCN5 directly regulates cardiomyocytes and thus is involved in atrial fibrillation.

Experimental example 1.4. Identification of the effect of suppression of atrial fibrosis using CCN5 in vitro

The heart of white Sprague Dawley (SD) rats was used to isolate fibroblasts from the atrium. Atrial fibroblasts were isolated by the method described in Experimental Method 6. After 2-3 days, the isolated atrial fibroblasts were treated with 100 nM angiotensin II simultaneously with control conditioned medium (CM-Con) or CCN5-containing conditioned medium (CM-CCN5) prepared in Experimental Example 8. After 48 hours the experiment was terminated (Fig. 6a). Fibroblasts that were treated with angiotensin II and CM-Con or CM-CCN5 were subjected to fluorescent immunocytochemistry in the same manner as in experimental method 8.

As a result, fibroblasts that were treated with 100 nM angiotensin II differentiated into myofibroblasts and expressed the myofibroblast-specific marker protein α-SMA, while fibroblasts that were simultaneously treated with CM-CCN5 did not express α-SMA at all (Fig. 6b). ).

In addition, in order to detect the effect of inhibition of atrial fibrosis, the cultured atrial fibroblasts were subjected to Western blotting according to the method described in Experimental Method 5. As a result, the fibroblasts that were treated with angiotensin II showed increased expression of α-SMA, Collagen I, and TGF-β1 proteins. while fibroblasts that were co-treated with CM-CCN5 showed markedly reduced expression of these proteins (FIGS. 6c-6f).

In addition, to detect the mRNA expression level of cultured atrial fibroblasts, the suppression effect of atrial fibrosis was monitored by qRT-PCR. As a result, it was found that in fibroblasts that were treated with angiotensin II and simultaneously with CM-CCN5, the expression levels of α-SMA, Collagen I and TGF-β1 mRNA were reduced to the control level (Fig. 6g-6i). Based on these results, it was found that CCN5 not only regulates fibrosis and thus inhibits atrial fibrillation, but also directly regulates cardiomyocytes and thus is involved in atrial fibrillation.

Experimental example 1.5. Detection of the suppression effect of AAV-CCN5 atrial fibrosis in a mouse model of atrial fibrillation

In the same manner as in experimental method 1.2, angiotensin II was infused into 8-10 week old mice at a concentration of 3 mg/kg/day. At time 2 weeks, AAV9-Control (control) or AAV9-CCN5 (comparative group) was injected into the tail vein in an amount of 5×10 ¹¹ viral genomes (vgs). After 4 weeks, the atrial tissue was subjected to Masson trichrome staining and molecular analysis (Fig. 7a).

First, to determine whether the AAV9 virus was well expressed in a heart-specific manner, Western blotting was used to detect CCN5 expression in atrial tissue. As a result, it was found that the CCN5 protein was overexpressed in the group of mice injected with AAV9-CCN5. In addition, from the results obtained by identifying the expression level of CCN5 mRNA in atrial tissue, it was found that CCN5 mRNA was similarly increased by injection of AAV9-CCN5 (FIGS. 7b and 7c).

From the results obtained by determining the degree of accumulation of collagen in the atrium by trichrome staining, it was found that in mice injected with angiotensin II and then AAV9-Control, collagen accumulated in approximately 6% of the atrial tissue, while in the group of mice injected with AAV9-CCN5 , the degree of collagen accumulation was reduced to about 3% (Fig. 7d and 7e).

In addition, qRT was performed to determine mRNA expression levels of fibrosis-associated marker genes, α-SMA, Collagen I, and TGF-β1, and inflammation-associated marker genes, IL-1β, RANTES, F4/80, and MCP-1. -PCR using atrial tissue in the same manner as in Experimental Method 3. As a result, AAV9-CCN5 was found to significantly decrease these marker genes, which were upregulated by angiotensin II (Fig. 7f-7l). Based on these results, it was found that even in a mouse model with heart-specific overexpression of CCN5 using AAV9-CCN5, angiotensin II-induced atrial fibrosis was effectively suppressed.

Experimental example 1.6. Identification of atrial fibrillation suppression effect exhibited by AAV-CCN5

The mouse model was generated in the same manner as in experimental method 4.2, and cardiophysiological experiments were performed at the end of week 6 (Fig. 8a). First, the electrocardiogram was checked, and as a result, it was found that in the group of mice injected with AAV9-Control, atrial function was attenuated by angiotensin II and thus atrial fibrillation was permanently induced after electrical stimulation. On the other hand, it was observed that in the group of mice injected with AAV9-CCN5, the heartbeat returned to normal after electrical stimulation. Even in experiments in which the Ca ²⁺ action potential (optical signal) was measured, the AAV9-Control injected group consistently showed conduction disturbances after pacing, while the AAV9-CCN5 injected group showed normal atrial activation after pacing (Fig. 8b and 8c).

As a result of analysis of the electrocardiogram in the group of mice injected with AAV9-Control, atrial fibrillation was induced in 4 out of 5 mice after electrical stimulation, while in the group of mice injected with AAV9-CCN5, 2 out of 5 mice had atrial abnormalities, and the rest showed normal atrial activation (Fig. 8d).

In electrocardiogram experiments where electrical stimulation was used to induce atrial fibrillation, a control (placebo) group of mice required approximately 28 Hz stimulation to induce atrial fibrillation; however, in control mice injected with angiotensin II and AAV9-Control, atrial fibrillation was sufficiently induced even with pacing as low as about 20 Hz. On the other hand, it was found that in the group of mice that were induced only when the stimulation was increased to about 25 Hz, it was similar to the level in the placebo group (Fig. 8e).

As described in Experimental Method 4.2, electrical activity in the left atrium was mapped to graphically represent the Ca ²⁺ action potential. The graph was analyzed at each moment of action potential duration 50 (APD ₅₀ ) and action potential duration 75 (APD ₇₅ ). As a result, it was found that the AAV9-CCN5 injected group developed action potential durations at the same rate as the placebo group compared to the AAV9-Control injected group (FIG. 8f).

This indicates the rate required for depolarization of the action potential, and it was found that in the group of mice injected with AAV9-CCN5, the rate required for depolarization, which was slowed down by angiotensin II, was restored to a placebo-like level (Fig. 8g) . Based on these results, it was found that even in a group of mice in which CCN5 overexpressed by injection of AAV9-CCN5, angiotensin II-induced atrial fibrillation was reliably suppressed.

Experimental Example 2 Determination of the Therapeutic Effect of AAV-CCN5 and AAV9-SERCA2a-P2A-CCN5 in a Mouse Model of Ventricular Arrhythmia

Experimental example 2.1. Creation of a mouse model of ventricular arrhythmia

A mouse model of ventricular arrhythmia was created in the manner described in Experimental Method 1.3. The male B6C3F1 WT mice (wild type, grey) used were 8-10 week old mice weighing 20 to 25 g. Angiotensin II was infused subcutaneously for 2 weeks at a concentration of 3 mg/kg. After 2 weeks, echocardiography revealed changes in cardiac function. Identified mice were randomly selected and injected with only the CCN5 vector (AAV9-CCN5) or a combination of CCN5 and SERCA2a vectors (AAV9-SERCA2a-P2A-CCN5) in an amount of 5×10 ¹¹ viral genomes (vgs). These mice were then housed for an additional 4 weeks for final functional evaluation.

First, to establish that the mouse ventricular arrhythmia model was well produced by angiotensin II, cardiac function in the control group of mice (placebo) and in the comparison group (AngII) were checked by echocardiography. As a result, in the group of mice injected subcutaneously with angiotensin II, there was a decrease in the shortening fraction compared to the control (placebo). However, the two groups did not show much difference in terms of mouse weight (Fig. 9a).

To determine ventricular tachycardia, optical mapping was performed by applying 10 Hz electrical stimulation (Fig. 9b) or 20 Hz electrical stimulation (Fig. 9c) to the right ventricle (RV) in the same manner as in experimental method 4.3. The greater the electrical stimulation, the more pronounced the difference was observed between the experimental group and the control. In the group of mice administered with angiotensin II, when electrical stimulation at 20 Hz was applied, the calcium ion channel or the like was not locally restored to its original state, and thus a break in stimulation transmission was observed. The calcium action potential was plotted and illustrated in FIG. 9d. The graph was analyzed at each time point of action potential duration 50 (APD ₅₀ ) and action potential duration 75 (APD ₇₅ ). As a result, it was found that a faster action potential duration was achieved by injection of angiotensin II (Fig. 9e). In addition, in the angiotensin II group, there was a significant decrease in the rate of duration of the action potential (Fig. 9f). As a result of the analysis of the conduction velocity and the velocity required for the depolarization of the action potential, it was found that such rates were significantly reduced by angiotensin II (Fig. 9g). Based on these results, the mouse model of ventricular arrhythmia was found to be significantly produced by angiotensin II.

Experimental example 2.2. Effects on β-adrenergic receptor antagonists in a mouse model of ventricular arrhythmia

Isoproterenol (ISO) is a drug that clinically acts on β-adrenergic receptors and thus increases myocardial contractility, thereby increasing cardiac output. In an angiotensin II-induced ventricular arrhythmia mouse model, a Ca ²⁺ action potential was established in the ventricle when isoproterenol was used to stimulate β-receptors (FIG. 10a). Electrical stimulation at 20 Hz was applied to the right ventricle and the resulting pattern of Ca ²⁺ repolarization is shown in the diagram.

As a result, it was found that in the mouse model injected with angiotensin II, the pattern of calcium repolarization was slower even with ISO treatment (Fig. 10b). As a result of the analysis of the duration of action potential 75 (APD ₇₅ ), it was found that in the mouse model injected with angiotensin II, ISO treatment did not give differences in the degree of duration of the action potential (Fig. 10c).

To determine ventricular tachycardia, optical mapping was performed by applying electrical stimulation at 20 Hz to the right ventricle (RV), just as in experimental method 4.3. As a result, a gap in the conduction block with stimulation was noted in the angiotensin II group of mice treated with ISO, compared to the control group of mice treated with ISO (Fig. 10d). As a result of the analysis of action potential duration 75 (APD ₇₅ ) compared with the placebo group, mice treated with angiotensin II showed a significantly increased action potential duration, but showed little change with ISO treatment (Fig. 10e). As a result of the conduction velocity analysis, it was found that the group of mice injected with angiotensin II showed a much slower velocity compared to placebo, and ISO treatment did not lead to restoration of the conduction velocity (Fig. 10f). From these results, it was found that angiotensin II injection rendered β-adrenergic receptors insensitive to the stimulus.

Experimental example 2.3. Determination of the therapeutic effect of AAV9-CCN5 and AAV9-SERCA2a-P2A-CCN5 in a mouse model of ventricular arrhythmia

The ventricular arrhythmia model was reliably obtained by the experimental method as described in Experimental Example 2.3. Angiotensin II was administered to mice for 2 weeks, and at the end of week 2, the AAV9-CCN5 or AAV9-SERCA2a-P2A-CCN5 vector was injected into the tail vein of the mouse. After 6 weeks, the experiment was terminated and experiments related to ventricular arrhythmia were performed.

Echocardiography was performed to determine cardiac function affected by AAV9-CCN5 and AAV9-SERCA2a-P2A-CCN5 in an angiotensin II-induced ventricular arrhythmia mouse model. Injection of AAV9-CCN5 or AAV9-SERCA2a-P2A-CCN5 caused a shortening fraction that was reduced by angiotensin II to show a shortening fraction that is the placebo level. In addition, as a result of analysis of the left ventricular wall thickness (LVSd), it was found that the thickness of the left ventricular wall, which was reduced by angiotensin II, was restored to almost normal levels of AAV9-SERCA2a-P2A-CCN5 (Fig. 11a and 11b).

The Ca ²⁺ action potential was shown using optical mapping. It was found that when the ventricle was exposed to electrical stimulation at 20 Hz, conduction regularly propagated in the AAV9-CCN5 or AAV9-SERCA2a-P2A-CCN5 treated group of mice, regardless of ISO treatment (FIGS. 11c and 11d). The calcium action potential was plotted and illustrated in FIG. 11e.

For the respective placebo groups, AngII, AngII+AAV9-CCN5, AngII+AAV9-SERCA2a-P2A-CCN5, ISO processing was performed and analysis was performed by repolarization mapping. As a result, it was found that injection of AAV9-CCN5 or AAV9-SERCA2a-P2A-CCN5 induced repolarization, which was slowed down by injection of angiotensin II to show a repolarization similar to placebo (Fig. 11f).

As a result of the analysis of the action potential duration of 75 (APD ₇₅ ) Ca ²⁺ , it was found that the duration of the action potential, which was increased by angiotensin II, was reduced by the injection of AAV9-CCN5 (Fig. 11g) and was reliably reduced to the placebo level by AAV9-SERCA2a -P2A-CCN5. In addition, it was found that the conduction velocity and depolarization rates, which were slowed down by angiotensin II, were normalized by injection of AAV9-CCN5 or AAV9-SERCA2a-P2A-CCN5 (FIGS. 11h and 11i). Based on these results, AAV9-CCN5 and AAV9-SERCA2a-P2A-CCN5 were found to be effective in the treatment of angiotensin II-induced ventricular arrhythmias.

In addition, programmed electrical pacing (PES) induced ventricular arrhythmia was identified in the angiotensin II injection model (Fig. 12a). As a result of pacing rate analysis to induce ventricular arrhythmia, ventricular arrhythmia was induced even by a low pacing rate in the AngII mice group, while ventricular arrhythmia was induced by a pacing rate that was equal to or greater than that of the placebo group in the injected mice group. AAV9-CCN5 or AAV9-SERCA2a-P2A-CCN5. As a result of the analysis of the incidence of ventricular arrhythmia, ventricular arrhythmia occurred with an episode rate of 5/5 in the AngII mice group, with an episode rate of 1/5 in the AngII+AAV9-CCN5 mice group, and with an episode rate of 0/5 in the AngII+AAV9-SERCA2a- mice group. P2A-CCN5 (FIGS. 12b and 12c).

Since cardiac arrhythmia is closely associated with cardiac fibrosis, cardiac fibrosis was determined by Masson trichrome staining. As a result, angiotensin II-induced cardiac fibrosis was found to be significantly reduced in the heart injected with AAV9-CCN5 or AAV9-SERCA2a-P2A-CCN5 (FIG. 13a).

In addition, this phenomenon is also closely associated with proteins associated with the channel that regulates the electrical impulses of the heart. Thus, the expression of Na _V 1.5 and connexin 43 proteins in heart tissue was determined using Western blotting. As a result, it was found that the decrease in the expression of the channel-associated proteins Na _V 1.5 and connexin 43, which was caused by angiotensin II, was restored by AAV9-SERCA2a-P2A-CCN5 (Fig. 13b). Based on these results, it was found that gene therapy using only the CCN5 vector or co-expression of SERCA2A and CCN5 can also suppress the occurrence of angiotensin II-induced ventricular tachycardia.

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Sequence listing

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

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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> 5

<211> 22

<212> PROTEIN

<213> Artificial sequence

<220>

<223> self-cleaving sequence derived from

teshovirus 1 swine

<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

twenty

<210> 6

<211> 66

<212> DNA

<213> Artificial sequence

<220>

<223> self-cleaving sequence derived from

teshovirus 1 swine

<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 Thosea asigna 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

twenty

<210> 8

<211> 63

<212> DNA

<213> Artificial sequence

<220>

<223> self-cleaving sequence derived from

thosea asigna 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

twenty

<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> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Alpha SMA Mouse Forward Primer

<400> 13

cccaccaga gtggagaa 18

<210> 14

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Alpha SMA mouse reverse primer

<400> 14

acatagctgg agcagcgtct 20

<210> 15

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Mouse Collagen I Forward Primer

<400> 15

catgttcagc tttgtggacc t 21

<210> 16

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Collagen I mice reverse primer

<400> 16

gacgctgact tcagggatgt 20

<210> 17

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer for mouse TGF-beta 1

<400> 17

tggagcaaca tgtggaactc 20

<210> 18

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Mouse TGF-beta 1 reverse primer

<400> 18

cagcagccgg ttaccaag 18

<210> 19

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer for mouse IL-1 beta

<400> 19

tccaggatga ggacatgatg agca 24

<210> 20

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Mouse IL-1 beta reverse primer

<400> 20

gaacgtcaca cacaccagca ggtta 25

<210> 21

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer for RANTES mouse

<400> 21

tgcagaggac tctgagacag c 21

<210> 22

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer for RANTES mouse

<400> 22

gagtggtgtc cgagccata 19

<210> 23

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer for F4/80 mice

<400> 23

cctggacgaa tcctgtgaag 20

<210> 24

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer for F4/80 mouse

<400> 24

ggtgggacca cagagagttg 20

<210> 25

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> MCP-1 Mouse Forward Primer

<400> 25

catccacgtg ttggctca 18

<210> 26

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> MCP-1 mouse reverse primer

<400> 26

gatcatcttg ctggtgaatg agt 23

<210> 27

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer for CCN5 mouse

<400> 27

atacaggtgc caggaaggtg 20

<210> 28

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Mouse CCN5 reverse primer

<400> 28

gttggatact cgggtggcta 20

<210> 29

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Mouse GAPDH Forward Primer

<400> 29

ctcatgacca cagtccatgc 20

<210> 30

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Mouse GAPDH reverse primer

<400> 30

ttcagctctg ggatgacctt 20

<210> 31

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer for mouse 18s rRNA

<400> 31

gtaacccgtt gaaccccatt 20

<210> 32

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer for mouse 18s pPPK

<400> 32

ccatccaatc ggtagtagcg 20

<210> 33

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Rat Alpha-SMA Forward Primer

<400> 33

tctgtctcta gcacacaact gtgaatg 27

<210> 34

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Rat alpha-SMA reverse primer

<400> 34

ttgacaggcc agggctagaa ggg 23

<210> 35

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> Rat Collagen I Forward Primer

<400> 35

aatgcacttt tggttttttgg tcacgt 26

<210> 36

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Rat Collagen I reverse primer

<400> 36

cagccctt tgccccaacc c 21

<210> 37

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer for rat TGF-beta 1

<400> 37

tgttcgcgct ctcggcagtg 20

<210> 38

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Rat TGF-beta 1 reverse primer

<400> 38

cggatggcct cgatgcgctt 20

<210> 39

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Rat GAPDH forward primer

<400> 39

acccagccca gcaaggatac tg 22

<210> 40

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Rat GAPDH reverse primer

<400> 40

attcgagaga agggagggct ccc 23

<210> 41

<211> 753

<212> DNA

<213> Homo sapiens

<400> 41

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

<210> 42

<211> 2994

<212> DNA

<213> Homo sapiens

<400> 42

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

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

<---

Claims (34)

1. The use of a pharmaceutical composition comprising as an active ingredient: a genetic construct that contains a nucleotide sequence encoding a CCN5 protein and a pharmaceutically acceptable carrier for the prevention or treatment of cardiac arrhythmias. 2. Use according to claim 1, wherein the CCN5 protein has the amino acid sequence shown in SEQ ID NO: 1. 3. Use according to claim 1, wherein the nucleotide sequence encoding the CCN5 protein is the sequence shown in SEQ ID NO: 2. 4. Use according to claim 1, wherein the genetic construct contains a promoter sequence operably linked to it. 5. Use according to claim 4, wherein the promoter is any promoter selected from the group consisting of CMV promoter, adenovirus late promoter, vaccinia 7.5K promoter, SV40 promoter, HSV tk promoter, RSV promoter, EF1alpha promoter, 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, and a human GM-CSF gene promoter. 6. Use according to claim 1, wherein the genetic construct further comprises a nucleotide sequence encoding a SERCA2a protein. 7. The use according to claim 6, wherein in the genetic construct, the nucleotide sequence encoding the SERCA2a protein is contained in the 5' to 3' direction in the order "nucleotide sequence encoding the SERCA2a protein -nucleotide sequence encoding the CCN5 protein". 8. Use according to claim 7, wherein the genetic construct contains a self-cleaving sequence located between the nucleotide sequence encoding the SERCA2a protein and the nucleotide sequence encoding the CCN5 protein. 9. Use according to claim 8, wherein the self-cleaving sequence is 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. 10. Use according to claim 8, wherein the self-cleaving sequence is a nucleotide sequence encoding peptide 2A derived from porcine teshovirus 1. 11. Use according to claim 8, wherein the self-cleaving sequence is the nucleotide sequence represented by SEQ ID NO: 6. 12. Use according to claim 1, wherein the cardiac arrhythmia is an atrial arrhythmia or a ventricular arrhythmia. 13. The use of a pharmaceutical composition, including as an active ingredient: an expression vector carrying a nucleotide sequence encoding a CCN5 protein and a pharmaceutically acceptable carrier for the prevention or treatment of cardiac arrhythmias. 14. Use according to claim 13, wherein the expression vector further comprises a nucleotide sequence encoding a SERCA2a protein. 15. Use according to claim 14, wherein in the expression vector, the nucleotide sequence encoding the SERCA2a protein is contained in the 5' to 3' direction, in the order "nucleotide sequence encoding the SERCA2a protein-nucleotide sequence encoding the CCN5 protein". 16. Use according to claim 15, wherein the expression vector further comprises a self-cleaving sequence located between the nucleotide sequence encoding the SERCA2a protein and the nucleotide sequence encoding the CCN5 protein. 17. Use according to claim 13, wherein the expression vector is any expression vector selected from the group consisting of a plasmid vector and a cosmid vector. 18. The use of a pharmaceutical composition containing as an active ingredient: a recombinant virus that contains a nucleotide sequence encoding a CCN5 protein and a pharmaceutically acceptable carrier for the prevention or treatment of cardiac arrhythmias. 19. Use according to claim 18, wherein the virus is any virus selected from the group consisting of adenovirus, adeno-associated viruses (AAV), retrovirus, lentivirus, herpes simplex virus, and vaccinia virus. 20. Use according to claim 18, wherein the recombinant virus further comprises a nucleotide sequence encoding a SERCA2a protein. 21. A method for the prevention or treatment of cardiac arrhythmia, including: administration of a pharmaceutical composition containing as an active ingredient: a genetic construct that contains a nucleotide sequence encoding a CCN5 protein, or an expression vector carrying a nucleotide sequence encoding a CCN5 protein, or a recombinant virus that contains a nucleotide sequence encoding a CCN5 protein, and a pharmaceutically acceptable carrier. 22. The use of a pharmaceutical composition comprising a CCN5 protein and a pharmaceutically acceptable carrier for the prevention or treatment of cardiac arrhythmias. 23. Use according to claim 22, wherein the pharmaceutical composition further comprises the SERCA2a protein. 24. A method for preventing or treating cardiac arrhythmias, comprising administering a CCN5 protein to a subject. 25. The method of claim 24, further comprising administering the SERCA2a protein to the subject. 26. The use of a pharmaceutical composition containing as an active ingredient: a genetic construct that contains a nucleotide sequence encoding a CCN5 protein, or an expression vector carrying a nucleotide sequence encoding a CCN5 protein, or a recombinant virus that contains a nucleotide sequence encoding a CCN5 protein, and a pharmaceutically acceptable carrier for the manufacture of a medicament for the prevention or treatment of cardiac arrhythmia.

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