Pharmaceutical composition for myocardial regeneration, suppression of myocardial fibrosis, or treatment of heart disease

Abstract

The present disclosure provides a pharmaceutical composition for myocardial regeneration, suppression of myocardial fibrosis, or treatment of heart disease, the pharmaceutical composition containing a polynucleotide encoding constitutively active IKKβ.

Description

Pharmaceutical composition for myocardial regeneration, inhibition of myocardial fibrosis or treatment of heart disease

[0001] This patent application claims priority to Japanese Patent Application No. 2023-149419, the entire contents of which are incorporated herein by reference. The present disclosure relates to pharmaceutical compositions for myocardial regeneration, suppression of myocardial fibrosis, or treatment of heart disease.

[0002] In cardiac diseases such as myocardial infarction and dilated cardiomyopathy, cardiomyocytes responsible for pumping function are damaged and eventually degenerate or die, resulting in myocardial remodeling due to fibrosis of the myocardial tissue. These changes are well known to lead to heart failure, arrhythmia, and subsequent cardiac death. Cardiomyocytes generally lack the ability to self-regenerate, and myocardial regeneration in mammals is only observed shortly after birth (Non-Patent Documents 1 and 2).

[0003] NF-κB is a transcription factor involved in inflammation, cell proliferation, and apoptosis. NF-κB activation is regulated by its inhibitory subunit, IκB, and requires phosphorylation, ubiquitination, and degradation of IκB. The IκB kinase (IKK) complex, which is responsible for the initiation step of NF-κB activation, consists of three subunits: IKKα, IKKβ (also known as IKK-2), and IKKγ. Myofibroblast-selective IKKβ knockout mice are known as a scleroderma model (Patent Document 1). IKKβ mutants that exhibit constitutive kinase activity are known (Non-Patent Document 3).

[0004] International Publication No. 2014 / 069597

[0005] Qi Xiao, et al., A p53-based genetic tracing system to follow postnatal cardiomyocyte expansion in heart regeneration, Development (2017) 144, 580-589Vanessa Hertig, et al., p38α MAPK inhibition translates to cell cycle re-entry of neonatal rat ventricular cardiomyocytes and de novo nestin expression in response to thrombin and after apex resection, Scientific Reports (2019) 9:8203Mercurio F, et al. IKK-1 and IKK-2: cytokine-activated IkappaB kinases essential for NF-kappaB activation. Science. 1997 Oct 31;278(5339):860-6.

[0006] An object of the present disclosure is to provide a pharmaceutical composition for myocardial regeneration, suppression of myocardial fibrosis or treatment of heart disease.

[0007] The present inventors have discovered that IKKβ, which exhibits constitutive kinase activity (constitutively active IKKβ), suppresses myocardial fibrosis and promotes myocardial regeneration, and is effective in treating cardiac diseases. Thus, in one aspect, the present disclosure provides a pharmaceutical composition for treating cardiac diseases, comprising a polynucleotide encoding a constitutively active IKKβ. In one aspect, the present disclosure provides a pharmaceutical composition for regenerating myocardium, comprising a polynucleotide encoding a constitutively active IKKβ. In one aspect, the present disclosure provides a pharmaceutical composition for suppressing myocardial fibrosis, comprising a polynucleotide encoding a constitutively active IKKβ.

[0008] The present disclosure provides a new means for myocardial regeneration, suppression of myocardial fibrosis, or treatment of heart diseases.

[0009] Figure 1 shows a method for generating CAIKKβ mice in which myofibroblasts express constitutively active IKKβ. Figure 2 shows representative images of Masson's trichrome stained heart sections 14 days after myocardial infarction in control and CAIKKβ mice. Figure 3 shows representative images of troponin T immunostained heart sections 14 days after myocardial infarction in control and CAIKKβ mice. Figure 4 shows the vector map of pAAV-SM22α-mIKKβ-CA-WPRE. Figure 5 shows echocardiographic images and time course of left ventricular contractility in mice administered SM22α-mIKKβCA-AAV9 two days after myocardial infarction. Figure 6 shows the survival rates of mice administered high- or low-dose SM22α-mIKKβCA-AAV9 and control mice two days after myocardial infarction. Logrank test: p = 0.000168 between 3 groups, p = 0.0000658 between administration and non-administration, dose 2 x 10 １１ vg vs 5x10 １１ :p=0.0499 Figure 7 shows representative images of heart sections stained with Masson's trichrome staining 142 days after myocardial infarction in mice, administered SM22α-mIKKβCA-AAV9 two days later. Figure 8 shows representative images of heart sections stained with Masson's trichrome staining 176 days after myocardial infarction in mice, administered SM22α-mIKKβCA-AAV9 two days later. Figure 9 shows representative images of heart sections stained with Masson's trichrome staining 142 days after myocardial infarction in mice, administered SM22α-mIKKβCA-AAV9 two days later. Figure 10 shows a representative image (left) of a heart section stained with Masson's trichrome staining 14 days after myocardial infarction in a mouse, and a representative image (right) of a heart section stained with Masson's trichrome staining 142 days after myocardial infarction in a mouse, which was administered SM22α-mIKKβCA-AAV9 two days after myocardial infarction. Figure 11 is an enlarged view of the boxed area in Figure 10.

[0010] Unless otherwise specified, terms used herein have the meanings commonly understood by those skilled in the art of organic chemistry, medicine, pharmacology, molecular biology, microbiology, etc. Definitions of some terms used herein are provided below, but these definitions take precedence over common understandings in this specification.

[0011] As used herein, when a numerical value is accompanied by the term "about," it is intended to encompass a range of ±10% of that value. For example, "about 20" is intended to include "18 to 22." A range of numerical values ​​includes all values ​​between and at the endpoints. "About" in reference to a range applies to both endpoints of the range. Thus, for example, "about 20 to 30" is intended to include "18 to 33."

[0012] IKKβ is one of the subunits that constitute the IκB kinase complex, which activates NF-κB. The IκB kinase complex contains IKKα, IKKβ, and IKKγ, and the kinase activity of the IκB kinase complex is activated upon stimulation by various cytokines. The amino acid sequences of human and mouse IKKβ are registered, for example, as GenBank accession numbers NP_001547.1 (SEQ ID NO: 1) and NP_001153246.1 (SEQ ID NO: 2), respectively. In the present disclosure, IKKβ includes products of its naturally occurring alleles.

[0013] In the present disclosure, constitutively active IKKβ refers to IKKβ that exhibits kinase activity in the absence of cytokine stimulation. For example, constitutively active IKKβ exhibits at least about 10% of the kinase activity, e.g., about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, about 97%, about 98%, about 99%, or about 100% or more, compared to cytokine-stimulated IKKβ. Kinase activity can be measured by known kinase assays, for example, the method described in Non-Patent Document 3.

[0014] Examples of constitutively active IKKβ include a mutant IKKβ (SEQ ID NO: 3) in which serine at positions 177 and 181 of SEQ ID NO: 1 is substituted with glutamic acid, and a mutant IKKβ (SEQ ID NO: 4) in which serine at positions 177 and 181 of SEQ ID NO: 2 is substituted with glutamic acid. In one embodiment, the constitutively active IKKβ has about 60% or more, preferably about 70% or more, more preferably about 80% or more, even more preferably about 90% or more, particularly preferably about 95% or more, and most preferably about 97%, about 98%, or about 99% or more identity to the amino acid sequence of SEQ ID NO: 3 or 4 when calculated using BLAST or the like (e.g., using the default BLAST parameters, i.e., initial conditions), and comprises or consists of an amino acid sequence in which the amino acids corresponding to positions 177 and 181 of SEQ ID NO: 3 or 4 are glutamic acid.

[0015] In one embodiment, the constitutively active IKKβ may comprise the amino acid sequence of SEQ ID NO: 3 or 4 in which one or more amino acids have been deleted, substituted, or added, as long as the amino acids corresponding to positions 177 and 181 of SEQ ID NO: 3 or 4 are glutamic acid. "Several" preferably means 2 to 7, more preferably 2 to 5, and most preferably 2 to 3 amino acids. Amino acid substitutions are preferably conservative substitutions between similar amino acid residues.

[0016] An amino acid position in a second amino acid sequence that corresponds to a certain amino acid position in a first amino acid sequence means a position in the second amino acid sequence that matches that position in the first amino acid sequence when the first amino acid sequence and the second amino acid sequence are aligned to maximize amino acid identity.

[0017] In some embodiments, the constitutively active IKKβ comprises the amino acid sequence of SEQ ID NO: 3 or 4. In some embodiments, the constitutively active IKKβ consists of the amino acid sequence of SEQ ID NO: 3 or 4.

[0018] In one embodiment, the polynucleotide encoding the constitutively active IKKβ comprises or consists of the nucleotide sequence of SEQ ID NO: 5, which encodes the amino acid sequence of SEQ ID NO: 3. In one embodiment, the polynucleotide encoding the constitutively active IKKβ comprises or consists of the nucleotide sequence of SEQ ID NO: 6, which encodes the amino acid sequence of SEQ ID NO: 4.

[0019] A polynucleotide encoding a constitutively active IKKβ may comprise a nucleotide sequence that has an identity of about 60% or more, preferably about 70% or more, more preferably about 80% or more, even more preferably about 90% or more, particularly preferably about 95% or more, and most preferably about 97%, about 98%, or about 99% or more with the nucleotide sequence of SEQ ID NO: 5 or 6 when calculated using BLAST or the like (e.g., when using the default, i.e., initial, parameters of BLAST), or may consist of such a nucleotide sequence, and may encode a constitutively active IKKβ in which the amino acids corresponding to positions 177 and 181 of SEQ ID NO: 3 or 4 are glutamic acid.

[0020] A polynucleotide encoding a constitutively active IKKβ hybridizes under stringent conditions to a polynucleotide having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 5 or 6, and may have a nucleotide sequence encoding a constitutively active IKKβ in which the amino acids corresponding to positions 177 and 181 of SEQ ID NO: 3 or 4 are glutamic acid. With regard to "hybridizing under stringent conditions," hybridization as used herein can be carried out in accordance with standard methods described, for example, in Molecular Cloning, T. Maniatis et al., CSH Laboratory (1983), etc. Furthermore, "stringent conditions" include, for example, conditions in which hybridization is performed at 45°C in a solution containing 6xSSC (a solution containing 1.5M NaCl and 0.15M trisodium citrate is made 10xSSC) or 50% formamide, followed by washing with 2xSSC at 50°C (Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6), and conditions that provide equivalent stringency.

[0021] The polynucleotide encoding a constitutively active IKKβ may be operably linked to a promoter. In the present disclosure, "operably linked" means that a regulatory sequence element such as a promoter and a polynucleotide encoding a constitutively active IKKβ are linked in a manner that allows expression of the constitutively active IKKβ, and they may be directly linked, or any nucleotide sequence may be present between them.

[0022] The promoter may be a constitutive promoter, an inducible promoter, or a tissue- or cell-specific promoter. Constitutive promoters include the CMV (cytomegalovirus) promoter, the PGK (phosphoglycerate kinase) promoter, the EF1α (elongation factor 1-alpha) promoter, the CAG promoter, the SV40 promoter, and the MSCV promoter. In one embodiment, the polynucleotide encoding the constitutively active IKKβ is operably linked to a myofibroblast-specific promoter. Examples of myofibroblast-specific promoters include promoters of SM22α, collagen, αSMA, desmin, osteopontin, FSP-1, HSP47, and the like.

[0023] Examples of human and mouse SM22α promoters include promoters consisting of the nucleotide sequences of SEQ ID NOs: 7 and 8, respectively. In one embodiment, the SM22α promoter may comprise a nucleotide sequence having about 60% or more, preferably about 70% or more, more preferably about 80% or more, even more preferably about 90% or more, particularly preferably about 95% or more, and most preferably about 97%, about 98%, or about 99% or more identity to the nucleotide sequence of SEQ ID NO: 7 or 8. In one embodiment, the SM22α promoter comprises the nucleotide sequence of SEQ ID NO: 7 or 8. In one embodiment, the SM22α promoter consists of the nucleotide sequence of SEQ ID NO: 7 or 8.

[0024] The polynucleotide encoding the constitutively active IKKβ may be DNA or RNA, and may contain one or more non-naturally occurring nucleotides.

[0025] In one embodiment, the polynucleotide encoding a constitutively active IKKβ is RNA. In this embodiment, the polynucleotide may comprise a 5' cap structure, a 5' untranslated region (UTR), a constitutively active IKKβ coding sequence, a 3' UTR, and a polyA sequence. Such a polynucleotide may be produced, for example, by in vitro transcription using a plasmid DNA as a template. For intracellular delivery, the polynucleotide may be administered encapsulated in a lipid particle or a polymer particle.

[0026] The polynucleotide encoding the constitutively active IKKβ may be contained in a vector. The vector can deliver a polynucleotide of a desired sequence into cells and can be a viral vector or a non-viral vector. Viral vectors include retroviruses, lentiviruses, adenoviruses, adeno-associated viruses (AAV), herpes simplex viruses (HSV), vaccinia viruses, Sendai viruses, or measles virus vectors. Non-viral vectors include plasmid vectors, liposome vectors, and artificial chromosomes (e.g., YACs, BACs, and PACs). Each vector can be prepared according to conventional methods.

[0027] The vector may contain one or more regulatory sequences that regulate the expression of constitutively active IKKβ, including promoters, enhancers, polyadenylation signals, and other expression control elements such as post-transcriptional regulatory elements (PREs).

[0028] The vector may contain an origin of replication (ori) for its replication. It may also contain a marker for selecting cells containing the vector. Examples of the selection marker include genes that confer resistance to drugs such as neomycin, kanamycin, puromycin, hygromycin, zeocin, and ampicillin.

[0029] In some embodiments, the vector is an AAV vector. There are many serotypes of AAV, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-rhlO, AAV-DJ, and AAVAnc80. In some embodiments, the vector is an AAV9 vector. AAV vectors may be in their natural form or in their recombinant form, with modified capsid proteins or the like.

[0030] AAV vectors can be prepared, for example, by introducing an AAV vector plasmid containing a sequence encoding a constitutively active IKKβ (or a sequence encoding a constitutively active IKKβ placed downstream of a promoter) and inverted terminal repeats (ITRs) at both ends into packaging cells together with an AAV helper plasmid containing the Rep and Cap genes required for AAV replication and particle formation, and an adenovirus helper plasmid containing adenovirus helper genes required for AAV propagation. Examples of packaging cells include 293T cells, 293 cells, HeLa cells, COS1 cells, and COS7 cells. Viral particles released from packaging cells can be recovered from the packaging cell culture supernatant by purification methods such as centrifugation, filtration, and column purification.

[0031] In the examples described below, myocardial infarction was performed on mice expressing constitutively active IKKβ in myofibroblasts, and suppression of myocardial infarction area and myocardial fibrosis was observed compared to controls. Furthermore, when wild-type mice underwent myocardial infarction and were administered a polynucleotide encoding constitutively active IKKβ, improvement in left ventricular contractility and prolonged survival were observed, as well as suppression of myocardial fibrosis and hyperplasia / thickening of the myocardial wall. Therefore, constitutively active IKKβ can be used to treat cardiac disease. Without being limited by theory, it is believed that expression of constitutively active IKKβ leads to suppression of myocardial fibrosis and myocardial regeneration.

[0032] Examples of cardiac diseases to be treated include cardiac diseases characterized by damage to the myocardium, such as myocardial infarction, dilated cardiomyopathy, hypertrophic cardiomyopathy, myocarditis, hypertensive cardiomyopathy and certain cardiomyopathies, especially myocardial infarction.

[0033] In this disclosure, "treat" or "treatment" means reducing or eliminating the cause of a disease, slowing or halting its progression, and / or reducing, alleviating, ameliorating or eliminating its symptoms in a subject suffering from the disease.

[0034] The subject for treatment of heart disease may be any species, typically a mammal (eg, human, mouse, rat, hamster, rabbit, cat, dog, cow, sheep, monkey), particularly a human.

[0035] The method of administration of the pharmaceutical composition is not particularly limited, and can be via common administration routes such as intracoronary administration, direct intramyocardial injection, oral administration, parenteral administration, injection, infusion, and infusion, and the composition can be in a dosage form suitable for each administration route. In some embodiments, the pharmaceutical composition is administered intravenously. In some embodiments, the pharmaceutical composition is administered via a coronary artery. In some embodiments, the pharmaceutical composition is administered by direct intramyocardial injection.

[0036] Oral dosage forms include granules, fine granules, powders, coated tablets, tablets, powders, capsules, microcapsules, chewable tablets, liquids, suspensions, and emulsions. Injectable dosage forms include those commonly used for pharmaceutical preparations, such as those for coronary administration, direct intramyocardial injection, intravenous injection, infusion, and formulations for extended release of active substances. Dosage forms for coronary administration, direct intramyocardial injection, intravenous injection, or infusion include aqueous and non-aqueous injection solutions (which may contain antioxidants, buffers, bacteriostats, isotonic agents, etc.) and aqueous and non-aqueous injection suspensions (which may contain suspending agents, thickeners, etc.). Injectable dosage forms may be provided in sealed ampoules or vials, or as lyophilized products that require only the addition of a sterile liquid (e.g., water for injection) immediately before use. Injectable solutions or suspensions may be prepared from powders, granules, or tablets.

[0037] These dosage forms are prepared by conventional formulation methods. Furthermore, various pharmaceutically acceptable pharmaceutical substances can be blended as needed for the formulation. The pharmaceutical substances can be appropriately selected depending on the dosage form of the preparation, and examples thereof include buffering agents, surfactants, stabilizers, preservatives, excipients, diluents, additives, disintegrants, binders, coating agents, lubricants, glidants, flavoring agents, sweeteners, solubilizers, etc.

[0038] The dosage and frequency of administration of the pharmaceutical composition can be appropriately determined by those skilled in the art depending on the animal species, health condition, age, body weight, administration route, administration form, etc. of the subject so that an effective amount of a polynucleotide encoding a constitutively active IKKβ is administered to the subject. The effective amount in a given situation can be easily determined by routine experimentation and is within the skill and judgment of an ordinary clinician. For example, when an AAV vector containing a polynucleotide encoding a constitutively active IKKβ is administered to a mouse, about 1 x 10 １１ vg~1x10 １２ vg, approx. 2x10 １１ vg~1x10 １２ vg, approx. 2x10 １１ vg~5x10 １１ vg, approx. 3x10 １１ vg~7x10 １１ vg or approximately 4x10 １１ vg~6x10 １１ vg, for example, about 2 x 10 １１ vg or about 5x10 １１ For example, when an AAV vector containing a polynucleotide encoding a constitutively active IKKβ is administered to a human, the amount of the AAV vector to be administered is about 1×10 １２ vg~1x10 １６ vg, approximately 1x10 １３ vg~1x10 １５ vg or approximately 1 x 10 １４ vg~5x10 １４ vg, for example, about 3 x 10 １４The pharmaceutical composition may be administered in a single dose, multiple doses, or continuous administration. When administered multiple times, the pharmaceutical composition may be administered, for example, once to several times a day, e.g., once, twice, or three times a day, on consecutive days or every few days, e.g., every 1, 2, 3, or 7 days. The administration period is not limited, and a drug-free period may be included. In one embodiment, the pharmaceutical composition is administered in a single dose. In one embodiment, the pharmaceutical composition is administered within 1 week, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day after myocardial infarction, e.g., within 2 days. In one embodiment, the pharmaceutical composition is administered simultaneously with, immediately before, or immediately after revascularization after myocardial infarction.

[0039] A polynucleotide encoding a constitutively active IKKβ can be used alone or in combination with one or more additional active ingredients, particularly active ingredients for the treatment or prevention of heart disease. For example, a pharmaceutical composition can contain one or more additional active ingredients in addition to a polynucleotide encoding a constitutively active IKKβ.

[0040] "Combined use" of ingredients refers not only to the use of a dosage form containing all ingredients or the use of a combination of dosage forms containing each ingredient separately, but also to the simultaneous administration of each ingredient or the administration of any ingredient with a delay, as long as they are used to treat heart diseases. Two or more additional active ingredients can also be used in combination. Active ingredients suitable for combination use include, for example, anti-inflammatory agents, vasodilators, thrombolytic agents, antiplatelet agents, anticoagulants, statin drugs, diuretics, cardiac inotropes, and drugs for treating heart failure.

[0041] In addition to administering a polynucleotide encoding a constitutively active IKKβ, other therapies besides drug therapy can also be performed. Suitable therapies include, for example, surgical procedures such as coronary intervention (PCI) and thrombus aspiration using a balloon catheter, a stent, or a drug-eluting stent (DES), gene therapy, regenerative medicine, and the like.

[0042] In one aspect, a method for treating cardiac disease is provided, comprising administering a polynucleotide encoding a constitutively active IKKβ to a subject in need of such treatment. In one aspect, a polynucleotide encoding a constitutively active IKKβ for use in treating cardiac disease is provided. In one aspect, a use of a polynucleotide encoding a constitutively active IKKβ for treating cardiac disease is provided. In one aspect, a use of a polynucleotide encoding a constitutively active IKKβ in the manufacture of a pharmaceutical composition for treating cardiac disease is provided.

[0043] In another aspect, a pharmaceutical composition for regenerating cardiac muscle is provided, comprising a polynucleotide encoding a constitutively active IKKβ. In the present disclosure, "regenerating cardiac muscle" or "cardiomyocyte regeneration" refers to the proliferation of cardiomyocytes in a subject suffering from at least a portion of the cardiac muscle being damaged, for example, a subject suffering from the above-mentioned cardiac diseases. This pharmaceutical composition can be used in the same manner as the pharmaceutical composition for treating the above-mentioned cardiac diseases.

[0044] In one aspect, a method for regenerating myocardium is provided, comprising administering a polynucleotide encoding a constitutively active IKKβ to a subject in need thereof. In one aspect, a polynucleotide encoding a constitutively active IKKβ for regenerating myocardium is provided. In one aspect, a use of a polynucleotide encoding a constitutively active IKKβ for regenerating myocardium is provided. In one aspect, a use of a polynucleotide encoding a constitutively active IKKβ in the manufacture of a pharmaceutical composition for regenerating myocardium is provided.

[0045] In another aspect, a pharmaceutical composition for suppressing myocardial fibrosis is provided, comprising a polynucleotide encoding a constitutively active IKKβ. In the present disclosure, "suppressing myocardial fibrosis" or "suppressing myocardial fibrosis" means preventing the onset of myocardial fibrosis, slowing or stopping the progression of myocardial fibrosis, or resolving or reducing myocardial fibrosis in a subject at risk of myocardial fibrosis or in a subject with fibrosis in part of the myocardium, such as a subject suffering from the above-mentioned cardiac diseases. This pharmaceutical composition can be used in the same manner as the pharmaceutical composition for treating the above-mentioned cardiac diseases.

[0046] In one aspect, a method for inhibiting myocardial fibrosis is provided, comprising administering a polynucleotide encoding a constitutively active IKKβ to a subject in need thereof. In one aspect, a polynucleotide encoding a constitutively active IKKβ for inhibiting myocardial fibrosis is provided. In one aspect, a use of a polynucleotide encoding a constitutively active IKKβ for inhibiting myocardial fibrosis is provided. In one aspect, a use of a polynucleotide encoding a constitutively active IKKβ in the manufacture of a pharmaceutical composition for inhibiting myocardial fibrosis is provided.

[0047] For example, the following embodiments are provided: [1] A pharmaceutical composition for treating cardiac disease, comprising a polynucleotide encoding a constitutively active IKKβ. [2] The pharmaceutical composition according to 1 above, wherein the cardiac disease is myocardial infarction, dilated cardiomyopathy, hypertrophic cardiomyopathy, myocarditis, hypertensive cardiomyopathy, or specific cardiomyopathy. [3] The pharmaceutical composition according to 1 or 2 above, wherein the cardiac disease is myocardial infarction. [4] A pharmaceutical composition for regenerating myocardium, comprising a polynucleotide encoding a constitutively active IKKβ. [5] A pharmaceutical composition for suppressing myocardial fibrosis, comprising a polynucleotide encoding a constitutively active IKKβ. [6] The pharmaceutical composition according to any one of 1 to 5 above, wherein the constitutively active IKKβ comprises an amino acid sequence having about 90% or more identity with the amino acid sequence of SEQ ID NO: 3, wherein the amino acids corresponding to positions 177 and 181 of SEQ ID NO: 3 are glutamic acid, or an amino acid sequence having about 90% or more identity with the amino acid sequence of SEQ ID NO: 4, wherein the amino acids corresponding to positions 177 and 181 of SEQ ID NO: 4 are glutamic acid. [7] The pharmaceutical composition according to any one of 1 to 6 above, wherein the constitutively active IKKβ comprises the amino acid sequence of SEQ ID NO: 3 or 4. [8] The pharmaceutical composition according to any one of 1 to 7 above, wherein the constitutively active IKKβ consists of the amino acid sequence of SEQ ID NO: 3 or 4. [9] The pharmaceutical composition according to any one of 1 to 8 above, wherein the polynucleotide encoding the constitutively active IKKβ is operably linked to a myofibroblast-specific promoter.

[10] The pharmaceutical composition according to 9 above, wherein the myofibroblast-specific promoter is the SM22α promoter.

[11] The pharmaceutical composition according to 10 above, wherein the SM22α promoter comprises a nucleotide sequence having about 90% or more identity to SEQ ID NO: 7 or 8.

[12] The pharmaceutical composition according to 10 or 11 above, wherein the SM22α promoter comprises the nucleotide sequence of SEQ ID NO: 7 or 8.

[13] The pharmaceutical composition according to any of 10 to 12 above, wherein the SM22α promoter consists of the nucleotide sequence of SEQ ID NO: 7 or 8.

[14] The pharmaceutical composition according to any of 1 to 13 above, wherein a polynucleotide encoding a constitutively active form of IKKβ is contained in a vector.

[15] The pharmaceutical composition according to 14 above, wherein the vector is a viral vector.

[16] The pharmaceutical composition according to any one of claims 14 or 15, wherein the vector is an adeno-associated virus (AAV) vector.

[17] The pharmaceutical composition according to any one of claims 16, wherein the AAV is AAV9.

[18] The pharmaceutical composition according to any one of claims 1 to 17, wherein the vector is an adeno-associated virus (AAV) vector.

[19] The pharmaceutical composition according to any one of claims 1 to 18 ...20] The pharmaceutical composition according to any one of claims 1 to 19, wherein the vector is an adeno-associated virus (AAV) vector.

[21] The pharmaceutical composition according to any one of claims 1 to 19, wherein the vector is an adeno-associated virus (AAV) vector.

[22] The pharmaceutical composition according to any one of claims 1 to 19, wherein the vector is an adeno-associated virus (AAV) vector.

[23] The pharmaceutical composition according to any one of claims 1 to 22, wherein the vector is an adeno-associated virus (AAV) vector.

[0048] All documents cited in this specification are incorporated herein by reference. The above descriptions are all non-limiting, and the present invention is defined in the appended claims, and various modifications are possible within the scope of the technical idea. The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0049] Generation of mice expressing constitutively active IKKβ in myofibroblasts (SM22αCre-CA (Constitutive Active) IKKβ mice). Stock Tg(Tagln-cre)1Her / J (Jackson Lab. strain# 004746, Boucher P., et al., LRP: role in vascular wall integrity and protection from atherosclerosis. Science 300(5617):329-32) and B6.Cg-Gt(ROSA)26Sortm4(Ikbkb)Rsky / J (Jackson Lab. strain# 008242, Sasaki Y., et al., Canonical NF-kappaB activity, dispensable for B cell development, replaces BAFF-receptor signals and promotes B cell proliferation upon activation. Immunity 24(6):729-39), and Cre(+) mice from the first generation were identified by genotyping PCR (forward primer: GCG GTC TGG CAG TAA AAA CTA TC (SEQ ID NO: 9); reverse primer: GTG AAA CAG CAT TGC TGT CAC TT (SEQ ID NO: 10)) to obtain SM22αCre-CAIKKβflox / wt mice (hereafter referred to as CAIKKβ mice) (Figure 1). Cre(-) mice were obtained as controls.

[0050] Preparation of a mouse myocardial infarction model: Male mice aged 8-12 weeks were placed under general anesthesia and intubated for respiratory management. The thoracic cavity was exposed through an intercostal incision, and the epicardium was incised. The left coronary artery was identified under direct vision and ligated with a 7.0 monofilament thread (PROLENE, ETHICON). After confirming changes in myocardial color and movement, the chest was closed.

[0051] Cardiac function after myocardial infarction was evaluated in unanesthetized mice using a mouse echocardiogram, Vevo2100 (Fujifilm).

[0052] Mouse hearts were sectioned transversely at the level of the coronary artery ligation, fixed in 4% paraformaldehyde, and embedded in paraffin or OCT. The embedded blocks were cut into 10 μm (paraffin blocks) or 6 μm (OCT blocks) thick slides, which were stained with hematoxylin and eosin (HE) or Masson's trichrome.

[0053] Mouse hearts were sectioned transversely at the level of the coronary artery ligation, fixed in 4% paraformaldehyde, and embedded in OCT. Slides were prepared from the embedded blocks at a thickness of 6 μm (OCT block) and immunostained using ThermoFisher Cardiac Troponin Monoclonal antibody (#MA512960).

[0054] Construction of SM22α-mIKKβCA-AAV9 and SM22α-tdTomato-AAV9 Gene synthesis of promoter / enhancer sequence and transgene sequence The mouse SM22α promoter / enhancer (546 nt) and the gene for FLAG-tagged mouse IKKbeta kinase active form (S177E / S181E, synonymous with CAIKKβ) (2,319 nt) were synthesized.

[0055] Insertion of Synthetic Gene into Cis-Plasmid Vector (1) Cis-Plasmid: pAAV-SM22α-tdTOMATO-WPRE The synthesized mouse SM22α promoter / enhancer DNA fragment was cleaved with restriction enzymes at the MluI and EcoRI sites and then inserted into the MluI and EcoRI sites of pAAV-CMV-WPRE (available from SignaGen) to replace the CMV promoter DNA sequence, thereby constructing pAAV-SM22α-WPRE. Next, pAAV-CMV-tdTOMATO-WPRE, available from SignaGen, was digested with restriction enzymes at the NheI and PmeI sites to obtain a DNA fragment containing the tdTOMATO sequence. This DNA fragment was inserted into the NheI and PmeI sites of the constructed pAAV-SM22α-WPRE to construct pAAV-SM22α-tdTOMATO-WPRE.

[0056] (2) cis-plasmid: pAAV-SM22α-mIKKβ-CA-WPRE pAAV-SM22α-WPRE constructed in (1) was digested with a restriction enzyme at the EcoRV site to generate blunt ends, and the blunt ends of a gene-synthesized FLAG-mouse IKK2CA (S177E-S181E) DNA fragment were inserted into the EcoRV site of pAAV-SM22α-WPRE by ligation to construct pAAV-SM22α-mIKKβ-CA-WPRE (SEQ ID NO: 11). Positions 156 to 665 of SEQ ID NO: 11 represent the mouse SM22α promoter / enhancer, and positions 698 to 3010 represent FLAG-mouse IKK2CA.

[0057] AAV packaging and purification AAV9 was packaged and purified using the following: (1) SM22α-tdTOMATO-AAV9 cis-plasmid: pAAV-SM22α-tdTOMATO (2) SM22α-mIKKβCA-AAV9 cis-plasmid: pAAV-SM22α-mIKKβ-CA

[0058] Administration of SM22α-mIKKβCA-AAV9 Two days after myocardial infarction, 2 × 10 １１ vg or 5x10 １１ vg of SM22α-mIKKβCA-AAV9 was administered to each mouse. As a control, SM22α-tdTomato-AAV9 or no AAV was administered.

[0059] Results Test 1: Myocardial Infarction in CAIKKβ Mice Control and CAIKKβ mice underwent myocardial infarction surgery. 14 days later, they were euthanized and their hearts were stained with Masson's trichrome. Representative results are shown in Figure 2. Compared with control mice (top row), the area stained blue, indicating fibrosis, was smaller in CAIKKβ mice (bottom row), suggesting that myocardial fibrosis was suppressed.

[0060] Control and CAIKKβ mice underwent myocardial infarction surgery, and 14 days later, they were euthanized. The hearts were immunostained with troponin T antibody. Representative results are shown in Figure 3. Troponin T was localized on the cell surface of cardiomyocytes from CAIKKβ mice (right) but not from control mice (left). Troponin T cell surface localization has been reported in cardiomyocytes during the cell cycle, such as in fish, where myocardium regenerates, and in the hearts of newborn mice (Non-Patent Documents 1 and 2). This suggests that cardiomyocyte proliferation and myocardial regeneration occurred in CAIKKβ mice after myocardial infarction.

[0061] Test 2: Effect of myofibroblast-specific CAIKKβ expression after myocardial infarction Wild-type mice (C57BL / 6J, 8-12 weeks old, male) underwent myocardial infarction surgery. Two days later, SM22α-mIKKβCA-AAV9 (5x10 １１ vg) was administered via the tail vein. The heart was observed over time by echocardiography starting one week after myocardial infarction surgery. Improvement in heart rate was observed in the treatment group compared to the control group. For objective evaluation, left ventricular fractional expiratory flow (EF) was calculated using the Teichholz method in left ventricular short-axis images at the center of the infarcted area. The results are shown in Figure 5. Consistent with the observation of heart rate, improvement in EF over time was observed.

[0062] The survival rate after myocardial infarction surgery is shown in Figure 6. The survival time was longest in mice administered with a high dose of SM22α-mIKKβCA-AAV9, followed by mice administered with a low dose of SM22α-mIKKβCA-AAV9, and was shortest in control mice (Logrank test: p = 0.000168 between 3 groups, p = 0.0000658 between groups with and without administration, dose 2 × 10 １１ vg vs 5x10 １１ : p=0.0499). Of the six mice administered high-dose SM22α-mIKKβCA-AAV9, two did not die during the observation period and survived for more than 150 days. Four mice started the experiment late, and observation was terminated 36 days after surgery (indicated by censored whiskers). Since most of the control mice died within 10 days, the improvement in survival rate was clear.

[0063] After myocardial infarction, the mice were euthanized and the hearts were stained with Masson's trichrome. Representative results 142 and 176 days after myocardial infarction are shown in Figures 7 and 8, respectively. １１ In mice administered PG, myocardial fibrosis was suppressed, and findings suggesting myocardial proliferation (arrows) were observed. Furthermore, thickening of the myocardial wall was observed in mice 142 days after myocardial infarction surgery (Figure 9: arrows at the right ventricular junction and center of the left ventricular septum).

[0064] Control mice were treated with SM22α-mIKKβCA-AAV9 (5x10 １１ Mice administered SM22α-mIKKβCA-AAV9 (vg) were euthanized 142 days after myocardial infarction surgery, and their hearts were stained with Masson's trichrome. Representative results are shown in Figures 10 and 11. The blue-stained areas (black in Figures 10 and 11), indicating fibrosis, were smaller in mice administered SM22α-mIKKβCA-AAV9 than in control mice.

[0065] These results indicate that constitutively active IKKβ suppresses myocardial fibrosis after myocardial infarction and promotes myocardial regeneration, and therefore constitutively active IKKβ is effective in treating heart diseases characterized by myocardial damage, such as myocardial infarction.

[0066] The present disclosure can be used in the medical field.

Claims

1. A pharmaceutical composition for treating heart disease, comprising a polynucleotide encoding a constitutively active IKKβ.

2. The pharmaceutical composition according to claim 1, wherein the constitutively active IKKβ comprises an amino acid sequence having at least about 90% identity with the amino acid sequence of SEQ ID NO:3, and in which the amino acids corresponding to positions 177 and 181 of SEQ ID NO:3 are glutamic acid, or an amino acid sequence having at least about 90% identity with the amino acid sequence of SEQ ID NO:4, and in which the amino acids corresponding to positions 177 and 181 of SEQ ID NO:4 are glutamic acid.

3. The pharmaceutical composition of claim 1, wherein the constitutively active IKKβ comprises the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:

4.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein a polynucleotide encoding a constitutively active IKKβ is operably linked to a myofibroblast-specific promoter.

5. The pharmaceutical composition according to claim 4, wherein the myofibroblast-specific promoter is the SM22α promoter.

6. The pharmaceutical composition according to claim 1, wherein the polynucleotide encoding constitutively active IKKβ is contained in a vector.

7. The pharmaceutical composition according to claim 6, wherein the vector is a viral vector.

8. The pharmaceutical composition according to claim 6, wherein the vector is an adeno-associated virus (AAV) vector.

9. The pharmaceutical composition according to claim 8, wherein the AAV is AAV9.

10. The pharmaceutical composition according to claim 1, wherein the cardiac disease is myocardial infarction, dilated cardiomyopathy, hypertrophic cardiomyopathy, myocarditis, hypertensive cardiomyopathy, or specific cardiomyopathy.

11. The pharmaceutical composition according to claim 1, wherein the heart disease is myocardial infarction.

12. The pharmaceutical composition according to claim 11, which is administered within two days after myocardial infarction.

13. A pharmaceutical composition for regenerating cardiac muscle, comprising a polynucleotide encoding a constitutively active IKKβ.

14. A pharmaceutical composition for suppressing myocardial fibrosis, comprising a polynucleotide encoding constitutively active IKKβ.

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