Nucleic acid medicine that suppresses the production of mRNA of the myostatin gene

Abstract

Provided is a novel method for inhibiting myostatin.　An antisense oligonucleotide having a base length of 15-30 that has a base sequence complementary to the target site of exon 1 of myostatin gene, a salt thereof or a solvate of the same, said antisense oligonucleotide being capable of suppressing the production of mRNA of myostatin gene. A medicine, a food, a feed, a drug for promoting the growth and / or enlargement of muscle cells, a drug for increasing muscle amount and / or inhibiting muscular depression, a drug for suppressing the production of mRNA of myostatin gene, and a myostatin function inhibitor, each comprising the aforesaid antisense oligonucleotide, a salt thereof or a solvate of the same.

Description

Technical Field

[0001] The present invention relates to a nucleic acid medicament that suppresses the production of mRNA of the myostatin gene.

Background Art

[0002] Myostatin is one of the TGF-beta type cell growth factors and is a protein encoded by the myostatin gene. Myostatin has the effect of suppressing the proliferation and hypertrophy of muscle cells. Therefore, inhibiting the function of myostatin has attracted attention as a therapeutic target for muscle atrophy because it promotes the proliferation and hypertrophy of muscle cells. In fact, antibodies against myostatin or antisense oligonucleotides (AO) that control the splicing of the myostatin gene to produce loss-of-function mRNA have been developed (Non-Patent Documents 1 and 2). However, a clinically useful method for inhibiting myostatin has not yet been established.

Prior Art Documents

Non-Patent Documents

[0003]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0004] An object of the present invention is to provide a new method for inhibiting myostatin.

Means for Solving the Problems

[0005] The myostatin (MSTN) gene has a simple structure with three exons. The inventors aimed to develop antisense oligonucleotides (AO) targeting exon 1 of the MSTN gene. Therefore, various AOs were synthesized using ENA complementary to sequences such as splicing factor binding sites within the exon as monomers. Each synthesized AO was introduced into rhabdomyosarcoma cells, and the MSTN mRNA expressed in the cells was semi-quantified by RT-PCR. Then, the AO that decreased the mRNA expression was identified (Figure 3). Furthermore, it was clarified that the introduction of this AO decreased the myostatin signal transduction activity of the cells (Figure 8). These results indicated that the identified AO has the effect of suppressing the expression of myostatin and consequently causing a decrease in myostatin signal transduction. The present invention was completed based on these findings.

[0006] The gist of the present invention is as follows. (1) An antisense oligonucleotide having a base sequence complementary to the target site of exon 1 of the myostatin gene, with a base length of 15 to 30, which can suppress the production of the mRNA of the myostatin gene, or a salt or solvate thereof. (2) The antisense oligonucleotide, salt, or solvate thereof according to (1), wherein the base sequence of exon 1 of the myostatin gene is the base sequence of SEQ ID NO: 1, and the target site of exon 1 of the myostatin gene is present within the region of base numbers 22 to 420 of the base sequence of SEQ ID NO: 1. (3) The antisense oligonucleotide, salt, or solvate thereof according to (1) or (2), wherein the base sequence of the antisense oligonucleotide contains a sequence consisting of at least 15 consecutive bases among the base sequences of any of SEQ ID NOs: 2 to 25 (however, t in the sequence may be u, and u may be t). (4) The antisense oligonucleotide, salt, or solvate thereof according to any of (1) to (3), wherein the base length of the antisense oligonucleotide is 18. (5) The base sequence of the antisense oligonucleotide is the base sequence of any one of SEQ ID NOs: 2 to 25 (provided that t in the sequence may be u, and u may be t), the antisense oligonucleotide, its salt or solvate described in (4). (6) The antisense oligonucleotide, its salt or solvate according to any one of (1) to (5), wherein at least one nucleotide is modified. (7) The sugar constituting the modified nucleotide is D-ribofuranose, and the hydroxyl group at the 2'-position of D-ribofuranose is modified, the antisense oligonucleotide, its salt or solvate described in (6). (8) The antisense oligonucleotide, its salt or solvate according to (7), wherein D-ribofuranose is 2'-O-alkylated and / or 2'-O, 4'-C-alkylenated. (9) A medicament comprising the antisense oligonucleotide according to any one of (1) to (8), its pharmaceutically acceptable salt or solvate. (10) The medicament according to (9) for preventing and / or treating a pathological condition and / or disease involving myostatin. (11) The medicament according to (10), wherein the pathological condition and / or disease involving myostatin is muscular atrophy. (12) The medicament according to (11), wherein the muscular atrophy is at least one selected from the group consisting of muscular dystrophy, myopathy, spinal muscular atrophy, sarcopenia and disuse muscular atrophy. (13) The medicament according to (10), wherein the pathological condition and / or disease involving myostatin is a pathological condition and / or disease in which a therapeutic effect is brought about by muscle mass recovery. (14) The medicament according to (13), wherein the pathological condition and / or disease in which a therapeutic effect is brought about by muscle mass recovery is at least one selected from the group consisting of cancer cachexia, diabetes, cardiovascular disease, kidney disease and bone disease. (15) The medicament according to (14), wherein the cardiovascular disease is heart failure and / or arteriosclerosis, the kidney disease is chronic renal failure, and the bone disease is inflammatory arthritis. (16) A food comprising the antisense oligonucleotide according to any one of (1) to (8), its food-acceptable salt or solvate. (17)A feed comprising an antisense oligonucleotide according to any one of (1) to (8), a salt or solvate thereof acceptable in the feed. (18)An agent for promoting the proliferation and / or hypertrophy of muscle cells, comprising an antisense oligonucleotide according to any one of (1) to (8), a salt or solvate thereof. (19)An agent for increasing muscle mass and / or suppressing muscle loss, comprising an antisense oligonucleotide according to any one of (1) to (8), a salt or solvate thereof. (20)An agent for suppressing the production of mRNA of the myostatin gene, comprising an antisense oligonucleotide according to any one of (1) to (8), a salt or solvate thereof. (21)An inhibitor of myostatin function, comprising an antisense oligonucleotide according to any one of (1) to (8), a salt or solvate thereof. (22)A method for preventing and / or treating a disease involving myostatin, comprising administering to a subject an effective amount of an antisense oligonucleotide according to any one of (1) to (8), a salt or solvate thereof. (23)A method for promoting the proliferation and / or hypertrophy of muscle cells, comprising administering to a subject an effective amount of an antisense oligonucleotide according to any one of (1) to (8), a salt or solvate thereof. (24)A method for increasing muscle mass and / or suppressing muscle loss, comprising administering to a subject an effective amount of an antisense oligonucleotide according to any one of (1) to (8), a salt or solvate thereof. (25)A method for suppressing the production of mRNA of the myostatin gene, comprising administering to a subject an effective amount of an antisense oligonucleotide according to any one of (1) to (8), a salt or solvate thereof. (26)A method for inhibiting the function of myostatin, comprising administering to a subject an effective amount of an antisense oligonucleotide according to any one of (1) to (8), a salt or solvate thereof. (27)An antisense oligonucleotide according to any one of (1) to (8), a salt or solvate thereof, for use in a method for preventing and / or treating a disease involving myostatin. (28) For use in a method of promoting the proliferation and / or hypertrophy of muscle cells, the antisense oligonucleotide, salt or solvate thereof according to any one of (1) to (8). (29) For use in a method of increasing muscle mass and / or suppressing muscle loss, the antisense oligonucleotide, salt or solvate thereof according to any one of (1) to (8). (30) For use in a method of suppressing the production of mRNA of the myostatin gene, the antisense oligonucleotide, salt or solvate thereof according to any one of (1) to (8). (31) For use in a method of inhibiting the function of myostatin, the antisense oligonucleotide, salt or solvate thereof according to any one of (1) to (8). [Advantages of the Invention]

[0007] By the AO of the present invention, the expression of myostatin can be suppressed and myostatin signal transduction can be reduced. This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2019-118446, which is the basis of the priority of the present application. [Brief Description of the Drawings]

[0008]

Figure 1

Figure 2

Figure 3

Figure 4a

Figure 4bc

Figure 5ab

Figure 6ab

Figure 7

Figure 8ab

[0009] a) Schematic diagram showing the effect on the in vitro myostatin transcriptional activity measurement system during AO2+1 treatment. Myostatin signal analysis was evaluated by the activity of luciferase whose expression was induced during AO2+1 treatment and without AO treatment.

[0010] b) Relative values are shown with the result without AO treatment (w / o) set to 1. *** P<0.001.

Figure 9

Figure 10

Figure 11

Mode for Carrying Out the Invention

[0011] Hereinafter, embodiments of the present invention will be described in more detail.

[0012] The present invention provides an antisense oligonucleotide having a base sequence complementary to the target site of exon 1 of the myostatin gene, the antisense oligonucleotide having a base length of 15 to 30 and capable of suppressing the production of mRNA of the myostatin gene, a salt thereof, or a solvate.

[0013] The base sequence of exon 1 of the human myostatin gene is shown in SEQ ID NO: 1. In the present invention, when the base sequence of exon 1 of the myostatin gene is the base sequence of SEQ ID NO: 1, the target site of exon 1 of the myostatin gene may be present within the region of base numbers 22 to 420 of the base sequence of SEQ ID NO: 1.

[0014] Furthermore, in the present invention, the base sequence of the antisense oligonucleotide preferably includes a sequence consisting of at least 15 consecutive bases among the base sequences of any one of SEQ ID NOs: 2 to 25 (provided that t in the sequence may be u and u may be t).

[0015] The base length of the antisense oligonucleotide may be 18, and the base sequence of the antisense oligonucleotide may be any one of the base sequences of SEQ ID NOs: 2 to 25 (provided that t in the sequence may be u and u may be t).

[0016] The nucleotides constituting the antisense oligonucleotide may be any of natural DNA, natural RNA, and modified forms thereof, but at least one is preferably a modified nucleotide.

[0017] Examples of the modified nucleotide include those with modified sugars (e.g., those with 2'-O-alkylated D-ribofuranose, 2'-O, 4'-C-alkylenated D-ribofuranose, etc., where the hydroxyl group at the 2' position of D-ribofuranose is modified), those with modified phosphodiester bonds (e.g., thioesterified), those with modified bases, and combinations thereof. Antisense oligonucleotides with at least one D-ribofuranose constituting them being 2'-O-alkylated or 2'-O, 4'-C-alkylenated have a high binding affinity for RNA and high resistance to nucleases, so higher therapeutic effects can be expected compared to natural nucleotides (i.e., oligo DNA, oligo RNA). Also, antisense oligonucleotides with at least one phosphodiester bond constituting them being thioesterified also have high resistance to nucleases, so higher therapeutic effects can be expected compared to natural nucleotides (i.e., oligo DNA, oligo RNA). Oligonucleotides containing both the above-mentioned modified sugars and modified phosphates have higher resistance to nucleases, so even higher therapeutic effects can be expected.

[0018] For antisense oligonucleotides, examples of sugar modifications include 2'-O-alkylation of D-ribofuranose (e.g., 2'-O-methylation, 2'-O-aminoethylation, 2'-O-propylation, 2'-O-allylation, 2'-O-methoxyethylation, 2'-O-butylation, 2'-O-pentylation, 2'-O-propynylation, etc.), 2'-O,4'-C-alkylation of D-ribofuranose (e.g., 2'-O,4'-C-ethylenation, 2'-O,4'-C-methylenation, 2'-O,4'-C-propylenation, 2'-O,4'-C-tetramethylenation, 2'-O,4'-C-pentamethylenation, etc.), 3'-deoxy-3'-amino-2'-deoxy-D-ribofuranose, 3'-deoxy-3'-amino-2'-deoxy-2'-fluoro-D-ribofuranose, and the like.

[0019] For antisense oligonucleotides, examples of modifications of the phosphodiester bond include phosphorothioate bonds, methylphosphonate bonds, methylthiophosphonate bonds, phosphorodithioate bonds, phosphoramidate bonds, and the like.

[0020] For antisense oligonucleotides, examples of base modifications include 5-methylation, 5-fluorination, 5-bromination, 5-iodination of cytosine, N4-methylation, 5-demethylation (uracil) of thymidine, 5-fluorination, 5-bromination, 5-iodination, N6-methylation, 8-bromination of adenine, N2-methylation, 8-bromination of guanine, and the like.

[0021] The antisense oligonucleotides of the present invention may be in the form of salts. When the antisense oligonucleotides of the present invention are used in medicine, the salts are preferably pharmaceutically acceptable salts, and examples of such salts include alkali metal salts such as sodium salt, potassium salt, and lithium salt; alkaline earth metal salts such as calcium salt and magnesium salt; metal salts such as aluminum salt, iron salt, zinc salt, copper salt, nickel salt, and cobalt salt; inorganic salts such as ammonium salt; amine salts such as t-octylamine salt, dibenzylamine salt, morpholine salt, glucosamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt, piperazine salt, tetramethylammonium salt, and tris(hydroxymethyl)aminomethane salt; halogen atom hydracid salts such as hydrofluoride salt, hydrochloride salt, hydrobromide salt, and hydroiodide salt; inorganic acid salts such as nitrate salt, perchlorate salt, sulfate salt, and phosphate salt; lower alkane sulfonate salts such as methanesulfonate salt, trifluoromethanesulfonate salt, and ethanesulfonate salt; aryl sulfonate salts such as benzenesulfonate salt and p-toluenesulfonate salt; organic acid salts such as acetate salt, malate salt, fumarate salt, succinate salt, citrate salt, tartrate salt, oxalate salt, and maleate salt; and amino acid salts such as glycine salt, lysine salt, arginine salt, ornithine salt, glutamate salt, and aspartate salt. These salts can be produced by known methods.

[0022] In addition, the antisense oligonucleotides may also exist as solvates (for example, hydrates), and such solvates may also be used.

[0023] Furthermore, the antisense oligonucleotides may be administered in the form of prodrugs, and examples of such prodrugs include amides, esters, carbamates, carbonates, ureides, and phosphates. These prodrugs can be produced by known methods.

[0024] The method for synthesizing the antisense oligonucleotide is not particularly limited, and a conventionally known method can be adopted. Examples of the synthesis method include a synthesis method by genetic engineering techniques, a chemical synthesis method, etc. Examples of the genetic engineering techniques include an in vitro transcription synthesis method, a method using a vector, and a method using a PCR cassette. The vector is not particularly limited, and examples thereof include non-viral vectors such as plasmids and viral vectors. The chemical synthesis method is not particularly limited, and examples thereof include the phosphoramidite method and the H-phosphonate method. For the chemical synthesis method, for example, a commercially available automatic nucleic acid synthesizer can be used. In the chemical synthesis method, amidite is generally used. The amidite is not particularly limited, and in the following examples, the antisense oligonucleotide was synthesized by the phosphoramidite method using ENA-2CE phosphoramidite and 2'OMe-2CE phosphoramidite.

[0025] For the phosphoramidite reagent, commercially available reagents can be used for natural nucleosides and 2'-O-methyl nucleosides (that is, 2'-O-methylguanosine, 2'-O-methyladenosine, 2'-O-methylcytosine, 2'-O-methyluridine). For 2'-O-alkylguanosine, adenosine, cytosine, and uridine with an alkyl group having 2 to 6 carbon atoms, it is as follows.

[0026] 2'-O-aminoethylguanosine, adenosine, cytosine, and uridine can be synthesized according to the literature (Blommers et al. Biochemistry (1998), 37, 17714-17725.).

[0027] 2'-O-propylguanosine, adenosine, cytosine, and uridine can be synthesized according to the literature (Lesnik, E.A. et al. Biochemistry (1993), 32, 7832-7838.).

[0028] 2'-O-allyl guanosine, adenosine, cytidine, and uridine can use commercially available reagents.

[0029] 2'-O-methoxyethyl guanosine, adenosine, cytidine, and uridine can be synthesized according to the patent (US6261840) or the literature (Martin, P. Helv. Chim. Acta. (1995) 78, 486-504.).

[0030] 2'-O-butyl guanosine, adenosine, cytidine, and uridine can be synthesized according to the literature (Lesnik, E.A. et al. Biochemistry (1993), 32, 7832-7838.).

[0031] 2'-O-pentyl guanosine, adenosine, cytidine, and uridine can be synthesized according to the literature (Lesnik, E.A. et al. Biochemistry (1993), 32, 7832-7838.).

[0032] 2'-O-propynyl guanosine, adenosine, cytidine, and uridine can use commercially available reagents.

[0033] For 2'-O, 4'-C-methylene guanosine, adenosine, 5-methylcytidine, and thymidine, they can be produced according to the method described in WO99 / 14226. For 2'-O, 4'-C-alkylene guanosine, adenosine, 5-methylcytidine, and thymidine with 2 to 5 carbon atoms in the alkylene group, they can be produced according to the method described in WO00 / 47599.

[0034] After coupling with phosphoramidite reagents, antisense oligonucleotides having phosphorothioate linkages can be synthesized by reacting with reagents such as sulfur, tetraethylthiuram disulfide (TETD, Applied Biosystems), Beaucage reagent (Glen Research), or xanthydrol (Tetrahedron Letters, 32, 3005 (1991), J. Am. Chem. Soc. 112, 1253 (1990), PCT / WO98 / 54198).

[0035] As the controlled pore glass (CPG) used in the synthesizer, commercially available products can be used for those with 2'-O-methyl nucleosides linked. For 2'-O, 4'-C-methylene guanosine, adenosine, 5-methylcytosine, and thymidine, according to the method described in WO99 / 14226, for 2'-O, 4'-C-alkylene guanosine, adenosine, 5-methylcytosine, and thymidine with 2 to 5 carbon atoms in the alkylene group, the nucleosides produced according to the method described in WO00 / 47599 can be bound to CPG according to the literature (Oligonucleotide Synthesis, Edited by M.J.Gait, Oxford University Press, 1984). By using modified CPG (described in Example 12b of JP-A-7-87982), oligonucleotides with a 2-hydroxyethyl phosphate group bound to the 3'-end can be synthesized. Also, by using 3'-amino-Modifier C3 CPG, 3'-amino-Modifier C7 CPG, Glyceryl CPG, (Glen Research), 3'-specer C3 SynBase CPG 1000, 3'-specer C9 SynBase CPG 1000 (link technologies), oligonucleotides with a hydroxyalkyl phosphate group or an aminoalkyl phosphate group bound to the 3'-end can be synthesized.

[0036] The antisense oligonucleotides of the present invention can be used in medicine. When used as a medicine, the antisense oligonucleotides may be in the form of a pharmaceutically acceptable salt, solvate or prodrug. Thus, the present invention provides a medicine comprising the above antisense oligonucleotides, a pharmaceutically acceptable salt or solvate thereof. The medicine is preferably for preventing and / or treating a pathological condition and / or disease involving myostatin, and examples of the pathological condition and / or disease involving myostatin include muscular atrophy (for example, muscular atrophy such as muscular dystrophy, myopathy, spinal muscular atrophy, sarcopenia, disuse muscular atrophy, etc.), pathological conditions and / or diseases in which a therapeutic effect is brought about by muscle mass recovery (for example, cancer cachexia, diabetes, cardiovascular diseases (such as heart failure, arteriosclerosis, etc.), kidney diseases (such as chronic renal failure, etc.), bone diseases (such as inflammatory arthritis, etc.)), but are not limited thereto. Since inhibition of myostatin results in an increase in skeletal muscle mass, it is considered possible to use it for the treatment of all diseases presenting muscular atrophy regardless of the cause of the muscular atrophy. An increase in skeletal muscle mass aims to increase the amount of exercise and also contributes to the improvement of whole-body metabolism. In addition, it can be expected to act on the myocardium and restore its function. On the other hand, it is also expected that myostatin inhibition acts on osteoclasts to suppress bone destruction, activates the ability of vascular endothelial cells to maintain homeostasis, induces apoptosis, enhances insulin sensitivity, etc.

[0037] The antisense oligonucleotide of the present invention, its pharmaceutically acceptable salt, solvate or prodrug (hereinafter referred to as the "active ingredient") can be administered orally or parenterally to mammals (e.g., humans, rabbits, dogs, cats, rats, mice) as a formulation in an appropriate dosage form, either alone or together with a pharmacologically acceptable carrier, diluent or excipient. The dosage varies depending on the administration subject, target disease, symptoms, administration route, etc. For example, when used for the prevention and treatment of muscular atrophy diseases (e.g., muscular dystrophy), the single dose of the active ingredient is usually about 0.1 to 50 mg / kg body weight, preferably about 0.5 mg / kg body weight, at a frequency of about once a week to once a month, preferably about once a year, and can be administered orally, intramuscularly, subcutaneously or intravenously (preferably, continuously or every other day). The same dosage can be administered in other cases of parenteral administration and oral administration. When the symptoms are particularly severe, the dosage can be increased according to the symptoms. For other diseases, the dosage can be appropriately increased or decreased with reference to the above dosage.

[0038] Formulations for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups, emulsions, suspensions, etc. Such formulations can be manufactured by conventional methods and may contain carriers, diluents or excipients commonly used in the pharmaceutical field. For example, carriers and excipients for tablets include lactose, starch, sucrose, magnesium stearate, etc.

[0039] Examples of formulations for parenteral administration include, for example, injections, suppositories, etc. The injection may be in dosage forms such as intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip infusions, etc. Such injections are prepared by a conventional method, that is, by dissolving, suspending or emulsifying the active ingredient in a sterile aqueous or oily liquid usually used for injections. Examples of aqueous liquids for injection include physiological saline, isotonic solutions containing glucose and other adjuvants, etc. Appropriate solubilizing agents, such as alcohols (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants (e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)), etc. may be used in combination. Examples of oily liquids include sesame oil, soybean oil, etc. Benzyl benzoate, benzyl alcohol, etc. may be used in combination as solubilizing agents. The prepared injection solution is usually filled into appropriate ampoules. Suppositories used for rectal administration can be prepared by mixing the active ingredient with a conventional suppository base.

[0040] The above oral or parenteral pharmaceutical formulations are preferably prepared in dosage forms of dosage units that are compatible with the dosage of the active ingredient. Examples of such dosage forms of dosage units include tablets, pills, capsules, injections (ampoules), suppositories, etc., and each dosage unit form preferably contains usually 0.1 to 1,000 mg of the active ingredient.

[0041] The antisense oligonucleotides of the present invention can also be used in food and feed. For example, the antisense oligonucleotides of the present invention are used as additives for food and feed, or as supplements for humans or animals. The antisense oligonucleotides of the present invention may be in the form of pharmaceutically acceptable salts, solvates or prodrugs that are acceptable as food or feed. Therefore, the present invention provides a food comprising the above antisense oligonucleotide, a pharmaceutically acceptable salt or solvate thereof. Further, the present invention provides a feed comprising the above antisense oligonucleotide, a pharmaceutically acceptable salt or solvate thereof. As an example of a pharmaceutically acceptable salt, solvate or prodrug that is acceptable as food or feed, pharmaceutically acceptable salts, solvates or prodrugs can be mentioned, and these have been described above.

[0042] Using the antisense oligonucleotide of the present invention, the proliferation and / or hypertrophy of muscle cells can be promoted. Muscle cells are contractile cells that form human and animal muscle tissues, and include skeletal muscle cells, smooth muscle cells, cardiomyocytes, and the like. The myostatin inhibition by the antisense oligonucleotide of the present invention is effective in myoblasts, and can induce the promotion of myoblast proliferation and differentiation, and as a result, can cause the proliferation / hypertrophy of muscle cells. Muscle cells also include progenitor cells such as myoblasts. The antisense oligonucleotide of the present invention may be in the form of a salt, solvate or prodrug. Examples of salts, solvates or prodrugs include pharmaceutically acceptable salts, solvates or prodrugs, which have been described above. The animal may be any animal that expresses myostatin, and examples of domestic and edible farm animals such as mammals such as cats, dogs, sheep, pigs, cows, chickens, and turkeys, and fish such as salmon, trout, cod, tuna, and sea bream can be exemplified. Therefore, the present invention provides an agent for promoting the proliferation and / or hypertrophy of muscle cells, which comprises the above antisense oligonucleotide, a salt or solvate thereof. Further, the present invention provides a method for promoting the proliferation and / or hypertrophy of muscle cells, which comprises administering the above antisense oligonucleotide, a salt or solvate thereof to a subject in an effective amount. The subject can be a human or an animal. Animals have been described above. The dosage form, dosage, administration route, administration frequency, etc. of the agent are preferably in accordance with the above-mentioned medicine, and can be appropriately changed so as to obtain a desired effect.

[0043] In addition, the antisense oligonucleotide of the present invention can be used to promote muscle formation and suppress muscle decline in humans and animals. The antisense oligonucleotide of the present invention may be in the form of a salt, solvate or prodrug. Examples of salts, solvates or prodrugs include pharmaceutically acceptable salts, solvates or prodrugs, which have been described above. The animal may be any animal that expresses myostatin, and examples of domesticated and edible farm animals such as mammals such as cats, dogs, sheep, pigs, cows, chickens, and turkeys, and fish such as salmon, trout, cod, tuna, and sea bream can be given. Therefore, the present invention provides a medicament for promoting muscle formation and / or suppressing muscle decline, which comprises the above antisense oligonucleotide, its salt or solvate. Further, the present invention provides a method for promoting muscle formation and / or suppressing muscle decline, which comprises administering the above antisense oligonucleotide, its salt or solvate to a subject in an effective amount. The subject can be a human or an animal. The animals have been described above. The dosage form, dosage, administration route, administration frequency, etc. of the medicament are preferably in accordance with the above-mentioned medicament and can be appropriately changed so as to obtain the desired effect.

[0044] Foods to which the antisense oligonucleotide of the present invention, its food-acceptable salt, solvate or prodrug is added can be any food such as plant-based foods, animal-based foods, fungal foods, fresh foods, processed foods, targeted foods, cooking and seasoning ingredients, beverages, health foods, space foods, pet foods, etc.

[0045] The food of the present invention may be added with general components such as protein, lipid, carbohydrate, and sodium, minerals such as potassium, calcium, magnesium, and phosphorus, trace elements such as iron, zinc, copper, selenium, and chromium, vitamins such as vitamin A, β-carotene, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin C, niacin, folic acid, vitamin D3, vitamin E, biotin, and pantothenic acid, coenzyme Q10, α-lipoic acid, galactooligosaccharide, dietary fiber, excipients (such as water, carboxymethyl cellulose, and lactose), sweeteners, flavor correctives (such as malic acid, citric acid, and amino acids), and fragrances. When the food of the present invention is made into a liquid preparation, water, physiological saline, soup, milk, fruit juice, etc. can be used as the liquid for dispersing or dissolving the food components. The food of the present invention may be in the form of powder, granule, tablet, liquid preparation, etc. In order to make it easily ingestible by patients and the elderly, it is preferably made into a gel-like product such as jelly.

[0046] The feed to which the antisense oligonucleotide of the present invention, an acceptable salt, solvate or prodrug thereof is added may be any feed such as single feeds like cereals, vegetable oil cakes, bran, manufacturing cakes, and animal feeds, and compound feeds formulated with a plurality of feed raw materials and feed additives.

[0047] The feed of the present invention contains antioxidants (such as ethoxyquin, dibutylhydroxytoluene, butylhydroxyanisole, etc.), mold inhibitors (such as propionic acid, calcium propionate, sodium propionate, etc.), binders (such as sodium alginate, sodium caseinate, sodium carboxymethylcellulose, propylene glycol, sodium polyacrylate, etc.), emulsifiers (such as glycerin fatty acid esters, sucrose fatty acid esters, sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene glycerin fatty acid esters, etc.), regulators (such as formic acid, etc.), amino acids, etc. (such as aminoacetic acid, DL-alanine, L-arginine, L-lysine hydrochloride, L-carnitine, guanidinoacetic acid, sodium L-glutamate, taurine, 2-deamino-2-hydroxymethionine, DL-tryptophan, L-tryptophan, L-threonine, L-valine, DL-methionine, L-lysine sulfate, etc.), vitamins (such as L-ascorbic acid, calcium L-ascorbate, sodium L-ascorbate, sodium calcium L-ascorbate-2-phosphate ester, magnesium L-ascorbate-2-phosphate ester, acetomenaphthone, inositol, dibenzoylthiamine hydrochloride, ergocalciferol, choline chloride, thiamine hydrochloride, pyridoxine hydrochloride, β-carotene, cholecalciferol, dl-α-tocopherol acetate, cyanocobalamin, thiamine nitrate, nicotinic acid, nicotinamide, para-aminobenzoic acid, calcium D-pantothenate, calcium DL-pantothenate, d-biotin, vitamin A powder, vitamin A oil, vitamin D powder, vitamin D3 oil, vitamin E powder, 25-hydroxycolecalciferol, menadione dimethylpyrimidinol bisulfite, sodium menadione bisulfite, folic acid, riboflavin, riboflavin butyrate, etc.), minerals (such as potassium chloride, iron citrate, calcium gluconate, sodium iron succinate citrate, magnesium oxide, aluminum hydroxide, zinc carbonate, cobalt carbonate, sodium bicarbonate, magnesium carbonate, manganese carbonate, zinc 2-deamino-2-hydroxymethionine, iron DL-threonine, calcium lactate, ferrous fumarate, peptide zinc, peptide iron, peptide copper, peptide manganese, potassium iodide, potassium iodate,Calcium iodate, zinc sulfate (dry), zinc sulfate (crystalline), zinc methionine sulfate, sodium sulfate (dry), magnesium sulfate (dry), magnesium sulfate (crystalline), cobalt sulfate (dry), cobalt sulfate (crystalline), iron sulfate (dry), copper sulfate (dry), copper sulfate (crystalline), manganese sulfate, potassium hydrogen phosphate (dry), sodium hydrogen phosphate (dry), potassium dihydrogen phosphate (dry), sodium dihydrogen phosphate (dry), sodium dihydrogen phosphate (crystalline), etc.), pigments, synthetic antibacterial agents, antibiotics, flavoring agents, flavor enhancers, enzymes, probiotics, organic acids, etc. may be added.

[0048] Food and feed intake should be carried out at an intake amount, frequency, and intake period such that the desired effect (e.g., promotion of muscle formation) is confirmed.

[0049] The antisense oligonucleotide of the present invention can suppress the production of mRNA of the myostatin gene. Therefore, the present invention provides a drug that suppresses the production of mRNA of the myostatin gene and contains the above antisense oligonucleotide. The drug of the present invention can be used as a medicine for humans and animals, as an additive or supplement to food and feed, as an animal growth promoter, or also as a reagent for experiments. The antisense oligonucleotide of the present invention may be in the form of a salt, solvate, or prodrug. Examples of salts, solvates, or prodrugs include pharmaceutically acceptable salts, solvates, or prodrugs, which have been described above.

[0050] The antisense oligonucleotide of the present invention can inhibit the function of myostatin. Therefore, a myostatin function inhibitor containing the above antisense oligonucleotide of the present invention is provided. The myostatin function inhibitor of the present invention can be used as a medicine for humans and animals, as an additive or supplement to food and feed, as an animal growth promoter, or also as a reagent for experiments. The antisense oligonucleotide may be in the form of a salt, solvate or prodrug. Examples of salts, solvates or prodrugs include pharmaceutically acceptable salts, solvates or prodrugs, which have been described above.

[0051] When used as a reagent for experiments, the expression of myostatin can be suppressed by treating cells, tissues or organs that express myostatin with the antisense oligonucleotide, salt or solvate thereof of the present invention. The antisense oligonucleotide, salt and solvate thereof of the present invention may be used in an amount effective for suppressing the expression of myostatin. Examples of cells that express myostatin include muscle cells, rhabdomyosarcoma cells, cancer cells such as those of the digestive tract, lung, and esophagus. In addition to naturally occurring cells, recombinant cells into which the myostatin gene has been introduced can also be exemplified. Examples of tissues and organs that express myostatin include skeletal muscle, myocardium, blood vessels, kidneys, digestive tract, uterus, liver, pancreas, and lungs. The expression of myostatin can be analyzed by analyzing myostatin mRNA in a sample by RT-PCR, detecting myostatin protein in the sample by Western blotting, or detecting it by mass spectrometry.

Example

[0052] Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited to these examples. 〔Examples 1 to 24〕Synthesis of antisense oligonucleotide (AO) Antisense oligonucleotides (AO) shown in Table 1 were synthesized. The sequence locations of AOs complementary to MSTN pre-mRNA are shown in Figs. 1 to 4. ENA (registered trademark) (2'-O,4'-C-Ethylene-bridged Nucleic Acids), a modified nucleic acid, was introduced into C (cytosine) and T (thymine) in the AO sequence to improve affinity and stability.

[0053] Synthesis of CaCuggaCCaGCaaCaau (MSTN_Ex1_AO1) (Example 1) Synthesis was carried out on a 1 μmol scale using an automated nucleic acid synthesizer (DNA / RNA synthesizer NTS H-6 manufactured by Nippon Techno Service Co., Ltd.). The concentrations of the solvent, reagents, and phosphoramidites in each synthesis cycle were the same as those in the case of natural oligonucleotide synthesis. As reagents, phosphoramidites of 2'-O-methylnucleosides (adenosine product No. 10-3100-10, guanosine product No. 10-3121-10) manufactured by Glen Research were used. The solvent used was from Wako Pure Chemical Industries, Ltd. As the non-natural phosphoramidites, the compounds of Example 22 (5'-O-dimethoxytrityl-2'-O,4'-C-ethylene-4-N-benzoyl-5-methylcytidine-3'-O-(2-cyanoethyl N,N-diisopropyl)phosphoramidite) and Example 9 (5'-O-dimethoxytrityl-2'-O,4'-C-ethylene-5-methyluridine-3'-O-(2-cyanoethyl N,N-diisopropyl)phosphoramidite) of JP 2000-297097 were used. Universal control pore glass (CPG) (product No. 25-5040 manufactured by Glen Research) was used as the solid-phase support, and the compounds shown in the notation were synthesized. However, the time required for amidite condensation was set to 15 minutes.

[0054] Protected oligonucleotide analogs having the target sequence were heat-treated with concentrated aqueous ammonia (55 °C, 8 h) to cleave the oligomers from the support and remove the protecting group cyanoethyl group on the phosphorus atom and the protecting group on the nucleobase. This ammonia solution was subjected to DMT removal in a cartridge using a Glen-Pak DNA Purification Cartridge (product No. 60-5100, manufactured by Glen Research) according to the Glen Research recommended protocol. The recovered solution was evaporated under reduced pressure, and the residue was purified by reverse-phase HPLC (LC-2a, manufactured by Shimadzu Corporation; column: Triart C18 (10 × 150 mm), manufactured by YMC; solution A: 0.1 M aqueous triethylamine acetate (TEAA), pH 7.0; solution B: acetonitrile; B%: 10% → 25% (30 min, linear gradient); 50 °C; 4.7 mL / min; 280 nm). After evaporation of the solvent, it was dissolved in 10 mM NaOH solution and subjected to ultrafiltration using a microsep centrifugal filtration device (product No. MCP003C, manufactured by Nippon Pall) to replace it with pure water, and the target compound was obtained after lyophilization.

[0055] Synthesis of augCaTTaCaCagCCCCu (MSTN_Ex1_AO2) (Example 2) A compound of Example 2 having the target sequence was synthesized in the same manner as the compound of Example 1. This compound was identified by negative-ion MALDI-TOFMS (calculated value: 6395.311, measured value: 6392.585).

[0056] The base sequence of this compound is complementary to the nucleotide numbers 240 - 257 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0057] Synthesis of gauuuaguguuuuguCuC (MSTN_Ex1_AO3) (Example 3) A compound of Example 3 having the target sequence was synthesized in the same manner as the compound of Example 1. This compound was identified by negative ion MALDI-TOFMS (calculated value: 6226.921, measured value: 6220.691).

[0058] The base sequence of this compound is a sequence complementary to nucleotides 265 - 282 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0059] Synthesis of aagTaCaTgCaTTaCaCa (MSTN_Ex1_AO2-6) (Example 4) A compound of Example 4 having the target sequence was synthesized in the same manner as the compound of Example 1. This compound was identified by negative ion MALDI-TOFMS (calculated value: 6441.351, measured value: 6435.8281).

[0060] The base sequence of this compound is a sequence complementary to nucleotides 246 - 263 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0061] Synthesis of TaCaTgCaTTaCaCagCC (MSTN_Ex1_AO2-3) (Example 5) A compound of Example 5 having the target sequence was synthesized in the same manner as the compound of Example 1. This compound was identified by negative ion MALDI-TOFMS (calculated value: 6445.375, measured value: 6439.7319).

[0062] The base sequence of this compound is a sequence complementary to nucleotides 243 - 260 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0063] Synthesis of CaTTaCaCagCCCCTCTT (MSTN_Ex1_AO2+3) (Example 6) A compound of Example 6 having the target sequence was synthesized in the same manner as the compound of Example 1. This compound was identified by negative-ion MALDI-TOFMS (calculated value: 6436.396, measured value: 6430.9077).

[0064] The base sequence of this compound is a sequence complementary to nucleotide numbers 237-254 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0065] Synthesis of TaCaCagCCCCTCTTTTT (MSTN_Ex1_AO2+6) (Example 7) A compound of Example 7 having the target sequence was synthesized in the same manner as the compound of Example 1. This compound was identified by negative-ion MALDI-TOFMS (calculated value: 6440.376, measured value: 6435.562).

[0066] The base sequence of this compound is a sequence complementary to nucleotide numbers 234-251 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0067] Synthesis of aCagCCCCTCTTTTTCCa (MSTN_Ex1_AO2+9) (Example 8) A compound of Example 8 having the target sequence was synthesized in the same manner as the compound of Example 1. This compound was identified by negative-ion MALDI-TOFMS (calculated value: 6439.392, measured value: 6434.6094).

[0068] The base sequence of this compound is a sequence complementary to nucleotide numbers 231-248 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0069] Synthesis of TgCaTTaCaCagCCCCTC (MSTN_Ex1_AO2+1) (Example 9) A compound of Example 9 having the target sequence was synthesized in the same manner as the compound of Example 1. This compound was identified by negative-ion MALDI-TOFMS (calculated value: 6449.399, measured value: 64,43.8228).

[0070] The base sequence of this compound is a sequence complementary to nucleotide numbers 239-256 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0071] Synthesis of gCaTTaCaCagCCCCTCT (MSTN_Ex1_AO2+2) (Example 10) A compound of Example 10 having the target sequence was synthesized in the same manner as the compound of Example 1. This compound was identified by negative-ion MALDI-TOFMS (calculated value: 6449.399, measured value: 6443.707).

[0072] The base sequence of this compound is a sequence complementary to nucleotide numbers 238-255 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0073] Synthesis of aTTaCaCagCCCCTCTTT (MSTN_Ex1_AO2+4) (Example 11) A compound of Example 11 having the target sequence was synthesized in the same manner as the compound of Example 1. This compound was identified by negative-ion MALDI-TOFMS (calculated value: 6437.38, measured value: 6431.9209).

[0074] The base sequence of this compound is a sequence complementary to nucleotide numbers 236 - 253 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0075] Synthesis of TTaCaCagCCCCTCTTTT (MSTN_Ex1_AO2+5) (Example 12) A compound of Example 12 having the target sequence was synthesized in the same manner as the compound of Example 1. This compound was identified by negative ion MALDI - TOFMS (calculated value: 6440.376, measured value: 6434.8232).

[0076] The base sequence of this compound is a sequence complementary to nucleotide numbers 235 - 252 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0077] Synthesis of TgTaCagTCTgagagaCa (MSTN_Ex1_AO4) (Example 13) A compound of Example 13 having the target sequence was synthesized in the same manner as the compound of Example 1. This compound was identified by negative ion MALDI - TOFMS (calculated value: 6487.336, measured value: 6481.8237).

[0078] The base sequence of this compound is a sequence complementary to nucleotide numbers 22 - 39 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0079] Synthesis of aaTgCaTgTaCagTCTga (MSTN_Ex1_AO4-6) (Example 14) A compound of Example 14 having the target sequence was synthesized in the same manner as the compound of Example 1. This compound was identified by negative-ion MALDI-TOFMS (calculated value: 6474.333, measured value: 6468.584).

[0080] The base sequence of this compound is a sequence complementary to nucleotides 28 - 45 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0081] Synthesis of TTgCTTTTgagTaaTgCC (MSTN_Ex1_AO5) (Example 15) A compound of Example 15 having the target sequence was synthesized in the same manner as the compound of Example 1. This compound was identified by negative-ion MALDI-TOFMS (calculated value: 6483.321, measured value: 6477.7007).

[0082] The base sequence of this compound is a sequence complementary to nucleotides 58 - 75 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0083] Synthesis of aaTCaaTaTaaTCTTTTT (MSTN_Ex1_AO6) (Example 16) A compound of Example 16 having the target sequence was synthesized in the same manner as the compound of Example 1. This compound was identified by negative-ion MALDI-TOFMS (calculated value: 6420.309, measured value: 6414.5815).

[0084] The base sequence of this compound is a sequence complementary to nucleotides 108 - 125 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0085] Synthesis of TTgCTCaCTgTTCTCaTT (MSTN_Ex1_AO7) (Example 17) The compound of Example 17, which has the same target sequence as the compound of Example 1, was synthesized. This compound was identified by negative ion MALDI-TOFMS (calculated value: 6458.343, measured value: 6452.9326).

[0086] The base sequence of this compound is complementary to nucleotides 203-220 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession no. NG_009800.1).

[0087] Synthesis of CTTgaagaTTTagTgTTT (MSTN_Ex1_AO3-6) (Example 18) The compound of Example 18 having the target sequence similar to that of the compound of Example 1 was synthesized. This compound was identified by negative ion MALDI-TOFMS (calculated value: 6482.293, measured value: 6476.7026).

[0088] The base sequence of this compound is complementary to nucleotides 271-288 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession no. NG_009800.1).

[0089] Synthesis of TCTaTTCTTgaagaTTTa (MSTN_Ex1_AO3-12) (Example 19) The compound of Example 19, which has the same target sequence as the compound of Example 1, was synthesized. This compound was identified by negative ion MALDI-TOFMS (calculated value: 6452.307, measured value: 6446.8232).

[0090] The base sequence of this compound is complementary to nucleotides 277-294 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession no. NG_009800.1).

[0091] Synthesis of TaCTgaggaTTTgTaTCT (MSTN_Ex1_AO8) (Example 20) A compound of Example 20 having the target sequence was synthesized in the same manner as the compound of Example 1. This compound was identified by negative-ion MALDI-TOFMS (calculated value: 6481.309, measured value: 6475.8228).

[0092] The base sequence of this compound is a sequence complementary to nucleotide numbers 303 - 320 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0093] Synthesis of CaTCTTTgCTgaTgTTag (MSTN_Ex1_AO9) (Example 21) A compound of Example 21 having the target sequence was synthesized in the same manner as the compound of Example 1. This compound was identified by negative-ion MALDI-TOFMS (calculated value: 6483.321, measured value: 6477.6953).

[0094] The base sequence of this compound is a sequence complementary to nucleotide numbers 342 - 359 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0095] Synthesis of gTTgTCTTaTaaCaTCTT (MSTN_Ex1_AO9-12) (Example 22) A compound of Example 22 having the target sequence was synthesized in the same manner as the compound of Example 1. This compound was identified by negative-ion MALDI-TOFMS (calculated value: 6454.319, measured value: 6448.7021).

[0096] The base sequence of this compound is a sequence complementary to nucleotide numbers 354 - 371 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0097] Synthesis of TgaTCaaTCagTTCCCgg (MSTN_Ex1_AO10) (Example 23) A compound of Example 23 having the target sequence was synthesized in the same manner as the compound of Example 1. This compound was identified by negative ion MALDI - TOFMS (calculated value: 6478.357, measured value: 6472.9458).

[0098] The base sequence of this compound is a sequence complementary to nucleotide numbers 394 - 411 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_*********.1).

[0099] Synthesis of aCaTCaTaCTgaTCaaTC (MSTN_Ex1_AO10-9) (Example 24) A compound of Example 24 having the target sequence was synthesized in the same manner as the compound of Example 1. This compound was identified by negative ion MALDI - TOFMS (calculated value: 6430.36, measured value: 6424.8286).

[0100] The base sequence of this compound is a sequence complementary to nucleotide numbers 403 - 420 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0101] (Table 1) The sequence of AO synthesized in this example. Capital letters are ENA nucleic acids, and small letters are 2' - OMe. TIFF0007715345000001.tif192154

[0102] [Test Example] Experimental method 1. Evaluation of MSTN mRNA level The change in MSTN mRNA expression by AO was evaluated by RT-PCR in human rhabdomyosarcoma cells (CRL-2016, ATCC) and human myoblasts (Wada et al. Development 2002, 129; 2987-2995).

[0103] AO transfection 1) 2 μl of AO (prepared as 50 pmol / μl with MilliQ sterilized water) was mixed with 100 μl of Opti-MEM medium (31985070, Thermo Fisher Scientific) respectively. For the untreated AO, 2 μl of MilliQ sterilized water was mixed. 2) In another tube, 4 μl of Lipofectamine TM 2000 Transfection Reagent (11668019, Thermo Fisher Scientific) was mixed with 100 μl of Opti-MEM medium (31985070, Thermo Fisher Scientific). 3) The solution of 1) and 2) was mixed and left at room temperature for 20 minutes. 4) Human rhabdomyosarcoma cells cultured in a 12-well plate were washed once with PBS, and then 800 μl of Opti-MEM medium (31985070, Thermo Fisher Scientific) was added to the well. 5) The solution of 3) was added to 4) (final concentration of AO: 100 nM), and after culturing at 37 °C under 5% CO2 for 3 hours, the medium was replaced with RPMI medium (22400-089, gibco) containing 10% FBS (10270-106, gibco), and the culture was continued. 6) Human myoblasts were cultured in DMEM medium (043-30085, Wako) containing 20% FBS (10270-106, gibco) and 2% Ultroser G, and AO was transfected at final concentrations of 0, 100, 200, and 400 nM.

[0104] RNA preparation 1) After culturing the cells transfected with each AO for 24 hours, they were washed once with PBS, and 300 μl of the RNA extraction reagent of the High Pure RNA Isolation Kit (#11828665001, Roche Life Science) was added to the cells. 2) After leaving at room temperature for 5 minutes, the RNA extraction reagent in the well was collected into a tube. 3) RNA was extracted according to the protocol of the High Pure RNA Isolation Kit (#11828665001, Roche Life Science), and finally 50 μl of RNA lysis solution was obtained.

[0105] Reverse transcription reaction 1) Random primers (#48190011, Thermo Fisher Scientific) and dNTPs (Takara) were added to 500 ng of RNA, and the mixture was incubated at 65°C for 5 minutes and at 25°C for 10 minutes. 2) M-MLV Reverse Transcriptase (#28025013, Thermo Fisher Scientific), RNaseOUT TM Recombinant Ribonuclease Inhibitor (#10777-019, Thermo Fisher Scientific), DTT (attached to MLVRT), and buffer (attached to MLVRT) were added to the solution in 1), and the mixture was incubated at 37°C for 55 minutes and at 70°C for 10 minutes to obtain cDNA.

[0106] PCR reaction and confirmation of reaction products 1) To 2 μl of the obtained cDNA, 1 μl of primer MSTN Ex1_F1 (sequence: 5'-agattcactggtgtggcaag-3': SEQ ID NO: 26), 1 μl of MSTN R2 (sequence: 5'-tgcatgacatgtctttgtgc-3': SEQ ID NO: 27), and TaKaRa Ex Taq (登録商標)DNA polymerase (#RR001A, Takara) 0.1 μl, dNTPs (attached to TaKaRa Ex Taq (登録商標) ), 1.6 μl, buffer (10x) 2 μl, and MilliQ sterilized water 12.3 μl were added. 2) Heated at 94°C for 3 minutes. 3) Performed 30 cycles of treatment at 94°C for 0.5 minute, 60°C for 0.5 minute, and 72°C for 1.5 minutes. 4) Heated at 72°C for 3 minutes. 5) The reaction products of the PCR reaction were added with Midri Green Direct DNA Stain (NE-MG06, Nippon Genetics) and Loading buffer (Takara), electrophoresed on a 2% agarose gel, and then visualized using a gel imaging device. Also, electrophoresis and quantification of the PCR reaction products were performed using an Agilent 2100 Bioanalyzer electrophoresis system (Agilent Technologies, Inc.). 6) For GAPDH, the above steps 1) - 5) were performed using primers GAPDH H_F (5'-cccttcattgacctcaac-3': SEQ ID NO: 28), GAPDH H_R (5'-ttcacacccatgacgaac-3': SEQ ID NO: 29). 7) For GDF11, the above steps 1) - 5) were performed using primers GDF11Ex1F4 (5'-ctgcagcagatcctggacct-3': SEQ ID NO: 34), GDF11Ex1R4 (5'-catgaacatgtactcgcact-3': SEQ ID NO: 35). 8) The PCR amplification products separated by agarose electrophoresis were semi-quantified using Image J and relatively compared with MSTN / GAPDH without AO treatment as 1. Similarly, in the PCR amplification products quantified using an Agilent 2100 Bioanalyzer electrophoresis system, MSTN / GAPDH and GDF11 / GAPDH without AO treatment were also relatively compared as 1.

[0107] 2. Evaluation of Myostatin Signaling The myostatin signal was evaluated by introducing a reporter gene (SBE4-Luc plasmid, #16495, Addgene) into human rhabdomyosarcoma cells (CRL-2061, ATCC) and human myoblasts (Wada et al. Development 2002,129;2987-2995) and measuring the luminescence of the induced luciferase expression.

[0108] AO transfection 1) 2 μl of AO2+1 (prepared as 50 pmol / μl in MilliQ sterilized water) was mixed with 100 μl of Opti-MEM medium (31985070, Thermo Fisher Scientific). 2) In a separate tube, 3 μg of SBE4-Luc plasmid, 1 μg of pSV-β-Galactosidase Control Vector (E108A, Promega), 4 μl of Lipofectamine TM 3000 Transfection Reagent (11668019, Thermo Fisher Scientific), and 8 μl of P3000 reagent (11668019, Thermo Fisher Scientific) were mixed with 100 μl of Opti-MEM medium (3198507, Thermo Fisher Scientific). 3) The solutions from 1) and 2) were mixed and left at room temperature for 15 minutes. 4) Human rhabdomyosarcoma cells cultured in a 12-well plate were washed once with PBS, and then 800 μl of Opti-MEM medium (31985070, Thermo Fisher Scientific) was added to the wells. 5) The solution from 3) was added to 4) (final AO concentration 100 nM), and after culturing at 37°C under 5% CO2 for 3 hours, the medium was replaced with RPMI medium (22400-089, gibco) containing 10% FBS (10270-106, gibco), and the culture was continued. 6) Human myoblasts were cultured in DMEM medium (043-30085, Wako) containing 20% FBS (10270-106, gibco) and 2% Ultroser G, and transfected with AO at final concentrations of 0, 50, 100, 150, 200, 300, and 400 nM.

[0109] Cell extract preparation 1) Cells 24 hours after transfection were washed once with PBS, and then 250 μl of Reporter Lysis Buffer from the Luciferase Assay System with Reporter Lysis Buffer (E4030, Promega) was added to the wells. 2) The cell lysate from 1) was centrifuged at 15000 rpm for 10 minutes at 4°C to obtain the supernatant. 3) Protein quantification in the cell extract was performed according to the protocol using the Qubit® Protein Assay Kit (#Q33211, Thermo Fisher Scientific).

[0110] Luciferase activity measurement 1) 100 μl of the cell extract and 100 μl of the luciferase substrate (Luciferase Assay System with Reporter Lysis Buffer, #E4030, Promega) were mixed on a 96-well plate. 2) The luciferase luminescence signal was measured using the multi-label plate reader ARVO TM X3 (PerkinElmer).

[0111] β-Galactosidase activity measurement 1) 100 μl of the cell extract and 100 μl of the β-galactosidase substrate (β-Galactosidase Enzyme Assay System with Reporter Lysis Buffer, #E2000, Promega) were mixed on a 96-well plate and incubated at 37°C for 1 hour. 2) After adding 100 μl of 1 M Sodium Carbonate to the well to stop the reaction, absorbance at 420 nm was measured using a multi-label plate reader ARVO TM X3 (PerkinElmer).

[0112] Activity evaluation After normalizing the luciferase activity value by dividing it by the β-galactosidase activity value, the relative value was evaluated with the result of measuring the extract from cells without AO treatment set as 1.

[0113] 3. Cell proliferation measurement Cell proliferation was evaluated using Cell Counting Kit-8 (347-07621, Dojindo Laboratories) for the proliferation of human myoblasts (Wada et al. Development 2002, 129; 2987-2995).

[0114] AO transfection 1) 0.4 μl of AO2+1 (prepared as 50 pmol / μl in MilliQ sterilized water) was mixed with 50 μl of Opti-MEM medium (31985070, Thermo Fisher Scientific). 2) In another tube, 0.4 μl of Lipofectamine TM 3000 Transfection Reagent (11668019, Thermo Fisher Scientific) was mixed with 50 μl of Opti-MEM medium (3198,5070, Thermo Fisher Scientific). 3) The solutions in 1) and 2) were mixed and left at room temperature for 15 minutes. 4) Human myoblasts cultured in a 96-well plate were washed once with PBS, then the solution in 3) was added to the well (final concentration of AO: 200 nM), and after culturing at 37°C under 5% CO2 for 3 hours, the medium was replaced with DMEM medium (043-30085, Wako) containing 20% FBS (10270-106, gibco) and 2% Ultroser G, and the culture was continued.

[0115] Measurement of viable cells Immediately after AO transfection (day 0), 2 days later, and 3 days later, 10 μl of Cell Counting Kit-8 (347-07621, Dojindo Laboratories) was added per well, and after culturing at 37 °C under 5% CO2 for 1 hour, the absorbance at 450 nm was measured to evaluate the cell count.

[0116] Nucleic acid sequence comparison by alignment Nucleic acid sequence alignment of the amino acid coding region of exon 1 of human myostatin (NM_005259.2), bovine myostatin (NC_037329.1), porcine myostatin (NC_010457.5), and canine myostatin (NM_001002959.1) was performed. For the alignment, the multiple sequence alignment program Clustal Omega of EMBL-EBI (https: / / www.ebi.ac.uk / Tools / msa / clustalo / ) was used. The sequences used for the alignment comparison of the myostatin exon 1 nucleic acid sequences of human, bovine, porcine, and canine are shown in SEQ ID NOs: 30 to 33.

[0117] Experimental results For the purpose of suppressing the expression of myostatin (MSTN), three 18-base AOs having a sequence complementary to exon 1 of MSTN pre-mRNA were prepared (FIG. 1, FIG. 2a). Each AO was prepared based on the prediction of the binding of splicing factors in MSTN pre-mRNA. After treating human rhabdomyosarcoma cells with each AO, the amount of MSTN mRNA was verified by RT-PCR. As a result, a significant decrease in MSTN mRNA was observed after 24 hours of treatment with AO1 and AO2 (FIG. 2).

[0118] Since AO2 decreased MSTN mRNA more than AO1, screening for the optimal AO sequence was performed with AO2 as the center. First, AOs were prepared by shifting the sequence of AO2 by three bases forward and backward, and six AOs targeting bases 231 to 263 of MSTN exon 1 (SEQ ID NO: 1) were verified (Figure 3a). As a result of verification in human rhabdomyosarcoma cells, a significant decrease in MSTN mRNA was observed after 24 hours of treatment with AO2-3, AO2, AO2+3, AO2+6, and AO2+9 (Figures 3b, c). Among them, since AO2+3 showed the most significant effect on decreasing MSTN mRNA, AOs were prepared by shifting AO2+3 by one base at a time, and seven AOs targeting bases 234 to 257 of MSTN exon 1 (SEQ ID NO: 1) were verified (Figure 3a). As a result of verification in human rhabdomyosarcoma cells, a significant decrease in MSTN mRNA was observed after 24 hours of treatment with AO2, AO2+1, AO2+2, AO2+3, AO2+4, AO2+5, and AO2+6 (Figures 3d, e). Through the above screening, AO2+1 was obtained as the AO most effective in suppressing MSTN expression.

[0119] Since it was possible to suppress MSTN expression with an AO targeting exon 1 of MSTN, AOs were prepared in addition to the sequence around AO2+1, and their effectiveness was compared with that of AO2+1. Twelve AOs targeting the range of bases 22 to 420 of MSTN exon 1 (SEQ ID NO: 1) were prepared, and 13 including AO2+1 were each treated on human rhabdomyosarcoma cells for 24 hours. As a result, an inhibitory trend in the expression of MSTN mRNA was observed for all AOs (Figure 4). In particular, AO6, AO7, AO3-6, AO3-12, AO8, AO9, AO9-12, AO10, and AO10-9 significantly decreased MSTN mRNA. Comparison of the reduction rates of these MSTN mRNAs showed that AO2+1 was the most effective. Furthermore, the effect of AO2+1 was also shown in human myoblasts (Figure 5). On the other hand, AO2+1 did not affect the expression of GDF11 (Figure 6).

[0120] When mature myostatin acts on receptors on the cell membrane, the transcription factors Smad2 / 3 are phosphorylated, translocate to the nucleus, and induce the expression of target genes (Figure 7). The activation of this myostatin signal was evaluated using an in vitro myostatin transcriptional activity measurement system (Figure 8). In the in vitro myostatin transcriptional activity measurement system, a plasmid with a luciferase gene arranged downstream of the Smad-binding promoter was used, and the myostatin signal was verified by measuring the activity of luciferase expressed from this plasmid. As a result of verifying the myostatin signal in human rhabdomyosarcoma cells and human myoblasts, since the luciferase activity was suppressed by the treatment with AO2+1, it was shown that AO2+1 suppresses the myostatin signal (Figures 8 and 9). Furthermore, AO2+1 showed a growth-promoting effect on human myoblasts (Figure 10).

[0121] Discussion Since the inhibition of myostatin function promotes myogenesis, it has attracted attention as a method for treating muscular atrophy diseases. In fact, clinical trials of muscular atrophy diseases such as Duchenne muscular dystrophy (DMD) have been conducted on antibodies and peptides that inhibit the function of myostatin. On the other hand, an off-target effect in which these antibodies and peptides target proteins other than myostatin has been regarded as a problem. For example, mature myostatin that activates the myostatin signal has 90% amino acid identity with mature GDF11 of the same TGF-beta family, and the myostatin inhibitory peptide also inhibits GDF11 (Osawa et al., PLoS One, 2015). In contrast, AO2+1 in this study does not affect the expression of GDF11 mRNA. Also, although loss-of-function AOs targeting the skipping of exon 2 of MSTN pre-mRNA have been developed so far, no clinically useful AO has been obtained. The AO of the present invention is an expression suppression type targeting exon 1 of MSTN pre-mRNA, and since its action and effect are different from those of the conventional loss-of-function AOs, it is expected to be effective as a therapeutic agent.

[0122] The AO2+1 of the present invention is particularly highly effective. It not only inhibits the expression of MSTN mRNA, but also suppresses the myostatin signal and promotes the proliferation of myoblasts. There are many diseases in which inhibition of myostatin is considered to be effective. As a medicine, it can be used for the prevention and / or treatment of muscular atrophy diseases (such as muscular dystrophy, spinal muscular atrophy, sarcopenia, disuse muscular atrophy), cardiovascular diseases (such as heart failure, arteriosclerosis, etc.), kidney diseases (such as chronic renal failure, etc.), bone diseases (such as inflammatory arthritis, etc.), cancer or diabetes. Furthermore, inhibition of myostatin leads to an increase in skeletal muscle mass, an increase in exercise volume, and also contributes to the improvement of whole-body metabolism. In addition, it is expected to act on cardiomyocytes and restore their function. Furthermore, it is also expected that myostatin inhibition acts on osteoclasts to suppress bone destruction, activates the ability of vascular endothelial cells to maintain homeostasis, induces apoptosis, and enhances sensitivity to insulin.

[0123] The target sequence of AO2+1 of the present invention is also conserved in cows and pigs, and it can also be used for promoting the growth of these livestock (Figure 11). Since the suppression of MSTN expression by administering AO to livestock does not correspond to the production of genetically modified organisms, it is easy to use. Similarly, it is considered to be effective for dogs as well, and it is considered to be applicable to the muscle decline of pet dogs (Figure 11). All publications, patents and patent applications cited in this specification are hereby incorporated herein by reference in their entirety.

Industrial Applicability

[0124] The present invention can be used as a nucleic acid medicine that suppresses the production of mRNA of the myostatin gene.

Sequence Listing Free-Text

[0125] <SEQ ID NO: 1> shows the nucleotide sequence information of MSTN exon 1. (Total 506 bases. The start codon (atg) is shown in □) TIFF0007715345000002.tif58146 <SEQ ID NOs: 2 to 25> shows the nucleotide sequences of AO synthesized in the examples. The nucleotides constituting the antisense oligonucleotide may be any of natural DNA, natural RNA, DNA / RNA chimeras, and modified forms thereof, and at least one may be a modified nucleotide. <SEQ ID NOs: 26 to 29> shows the nucleotide sequences of the primers used in the test examples. <SEQ ID NOs: 30 to 33> shows the sequences used for the alignment comparison of the myostatin exon 1 nucleic acid sequences of human, bovine, porcine, and canine. The alignment comparison was performed using Multiple Sequence Alignment of EMBL-EBI. Only the amino acid coding region of exon 1 was compared. The names in parentheses are those found in BLAST. The sequences within the box are the target sequences of AO2 + 1. Ex1 (ORF) TIFF0007715345000003.tif33160 TIFF0007715345000004.tif32160 TIFF0007715345000005.tif32160 TIFF0007715345000006.tif33160 <SEQ ID NOs: 34 and 35> shows the nucleotide sequences of the primers GDF11Ex1F4 and GDF11Ex1R4.

Claims

1. An antisense oligonucleotide having a base sequence of 18 bases in length, which is complementary to a target site in exon 1 of the myostatin gene, wherein the base sequence of the antisense oligonucleotide is the base sequence of SEQ ID NO: 10 (however, t in the sequence may be u, and u may be t), at least one nucleotide is modified, and the production of myostatin gene mRNA can be suppressed by controlling the splicing of the myostatin gene, said antisense oligonucleotide, its salt or solvate.

2. The antisense oligonucleotide, its salt or solvate according to Claim 1, wherein the sugar constituting the modified nucleotide is D-ribofuranose and the hydroxyl group at the 2'-position of D-ribofuranose is modified.

3. The antisense oligonucleotide, its salt or solvate according to Claim 2, wherein D-ribofuranose is 2'-O-alkylated and / or 2'-O, 4'-C-alkylenated.

4. A medicament comprising the antisense oligonucleotide according to any one of Claims 1 to 3, its pharmaceutically acceptable salt or solvate.

5. The medicament according to Claim 4, which is for preventing and / or treating a condition and / or disease in which myostatin is involved.

6. The medicament according to Claim 5, wherein the condition and / or disease in which myostatin is involved is muscular atrophy.

7. The medicament according to Claim 6, wherein the muscular atrophy is at least one selected from the group consisting of muscular dystrophy, myopathy, spinal muscular atrophy, sarcopenia and disuse muscular atrophy.

8. The medicament according to Claim 5, wherein the condition and / or disease in which myostatin is involved is a condition and / or disease in which a therapeutic effect is brought about by muscle mass recovery.

9. The medicament according to Claim 8, wherein the condition and / or disease in which a therapeutic effect is brought about by muscle mass recovery is at least one selected from the group consisting of cancer cachexia, diabetes, cardiovascular disease, kidney disease and bone disease.

10. The medicament according to Claim 9, wherein the cardiovascular disease is heart failure and / or arteriosclerosis, the kidney disease is chronic renal failure, and the bone disease is inflammatory arthritis.

11. A food comprising the antisense oligonucleotide according to any one of Claims 1 to 3, its food-acceptable salt or solvate.

12. A feed comprising the antisense oligonucleotide according to any one of claims 1 to 3, a salt or solvate thereof acceptable in the feed.

13. A medicament for promoting the proliferation and / or hypertrophy of muscle cells, comprising the antisense oligonucleotide according to any one of claims 1 to 3, a salt or solvate thereof.

14. A medicament for increasing muscle mass and / or suppressing muscle loss, comprising the antisense oligonucleotide according to any one of claims 1 to 3, a salt or solvate thereof.

15. A medicament for suppressing the production of myostatin gene mRNA by controlling the splicing of the myostatin gene, comprising the antisense oligonucleotide according to any one of claims 1 to 3, a salt or solvate thereof.

16. A myostatin function inhibitor, comprising the antisense oligonucleotide according to any one of claims 1 to 3, a salt or solvate thereof.

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