Compound and pharmaceutical composition for regulating myoregulin expression

Abstract

Provided is a modified oligonucleotide having an activity of inhibiting myoregulin expression and comprising 12-30 linked nucleosides or a pharmaceutically acceptable salt of the modified oligonucleotide. The modified oligonucleotide comprises a base sequence 100% complementary to eight or more consecutive bases in a specific region of a base sequence represented by SEQ ID NO: 1 in the sequence listing. The full-length base sequence of the modified oligonucleotide is at least 85% complementarity to an equal length portion of the base sequence represented by SEQ ID NO: 1 in the sequence listing.

Description

Compounds and pharmaceutical compositions for regulating myoregulin expression

[0001] The present invention relates to a compound for reducing at least one of the pre-mRNA level, mRNA level and protein level of Myoregulin (MRLN) in an animal, and a pharmaceutical composition containing said compound.

[0002] Myoregulin (MRLN) is a micropeptide encoded by long noncoding RNA, specifically expressed in skeletal muscle, and discovered to regulate calcium levels within the sarcoplasmic reticulum (Non-Patent Document 1). It has been reported that treatment of cells derived from Duchenne muscular dystrophy (DMD) patients with MRLN siRNA, which suppresses MRLN mRNA expression, increased calcium uptake into the sarcoplasmic reticulum (Patent Document 1). It has also been reported that administration of MRLN antisense oligonucleotides to DMD model mice (mdx mice) and sarcoglycanopathy model mice (sarcoglycan β knockout mice) reduced MRLN gene expression, resulting in a decrease in creatine kinase levels, a clinical indicator of muscular dystrophy and caused by muscle damage and muscle cell death, in both model mice (Patent Document 1). Sarcoglycanopathy is a general term for limb girdle muscular dystrophy (LGMD) types 2C to 2F.

[0003] Thus, suppressing the expression level of MRLN can increase the amount of calcium uptake into the sarcoplasmic reticulum, and is therefore effective in treating or preventing muscle diseases such as muscular dystrophy, which are accompanied by muscle damage or muscle cell death due to abnormal calcium influx into the cytoplasm and / or decreased muscle contractility.

[0004] Under these circumstances, there is a need for an excellent antisense oligonucleotide that suppresses the expression level of MRLN for the treatment of muscle diseases. The MRLN antisense oligonucleotides reported so far were designed based on the nucleotide sequence of the mouse MRLN gene (Patent Document 1), and it is difficult to apply them clinically for the treatment or prevention of human muscle diseases.

[0005] WO2022 / 014703

[0006] Cell, 160(4):595-606 (2015)

[0007] The present invention provides compounds for inhibiting MRLN expression.

[0008] The present inventors have conducted extensive research and discovered modified oligonucleotides that suppress MRLN expression, thereby completing the present invention.

[0009] The present invention includes the following aspects: [1] A modified oligonucleotide consisting of 12 to 30 linked nucleosides or a pharmaceutically acceptable salt thereof, which has activity of inhibiting Myoregulin expression, wherein the modified oligonucleotide is located at positions 15 to 41, 62 to 108, 448 to 471, 649 to 666, 848 to 863, 1104 to 1119, 1232 to 1254, 1691 to 1719, 1735 to 1751, 2195 to 2211, 3195 to 3211, 3215 to 3215, 3216 to 3217, 3218 to 3219, 3220 to 3221, 3222 to 3222, 3223 to 3224, 3225 to 3225, 3226 to 3227, 3228 to 3229, 3230 to 3231, 3232 to 3232, 3233 to 3234, 3235 to 3235, 3236 to 3237, 3238 to 3239, 3240 to 3241, 3242 to 3243, 3244 to 3245, 3246 to 3248, 3248 to 3249, 3249 to 3251, 3249 to 3252, 3249 to 3253, 3249 to 3254, 3255 to 3255, 3256 to 3256, 3257 to 3258, 3259 to 32 2289-2310, 2479-2512, 2616-2631, 2692-2716, 2751-2766, 2893-2909, 2979-2995, 3431-3447, 3564-3585, 3601-3 617th, 3721-3736, 4074-4089, 4127-4142, 4156-4171, 4277-4292, 4305-4322, 4500-4515, 4613-4628, 5039-5055, 50 63-5079th, 5103-5119, 5522-5540, 5727-5742, 5772-5803, 5998-6013, 6272-6288, 6695-6711, 6763-6778, 7224-724 7th, 8013-8028, 9844-9864, 14649-14665, 14903-14920, 14951-14995, 16113-16129, 16235-16254, 16256-16273, 162 A modified oligonucleotide or a pharmaceutically acceptable salt thereof, comprising a base sequence that is 100% complementary to any 8 or more consecutive bases in any of the base sequences at positions 81 to 16306, 16316 to 16336, 16514 to 16536, 16578 to 16593, 16610 to 16637, or 16724 to 16739, and the full-length base sequence of the modified oligonucleotide is 85% or more complementary to an equal-length portion of the base sequence of SEQ ID NO: 1 in the Sequence Listing.[2] The modified oligonucleotide is selected from the group consisting of positions 1232 to 1254, 1691 to 1719, 1735 to 1751, 2195 to 2211, 2289 to 2308, 3431 to 3446, 4127 to 4142, 4156 to 4171, 4500 to 4515, 5522 to 5537, 5727 to 5742, 5772 to 5803, 5998 to 6013, 6272 to 6287, 6763 to 6778, 8013 to 8028, 98 The modified oligonucleotide or a pharmaceutically acceptable salt thereof according to [1], comprising a base sequence that is 100% complementary to any 8 or more consecutive bases in any of the base sequences at positions 46 to 9864, 14963 to 14978, 16235 to 16254, 16256 to 16273, 16281 to 16306, or 16316 to 16331, and the full-length base sequence of the modified oligonucleotide is 85% or more complementary to an equal-length portion of the base sequence of SEQ ID NO: 1 in the Sequence Listing. [3] A modified oligonucleotide consisting of 12 to 30 linked nucleosides, or a pharmaceutically acceptable salt thereof, which has activity of inhibiting Myoregulin expression, wherein the modified oligonucleotide comprises a base sequence that is 100% complementary to any 8 or more consecutive bases in any of the base sequences at positions 15 to 41, 62 to 91, 100 to 129, 160 to 225, 227 to 244, 252 to 277, 287 to 307, 485 to 507, 549 to 564, 581 to 608, or 695 to 710 from the 5' end of the base sequence of SEQ ID NO: 2 in the Sequence Listing, and the full-length base sequence of the modified oligonucleotide is 85% or more complementary to an equal-length portion of the base sequence of SEQ ID NO: 2 in the Sequence Listing.[4] The modified oligonucleotide or a pharmaceutically acceptable salt thereof according to [3], wherein the modified oligonucleotide comprises a base sequence that is 100% complementary to any 8 or more consecutive bases in any of the base sequences at positions 172 to 187, 201 to 225, 227 to 244, 252 to 277, or 287 to 302 from the 5' end of the base sequence of SEQ ID NO: 2 in the Sequence Listing, and the full-length base sequence of the modified oligonucleotide is 85% or more complementary to the equal-length portion of the base sequence of SEQ ID NO: 2 in the Sequence Listing. [5] A modified oligonucleotide or a pharmaceutically acceptable salt thereof having an activity of suppressing Myoregulin expression, wherein the base sequence of the modified oligonucleotide is: CCAGAATTATCCCGCT (complementary sequence of positions 16238 to 16253 of SEQ ID NO: 1) (SEQ ID NO: 275 in the Sequence Listing), TCCAGAATTATCCCGC (complementary sequence of positions 16239 to 16254 of SEQ ID NO: 1) (SEQ ID NO: 276 in the Sequence Listing), AGTTTTTACCAGTCAT (complementary sequence of positions 16256 to 16271 of SEQ ID NO: 1) (SEQ ID NO: 277 in the Sequence Listing), CCAGTTTTTTACCAGTC (complementary sequence of positions 16258 to 16273 of SEQ ID NO: 1) (SEQ ID NO: 278 in the Sequence Listing), A modified oligonucleotide or a pharmaceutically acceptable salt thereof, which is any base sequence selected from the group consisting of GACTTTTTGGGAGTAGT (complementary sequence of positions 16289 to 16304 of SEQ ID NO: 1) (SEQ ID NO: 279 in the Sequence Listing), AGACTTTTTGGGAGTAG (complementary sequence of positions 16290 to 16305 of SEQ ID NO: 1) (SEQ ID NO: 229 in the Sequence Listing), and GAAGTCTTCCCACAAT (complementary sequence of positions 16316 to 16331 of SEQ ID NO: 1) (SEQ ID NO: 231 in the Sequence Listing), or a base sequence consisting of 17 or 18 consecutive bases containing the base sequence, and wherein the full-length base sequence of the modified oligonucleotide is 100% complementary to the isolength portion of the base sequence of SEQ ID NO: 1.[6] A modified oligonucleotide or a pharmaceutically acceptable salt thereof having activity of suppressing Myoregulin expression, wherein the base sequence of the modified oligonucleotide is either one of the base sequences: TTATCCCGCTCCCTGA (complementary sequence of positions 203 to 218 of SEQ ID NO: 2) (SEQ ID NO: 273 in the Sequence Listing) or ATTATCCCGCTCCCTG (complementary sequence of positions 204 to 219 of SEQ ID NO: 2) (SEQ ID NO: 280 in the Sequence Listing), or a base sequence consisting of 17 or 18 consecutive bases containing the base sequence, and wherein the base sequence of the entire length of the modified oligonucleotide is 100% complementary to an equal-length portion of the base sequence of SEQ ID NO: 2. [7] The modified oligonucleotide or a pharmaceutically acceptable salt thereof according to any of [1] to [6], wherein the modified oligonucleotide is single-stranded. [8] The modified oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of [1] to [7], wherein at least one nucleoside constituting the modified oligonucleotide contains a modified sugar. [9] The modified oligonucleotide or a pharmaceutically acceptable salt thereof according to [8], wherein the modified sugar is a bicyclic sugar.

[10] The modified oligonucleotide or a pharmaceutically acceptable salt thereof according to [9], wherein the bicyclic sugar is selected from the group consisting of LNA, ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Oxz], and ALNA[Trz] sugar moieties.

[11] The modified oligonucleotide or a pharmaceutically acceptable salt thereof according to [9] or

[10] , wherein the bicyclic sugar is the sugar moiety of ALNA[Ms].

[12] The modified oligonucleotide or a pharmaceutically acceptable salt thereof according to [8], wherein the modified sugar is a substituted sugar.

[13] The modified oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of [1] to

[12] , wherein at least one nucleoside constituting the modified oligonucleotide contains a modified base.

[14] The modified oligonucleotide or a pharmaceutically acceptable salt thereof according to

[13] , wherein the modified base is 5-methylcytosine.

[15] The modified oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of [1] to

[14] , wherein at least one internucleoside linkage constituting the modified oligonucleotide is a modified internucleoside linkage.

[16] The modified oligonucleotide or a pharmaceutically acceptable salt thereof according to

[15] , wherein the modified internucleoside linkage is a phosphorothioate internucleoside linkage.

[17] The modified oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of [1] to

[16] , wherein the modified oligonucleotide comprises 1) a gap segment, 2) a 5' wing segment, and 3) a 3' wing segment, wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment, and all of the sugar moieties of the nucleosides constituting the 5' wing segment and the 3' wing segment are modified sugars.

[18] A pharmaceutical composition comprising the modified oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of [1] to

[17] .

[19] The pharmaceutical composition according to

[18] , for treating or preventing a muscular disease.

[20] The pharmaceutical composition of

[19] , wherein the muscle disease is selected from the group consisting of muscular dystrophy, inclusion body myositis, amyotrophic lateral sclerosis, disuse muscular atrophy, and sarcopenia.

[21] The pharmaceutical composition of

[20] , wherein the muscle disease is muscular dystrophy.

[22] The pharmaceutical composition of

[21] , wherein the muscular dystrophy is selected from the group consisting of Duchenne muscular dystrophy, Becker muscular dystrophy, and sarcoglycanopathy.

[23] The pharmaceutical composition of

[19] , wherein the muscle disease is Duchenne muscular dystrophy.

[24] The pharmaceutical composition of

[19] , wherein the muscle disease is Becker muscular dystrophy.

[25] The pharmaceutical composition of

[19] , wherein the muscle disease is sarcoglycanopathy.

[26] A method for treating or preventing a muscle disease, comprising the step of administering to a subject an effective amount of the modified oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of [1] to

[17] .

[27] Use of the modified oligonucleotide according to any one of [1] to

[17] or a pharmaceutically acceptable salt thereof for the treatment or prevention of a muscular disease.

[28] Use of the modified oligonucleotide according to any one of [1] to

[17] or a pharmaceutically acceptable salt thereof in the manufacture of a pharmaceutical composition for treating or preventing a muscular disease.

[29] The modified oligonucleotide according to any one of [1] to

[17] or a pharmaceutically acceptable salt thereof for use in the manufacture of a pharmaceutical composition for treating or preventing a muscular disease.

[0010] The modified oligonucleotides of the present invention have excellent inhibitory activity against MRLN expression.

[0011] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed. Herein, the use of the singular includes the plural unless expressly stated otherwise. Herein, the use of the term "including" as well as other forms such as "includes" and "included" is not limiting.

[0012] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. All documents or portions of documents cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are expressly incorporated herein by reference in their entirety and with respect to the portions of the documents discussed herein.

[0013] Definitions Unless specific definitions are provided, the nomenclatures used in connection with analytical chemistry, organic synthetic chemistry, and medicinal and pharmaceutical chemistry described herein, and the procedures and techniques thereof, are well known and commonly used in the art. Standard techniques can be used for the chemical synthesis and chemical analysis used herein. Where permitted, all patents, applications, published applications, and other publications referred to throughout this disclosure, GenBank accession numbers and related sequence information and other data available through databases such as the National Center for Biotechnology Information (NCBI), are incorporated by reference in their entirety with respect to portions of the documents discussed herein. This specification is also filed with a Sequence Listing in electronic format, and the information in the Sequence Listing set forth in said electronic format is incorporated by reference in its entirety herein.

[0014] Unless otherwise indicated, the following terms have the following meanings:

[0015] "Base" means a heterocyclic moiety capable of pairing with a base of another nucleic acid.

[0016] "Contiguous bases" means bases that are adjacent to each other.

[0017] "Base sequence" means the order of consecutive bases.

[0018] "Nucleoside" means a molecule consisting of a sugar and a base linked together.

[0019] "Linked nucleosides" means nucleosides that are joined by internucleoside linkages in a contiguous sequence.

[0020] "Nucleotide" refers to a molecule in which a phosphate group is attached to the sugar moiety of a nucleoside. Naturally occurring nucleotides have a sugar moiety of ribose or deoxyribose.

[0021] "Oligonucleotide" means a polymer of nucleosides in which each nucleoside is joined by an internucleoside linkage.

[0022] "Internucleoside linkage" refers to the chemical bond between nucleosides.

[0023] A "modified internucleoside linkage" refers to a substitution or any change from a naturally occurring internucleoside linkage (i.e., a phosphodiester internucleoside linkage), such as, but not limited to, a phosphorothioate internucleoside linkage.

[0024] "Modified base" refers to any base other than adenine, cytosine, guanine, thymidine, or uracil, for example, but not limited to, 5-methylcytosine.

[0025] "Modified nucleoside" means a nucleoside having, independently, a modified sugar or modified base.

[0026] "Modified oligonucleotide" means an oligonucleotide containing at least one modified nucleoside and / or modified internucleoside linkage.

[0027] "Sugar" or "sugar moiety" means a naturally occurring sugar moiety or a modified sugar moiety.

[0028] "Modified sugar" means a substitution or change from a natural sugar. Modified sugars include, for example, substituted sugars or bicyclic sugars.

[0029] "Substituted sugar" means a furanosyl sugar other than the natural sugars of RNA or DNA, but does not include bicyclic sugars.

[0030] "Bicyclic sugar" means a furanosyl modified by bridging two different carbon atoms of the four carbon atoms forming the furanosyl ring.

[0031] "Single-stranded oligonucleotide" means an oligonucleotide that is not hybridized to a complementary strand.

[0032] "Identical length portion" refers to a portion of the base sequence of a second nucleic acid that has a length equal to the length of the base sequence of a first nucleic acid. In certain embodiments, the first nucleic acid is a modified oligonucleotide and the second nucleic acid is MRLN pre-mRNA or MRLN mRNA.

[0033] "Complementary" refers to the capacity for pairing between the bases of a first nucleic acid and a second nucleic acid. In certain embodiments, adenine is complementary to thymidine or uracil. In certain embodiments, cytosine is complementary to guanine. In certain embodiments, 5-methylcytosine is complementary to guanine.

[0034] "100% complementary" means that the base sequence of a first nucleic acid is completely complementary to the base sequence of a second nucleic acid. In certain embodiments, the first nucleic acid is a modified oligonucleotide and the target nucleic acid is the second nucleic acid.

[0035] A "mismatch" refers to the failure of a base of a first nucleic acid to pair with the corresponding base of a second or target nucleic acid.

[0036] "Target nucleic acid," "target RNA," and "target RNA transcript" refer to a nucleic acid, RNA, or RNA transcript, respectively, that can be targeted by a modified oligonucleotide. In certain embodiments, the target nucleic acid comprises a region of MRLN mRNA or MRLN pre-mRNA.

[0037] "Pharmaceutically acceptable salts" refers to physiologically and pharmaceutically acceptable salts of modified oligonucleotides of the present invention, including salts formed with inorganic ions, such as metal ions, on phosphorothioates or phosphodiesters or on functional groups (e.g., amino groups) in the modified bases.

[0038] "Amelioration" refers to a lessening of at least one indicator, sign, or symptom of the associated disease, disorder, or condition. The severity of the indicator can be determined by subjective or objective measures known to those skilled in the art.

[0039] "Animal" refers to a human or non-human animal, including, but not limited to, mice, rats, rabbits, dogs, cats, pigs, and non-human primates, including, but not limited to, monkeys and chimpanzees.

[0040] "Effective amount" refers to an amount of a modified oligonucleotide of the present invention that is sufficient to achieve a desired physiological outcome in an individual in need of the drug. The effective amount may vary from individual to individual, depending on the health and physical condition of the individual being treated, the taxonomic group of the individual being treated, the formulation of the composition, an evaluation of the individual's medical condition, and other relevant factors.

[0041] "Individual" means a human or non-human animal selected for treatment or therapy.

[0042] "Prevent" means to delay or forestall the onset or occurrence of a disease, disorder, or adverse health condition, or one or more symptoms associated with the disease, disorder, or adverse health condition, for a period of time ranging from minutes to indefinite. Prevent can also mean reducing the risk of developing a disease, disorder, or adverse health condition.

[0043] "Treating" means alleviating or eliminating a disease, disorder or adverse condition, or one or more symptoms associated with the disease, disorder or adverse condition, or partially eliminating or eradicating one or more causes of the disease, disorder or adverse condition itself.

[0044] Specific Embodiments Namely, compounds for inhibiting the expression of MRLN, methods of using the compounds, and pharmaceutical compositions containing the compounds are provided.

[0045] (1) Modified Oligonucleotides The modified oligonucleotides of the present invention (hereinafter sometimes referred to as "compounds of the present invention" or "modified oligonucleotides of the present invention") are modified oligonucleotides having the activity of suppressing MRLN expression. In the present invention, suppression of MRLN expression (level) means suppression of at least one of the pre-mRNA level transcribed from the MRLN gene, the mRNA level spliced ​​from the pre-mRNA, and the MRLN protein level translated from the mRNA (these levels may be collectively referred to as "MRLN expression level"). The MRLN pre-mRNA is not particularly limited and may include sequence variations, but examples include the sequence shown in GenBank Accession No. NC_000010.11:c59753455-59736692 (SEQ ID NO: 1 in the Sequence Listing). Furthermore, the MRLN mRNA is not particularly limited and may include sequence variations, such as the sequence set forth in GenBank Accession No. NM_001304731.2 (SEQ ID NO: 2 in the Sequence Listing). The RNA sequence is determined by replacing T with U in SEQ ID NO: 1. Furthermore, MRLN mRNA and pre-mRNA may include sequence variants such as various splicing variants and single nucleotide polymorphisms (SNPs).

[0046] The degree of MRLN expression suppression by the compound of the present invention may be any degree that reduces at least one of MRLN pre-mRNA level, mRNA level, and protein level compared to when the compound is not administered, thereby preventing and / or ameliorating symptoms associated with muscular disorders. Specifically, for example, in the in vitro MRLN expression measurement method described below, after contacting cells with the compound of the present invention, the MRLN expression level is reduced by 70% or less, preferably 50% or less, more preferably 40% or less, even more preferably 30% or less, and particularly preferably 20% or less, compared to when the cells are not contacted or when the cells are contacted with a negative control substance. For example, the degree of MRLN expression suppression may be such that when RD cells (human rhabdomyosarcoma cells) are treated with 100 nM of the compound of the present invention by lipofection, the MRLN expression level is reduced by 50% or less. Alternatively, when RD cells are treated with 100 nM of the compound of the present invention by the lipofection method or 1000 nM of the compound of the present invention by the Gymnosis method, the MRLN expression level may be reduced to 50% or less in either case.

[0047] The activity of the compound of the present invention can be verified by any method that can verify that the compound of the present invention suppresses MRLN expression, and examples thereof include the methods described below in the section "Method for evaluating the compound of the present invention."

[0048] The compound of the present invention is a modified oligonucleotide consisting of 12 to 30 linked nucleosides, preferably 16 to 20 linked nucleosides, more preferably 16 to 18 linked nucleosides, and particularly preferably 16, 17, or 18 linked nucleosides, which has activity of suppressing MRLN expression. The modified oligonucleotides are selected from the group consisting of positions 15 to 41, 62 to 108, 448 to 471, 649 to 666, 848 to 863, 1104 to 1119, 1232 to 1254, 1691 to 1719, 1735 to 1751, 2195 to 2211, 2289 to 2310, 2479 to 2512, 2616 to 2631, 2692 to 2716, 2751 to 2766, 2893 to 2909, 2979 to 2980, 3000 to 3000, 3010 to 3012, 3013 to 3014, 3015 to 3016, 3017 to 3018, 3019 to 3020, 3021 to 3022, 3023 to 3024, 3025 to 3026, 3027 to 3028, 3029 to 3030, 3031 to 3032, 3033 to 3034, 3035 to 3036, 3037 to 3038, 3039 to 3040, 3041 to 3042, 3043 to 3044, 3045 to 3046, 3047 to 3048, 3048 to 3049, 3049 to 3050, 3051 to 3051, 3052 to 3052, 3053 to 3054, 3055 to 995th, 3431-3447, 3564-3585, 3601-3617, 3721-3736, 4074-4089, 4127-4142, 4156-4171, 4277-4292, 4305-4322 4500-4515, 4613-4628, 5039-5055, 5063-5079, 5103-5119, 5522-5540, 5727-5742, 5772-5803, 5998-6013, 62 72-6288th, 6695-6711, 6763-6778, 7224-7247, 8013-8028, 9844-9864, 14649-14665, 14903-14920, 14951-14995 , 16113-16129, 16235-16254, 16256-16273, 16281-16306, 16316-16336, 16514-16536, 16578-16593, 16610-16637 Any base sequence (hereinafter sometimes referred to as "MRLN complementary base sequence") that is 100% complementary to any 8 or more, preferably 12 or more, more preferably 14 or more, even more preferably 16 or more, and / or 20 or less, preferably 18 or less, more preferably 17 or less, even more preferably 16 or less, particularly preferably 16 consecutive bases in the base sequence at positions 16724 to 16739 of the MRLN-complementary base sequence may be used.

[0049] The compound of the present invention is a modified oligonucleotide consisting of 12 to 30 linked nucleosides, preferably 16 to 20 linked nucleosides, more preferably 16 to 18 linked nucleosides, and particularly preferably 16, 17, or 18 linked nucleosides, which has activity of suppressing MRLN expression. The modified oligonucleotides are selected from the following positions from the 5' end of the base sequence of SEQ ID NO: 1 in the Sequence Listing: 1232 to 1254, 1691 to 1719, 1735 to 1751, 2195 to 2211, 2289 to 2308, 3431 to 3446, 4127 to 4142, 4156 to 4171, 4500 to 4515, 5522 to 5537, 5727 to 5742, 5772 to 5803, 5998 to 6013, 6272 to 6287, 6763 to 6778, 8013 to 8028, 9846 to 9864, 1499 to 150 ...501 to 1502, 1503 to 1504, 1505 to 1506, 1507 to 1508, 1509 to 1 Any base sequence may be used as long as it contains a base sequence that is 100% complementary to any 8 or more, preferably 12 or more, more preferably 14 or more, even more preferably 16 or more, and / or 20 or less, preferably 18 or less, more preferably 17 or less, even more preferably 16 or less, particularly preferably 16 consecutive bases in the base sequence of any of positions 63 to 14978, 16235 to 16254, 16256 to 16273, 16281 to 16306, or 16316 to 16331.

[0050] The compound of the present invention is a modified oligonucleotide having an activity of suppressing MRLN expression, which is composed of 12 to 30 linked nucleosides, preferably 16 to 20 linked nucleosides, more preferably 16 to 18 linked nucleosides, and particularly preferably 16, 17, or 18 linked nucleosides. The modified oligonucleotide may be any, so long as it contains a nucleotide sequence that is 100% complementary to any 8 or more, preferably 12 or more, more preferably 14 or more, even more preferably 16 or more, and / or 20 or less, preferably 18 or less, more preferably 17 or less, even more preferably 16 or less, and particularly preferably 16 consecutive bases in the nucleotide sequence from positions 16256 to 16273 from the 5' end of SEQ ID NO: 1 in the Sequence Listing.

[0051] The compound of the present invention is a modified oligonucleotide consisting of 12 to 30 linked nucleosides, preferably 16 to 20 linked nucleosides, more preferably 16 to 18 linked nucleosides, and particularly preferably 16, 17, or 18 linked nucleosides, which has activity of suppressing MRLN expression. The modified oligonucleotide may be any, as long as it contains a base sequence that is 100% complementary to any 8 or more, preferably 12 or more, more preferably 14 or more, even more preferably 16 or more, and / or 20 or less, preferably 18 or less, more preferably 17 or less, even more preferably 16 or less, and particularly preferably 16 consecutive bases in the base sequence of any of positions 15 to 41, 62 to 91, 100 to 129, 160 to 225, 227 to 244, 252 to 277, 287 to 307, 485 to 507, 549 to 564, 581 to 608, or 695 to 710 from the 5' end of the base sequence of SEQ ID NO: 2 in the Sequence Listing.

[0052] The compound of the present invention is a modified oligonucleotide having an activity of inhibiting MRLN expression, which is composed of 12 to 30 linked nucleosides, preferably 16 to 20 linked nucleosides, more preferably 16 to 18 linked nucleosides, and particularly preferably 16, 17, or 18 linked nucleosides. The modified oligonucleotide may be any, as long as it contains a nucleotide sequence that is 100% complementary to any 8 or more, preferably 12 or more, more preferably 14 or more, even more preferably 16 or more, and / or 20 or less, preferably 18 or less, more preferably 17 or less, even more preferably 16 or less, and particularly preferably 16 consecutive bases in the nucleotide sequence of any of positions 172 to 187, 201 to 225, 227 to 244, 252 to 277, or 287 to 302 from the 5' end of the nucleotide sequence of SEQ ID NO: 2 in the Sequence Listing.

[0053] The modified oligonucleotide of the present invention has a total length of 12 to 30 linked nucleosides, preferably 16 to 20 linked nucleosides, more preferably 16 to 18 linked nucleosides, and particularly preferably 16, 17, or 18 linked nucleosides. The modified oligonucleotide may have an additional sequence at the 5'-end and / or 3'-end in addition to the MRLN-complementary nucleotide sequence, so long as the modified oligonucleotide of the present invention has activity to inhibit MRLN expression, provided that its full-length nucleotide sequence is 85% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 100% complementary to the isotopic portion of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 in the Sequence Listing. Furthermore, the additional sequence may be any sequence, so long as the modified oligonucleotide of the present invention has activity to inhibit MRLN expression.

[0054] The full-length base sequence of the modified oligonucleotide of the present invention has 85% or more complementarity to the isolength portion of SEQ ID NO: 1 or 2 in the Sequence Listing, and this complementarity is preferably 90% or more, more preferably 95% or more, and even more preferably 100%. In other words, the base sequence of the modified oligonucleotide of the present invention is desirably completely complementary to the isolength portion of the base sequence of the modified oligonucleotide in the base sequence of MRLN pre-mRNA or mRNA, but may have one or more mismatched bases, and those having 85% or more, preferably 90% or more, and more preferably 95% or more complementarity are used. The mismatched bases may be consecutive.

[0055] The percent complementarity of the modified oligonucleotides of the present invention with MRLN pre-mRNA or mRNA can be routinely determined using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656). Percent homology, sequence identity or complementarity can be determined, for example, by the GAP program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.) using default settings using the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489).

[0056] Specifically, the base sequence of the modified oligonucleotide of the present invention is positions 15 to 30, 21 to 36, 22 to 37, 24 to 39, 26 to 41, 62 to 77, 69 to 84, 76 to 91, 91 to 106, 92 to 107, 93 to 108, 448 to 463, 449 to 464, 456 to 471, 649 to 664, 650 to 665, 651 to 666, 848 to 863, 1104 to 1119, 1232 to 1247, 1233 to 1248, 1239 to 1254, 1691 to 1706, 1704 to 1719, 1735 to 1750, 1736 to 1751, 2195 to 2210, 2196 to 2211, 2289 to 2304, 2290 to 2305, 2293 to 2308, 2295 to 2310, 2497 to 2512, 2616 to 2631, 2692 to 2707, 2700 to 2715, 2701 to 2716, 2751 to 2766, 2893 to 2908, 2894 to 2909, 2979 to 2994, 2980 to 2995, 3431 to 3446, 3432 to 3447, 3564 to 3579, 3569 to 3584, 3570 to 3585, 3601 to 3616, 3602 to 3617, 3721 to 3736, 4074 to 4089, 4127 to 4142, 4156 to 4171, 4277 to 4292, 4305 to 4320, 4306 to 4321, 4307 to 4322, 4500 to 4515, 4613 to 4628, 5039 to 5054, 5040 to 5055, 5063 to 5078, 5064 to 5079, 5103 to 5118, 5104 to 5119, 5522 to 5537, 5525 to 5540, 5727 to 5742, 5772 to 5787, 5773 to 5788, 5774 to 5789, 5775 to 5790, 5776 to 5791, 5780 to 5795, 5781 to 5796, 5782 to 5797, 5784 to 5799, 5785 to 5800, 5786 to 5801, 5788 to 5803, 5998 to 6013, 6272 to 6287, 6273 to 6288, 6695 to 6710, 6696 to 6711, 6763 to 6778, 7224 to 7239, 7232 to 7247, 8013 to 8028, 9844 to 9859, 9845 to 9860, counted from the 5'-end of the base sequence of SEQ ID NO: 1 in the Sequence Listing.9846-9861, 9848-9863, 9849-9864, 14649-14664, 14650-14665, 14903-14918, 14904-14919, 14905-14920 , 14951-14966, 14952-14967, 14953-14968, 14956-14971, 14957-14972, 14958-14973, 14959-14974, 14960 ~14975th, 14961-14976, 14962-14977, 14963-14978, 14964-14979, 14965-14980, 14966-14981, 14967-14982 14968-14983, 14980-14995, 16113-16128, 16114-16129, 16235-16250, 16236-16251, 16237-16252, 16238 ~16253rd, 16239-16254, 16256-16271, 16258-16273, 16281-16296, 16282-16297, 16283-16298, 16285-16300 16287-16302, 16289-16304, 16290-16305, 16291-16306, 16316-16331, 16321-16336, 16514-16529, 1651 It is a base sequence consisting of 16 consecutive bases that is 85% or more, preferably 90% or more, more preferably 95% or more, and particularly preferably 100% complementary to the base sequence of any of positions 7 to 16532, 16521 to 16536, 16578 to 16593, 16610 to 16625, 16612 to 16627, 16614 to 16629, 16616 to 16631, 16622 to 16637, or 16724 to 16739. Alternatively, it is a base sequence consisting of 17, 18, 19, or 20, preferably 17 or 18, consecutive bases that includes the complementary base sequence consisting of 16 consecutive bases. Furthermore, the full-length base sequence of the modified oligonucleotide is 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, orThe base sequence is 98% or more or 99% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 100% complementary.

[0057] Specifically, the base sequence of the modified oligonucleotide of the present invention is, from the 5' end of the base sequence of SEQ ID NO: 1 in the Sequence Listing, positions 1232 to 1247, 1233 to 1248, 1239 to 1254, 1691 to 1706, 1704 to 1719, 1735 to 1750, 1736 to 1751, 2195 to 2210, 2196 to 2211, 2289 to 2304, 2290 to 2305, 2293 to 2308, 34 31st to 3446th, 4127th to 4142nd, 4156th to 4171st, 4500th to 4515th, 5522nd to 5537th, 5727th to 5742nd, 5772nd to 5787th, 5773rd to 5788th, 5774th to 5789th, 5775-5790, 5776-5791, 5780-5795, 5781-5796, 5782-5797, 5784-5799, 5785-5800, 5786-5801 5788-5803, 5998-6013, 6272-6287, 6763-6778, 8013-8028, 9846-9861, 9848-9863, 9849-9864, 1 4963-14978th, 16235-16250, 16236-16251, 16237-16252, 16238-16253, 16239-16254, 16256-16271, 16 It is a base sequence consisting of 16 consecutive bases that is 85% or more, preferably 90% or more, more preferably 95% or more, and particularly preferably 100% complementary to the base sequence of any of positions 258 to 16273, 16281 to 16296, 16282 to 16297, 16283 to 16298, 16289 to 16304, 16290 to 16305, 16291 to 16306, or 16316 to 16331. Alternatively, it is a base sequence consisting of 17, 18, 19, or 20, preferably 17 or 18, consecutive bases that includes the complementary base sequence consisting of 16 consecutive bases.Furthermore, the full-length base sequence of the modified oligonucleotide is a base sequence that is 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 100% complementary to the isotopic portion of the base sequence of SEQ ID NO: 1.

[0058] Specifically, the base sequence of the modified oligonucleotide of the present invention is a base sequence consisting of 16 consecutive bases that is 85% or more, preferably 90% or more, more preferably 95% or more, and particularly preferably 100% complementary to the base sequence of either positions 16256 to 16271 or 16258 to 16273 from the 5' end of the base sequence of SEQ ID NO: 1 in the Sequence Listing. Alternatively, the modified oligonucleotide is a base sequence consisting of 17, 18, 19, or 20, preferably 17 or 18, consecutive bases that includes the complementary base sequence consisting of 16 consecutive bases. Furthermore, the full-length base sequence of the modified oligonucleotide is a base sequence that is 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 100% complementary to the equal-length portion of the base sequence in SEQ ID NO: 1.

[0059] The base sequence of the modified oligonucleotide of the present invention is specifically, from the 5' end of the base sequence of SEQ ID NO: 2 in the Sequence Listing, positions 15 to 30, 21 to 36, 22 to 37, 24 to 39, 26 to 41, 62 to 77, 69 to 84, 76 to 91, 100 to 115, 102 to 117, 112 to 127, 113 to 128, 114 to 129, 160 to 175, 161 to 176, 162 to 177, 163 to 178, 164 to 179, 165 to 170, 166 to 171, 167 to 172, 168 to 173, 169 to 180, 181 to 182, 182 to 183, 183 to 184, 184 to 185, 185 to 186, 186 to 187, 187 to 188, 189 to 200, 200 to 201, 201 to 202, 202 to 203, 203 to 204, 204 to 205, 205 to 206, 206 to 207, 207 to 208, 208 to 210, 209 to 211, 210 to 212, 211 to 213, 212 to 214, 213 to 215, 214 to 216, 215 to 217, 216 to 218, 217 to 2 2nd to 177th, 165th to 180th, 166th to 181st, 167th to 182nd, 168th to 183rd, 169th to 184th, 170th to 185th, 171st to 186th, 172nd to 187th, 173rd to 18th 8th, 174-189, 175-190, 176-191, 177-192, 189-204, 199-214, 200-215, 201-216, 202-217, 20 3rd to 218th, 204th to 219th, 206th to 221st, 207th to 222nd, 208th to 223rd, 209th to 224th, 210th to 225th, 227th to 242nd, 229th to 244th, 252nd to 26th 7th place, 253rd-268th place, 254th-269th place, 256th-271st place, 258th-273rd place, 260th-275th place, 261st-276th place, 262nd-277th place, 287th-302nd place, 292nd-307th place, 4th place It is a base sequence consisting of 16 consecutive bases that is 85% or more, preferably 90% or more, more preferably 95% or more, and particularly preferably 100% complementary to the base sequence of any of positions 85 to 500, 488 to 503, 492 to 507, 549 to 564, 581 to 596, 583 to 598, 585 to 600, 587 to 602, 593 to 608, or 695 to 710. Alternatively, it is a base sequence consisting of 17, 18, 19, or 20, preferably 17 or 18, consecutive bases that includes the complementary base sequence consisting of 16 consecutive bases. Furthermore, the full-length base sequence of the modified oligonucleotide is a base sequence that is 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 100% complementary to the base sequence of the isolength portion in SEQ ID NO: 2.

[0060] Specifically, the base sequence of the modified oligonucleotide of the present invention is a base sequence consisting of 16 consecutive bases that is 85% or more, preferably 90% or more, more preferably 95% or more, and particularly preferably 100% complementary to any of the base sequences at positions 172 to 187, 201 to 216, 203 to 218, 204 to 219, 206 to 221, 207 to 222, 208 to 223, 209 to 224, 210 to 225, 227 to 242, 229 to 244, 252 to 267, 253 to 268, 254 to 269, 260 to 275, 261 to 276, 262 to 277, or 287 to 302 from the 5' end of the base sequence of SEQ ID NO: 2 in the Sequence Listing. Alternatively, the modified oligonucleotide may be a base sequence consisting of 17, 18, 19, or 20, preferably 17 or 18, consecutive bases, including the base sequence consisting of 16 complementary consecutive bases. The full-length base sequence of the modified oligonucleotide is a base sequence that is 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 100% complementary to the base sequence of the same length portion in SEQ ID NO: 2.

[0061] In such modified oligonucleotides, the base sequence consisting of 17, 18, 19, or 20, preferably 17 or 18, consecutive bases is a base sequence in which 1 to 4, preferably 1 and / or 2, nucleosides are linked via internucleoside linkages at the 5'- or 3'-end of the complementary base sequence consisting of 16 consecutive bases. The nucleosides and / or internucleoside linkages may be modified nucleosides and / or modified internucleoside linkages.

[0062] Specific examples of the base sequence of the modified oligonucleotide of the present invention include: CCAGAATTATCCCGCT (complementary sequence of positions 16238 to 16253 of SEQ ID NO: 1) (SEQ ID NO: 275 in the Sequence Listing), TCCAGAATTATCCCGC (complementary sequence of positions 16239 to 16254 of SEQ ID NO: 1) (SEQ ID NO: 276 in the Sequence Listing), AGTTTTTACCAGTCAT (complementary sequence of positions 16256 to 16271 of SEQ ID NO: 1) (SEQ ID NO: 277 in the Sequence Listing), CCAGTTTTTTACCAGTC (complementary sequence of positions 16258 to 16273 of SEQ ID NO: 1) (SEQ ID NO: 278 in the Sequence Listing), GACTTTTGGGAGTAGT (complementary sequence of positions 16289 to 16304 of SEQ ID NO: 1) (SEQ ID NO: 279 in the Sequence Listing), Examples of the base sequence include the base sequence set forth in AGACTTTTTGGGAGTAG (complementary sequence of positions 16290 to 16305 of SEQ ID NO: 1) (SEQ ID NO: 229 in the Sequence Listing) or GAAGTCTTCCCACAAT (complementary sequence of positions 16316 to 16331 of SEQ ID NO: 1) (SEQ ID NO: 231 in the Sequence Listing).Also examples include base sequences consisting of 17, 18, 19 or 20, preferably 17 or 18, consecutive bases containing the base sequence. Furthermore, the full-length base sequence of the modified oligonucleotide is a base sequence that is 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 100% complementary to the base sequence of the same length portion in SEQ ID NO: 1.

[0063] Another example of a specific base sequence of the modified oligonucleotide of the present invention includes the base sequence set forth as follows: TTATCCCGCTCCCTGA (complementary sequence of positions 203 to 218 of SEQ ID NO: 2) (SEQ ID NO: 273 in the Sequence Listing) or ATTATCCCGCTCCCTG (complementary sequence of positions 204 to 219 of SEQ ID NO: 2) (SEQ ID NO: 280 in the Sequence Listing). Alternatively, the modified oligonucleotide may include a base sequence comprising the above base sequence and consisting of 17, 18, 19, or 20, preferably 17 or 18, consecutive bases. Furthermore, the full-length base sequence of the modified oligonucleotide is a base sequence that is 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 100% complementary to the base sequence of the isolength portion in SEQ ID NO: 2.

[0064] The base sequence of such a modified oligonucleotide is preferably TCCAGAATTAATCCCGC (complementary sequence of positions 16239 to 16254 of SEQ ID NO: 1) (SEQ ID NO: 276 in the Sequence Listing), AGTTTTTACCAGTCAT (complementary sequence of positions 16256 to 16271 of SEQ ID NO: 1) (SEQ ID NO: 277 in the Sequence Listing), CCAGTTTTTTACCAGTC (complementary sequence of positions 16258 to 16273 of SEQ ID NO: 1) (SEQ ID NO: 278 in the Sequence Listing), AGACTTTTGGGAGTAG (complementary sequence of positions 16290 to 16305 of SEQ ID NO: 1) (SEQ ID NO: 229 in the Sequence Listing), or GAAGTCTTCCCACAAT (complementary sequence of positions 16316 to 16331 of SEQ ID NO: 1) (SEQ ID NO: 231 in the Sequence Listing). or a base sequence comprising 17, 18, 19, or 20, preferably 17 or 18, consecutive bases containing the base sequence. The full-length base sequence of the modified oligonucleotide is a base sequence that is 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 100% complementary to the base sequence of the same length portion in SEQ ID NO: 1.

[0065] Further preferred examples of the base sequence of such modified oligonucleotides include the base sequences set forth below: AGTTTTTACCAGTCAT (complementary sequence of positions 16256 to 16271 of SEQ ID NO: 1) (SEQ ID NO: 277 in the Sequence Listing), CCAGTTTTTTACCAGTC (complementary sequence of positions 16258 to 16273 of SEQ ID NO: 1) (SEQ ID NO: 278 in the Sequence Listing), or AGACTTTTTGGGAGTAG (complementary sequence of positions 16290 to 16305 of SEQ ID NO: 1) (SEQ ID NO: 229 in the Sequence Listing). Alternatively, examples include base sequences comprising these base sequences and consisting of 17, 18, 19, or 20, preferably 17 or 18, consecutive bases. Furthermore, the full-length base sequence of the modified oligonucleotide is a base sequence that is 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 100% complementary to the isotopic portion of the base sequence of SEQ ID NO: 1.

[0066] Among the bases contained in the base sequences of these modified oligonucleotides, one or more cytosines may be 5-methylcytosine, a modified base described below. Specific examples of the base sequences of such modified oligonucleotides include the base sequences set forth in SEQ ID NOs: 213, 214, 218, 219, 228, 229, and 231, or base sequences comprising said base sequence and consisting of 17, 18, 19, or 20, preferably 17 or 18, consecutive bases. Furthermore, the full-length base sequence of the modified oligonucleotide is a base sequence that is 90% or more, preferably 95% or more, and more preferably 100% complementary to the base sequence of the isomorphic portion of SEQ ID NO: 1. Alternatively, the modified oligonucleotide may be the base sequence set forth in SEQ ID NO: 273 or 274, or a base sequence comprising said base sequence and consisting of 17, 18, 19, or 20, preferably 17 or 18, consecutive bases. The full-length base sequence of the s-modified oligonucleotide is 90% or more, preferably 95% or more, and more preferably 100% complementary to the base sequence of the isolength portion in SEQ ID NO: 2. Preferred base sequences include the base sequence shown in SEQ ID NO: 214, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 229, and SEQ ID NO: 231, or a base sequence consisting of 17, 18, 19, or 20, preferably 17 or 18, consecutive bases containing the base sequence, and the full-length base sequence of the modified oligonucleotide is 90% or more, preferably 95% or more, and more preferably 100% complementary to the base sequence of the isolength portion in SEQ ID NO: 1. More preferably, it is a base sequence shown in SEQ ID NO: 218, SEQ ID NO: 219, or SEQ ID NO: 229, or a base sequence consisting of 17, 18, 19, or 20, preferably 17 or 18, consecutive bases including said base sequence, and the full-length base sequence of the modified oligonucleotide is 90% or more, preferably 95% or more, and more preferably 100% complementary to the equal-length portion of the base sequence in SEQ ID NO: 1.

[0067] The modified oligonucleotide of the present invention may be a double-stranded modified oligonucleotide, but a single-stranded modified oligonucleotide is preferably used.

[0068] The modified oligonucleotide of the present invention may be one in which at least one nucleoside constituting the oligonucleotide contains a modified base, at least one nucleoside constituting the oligonucleotide contains a modified sugar, or at least one internucleoside linkage constituting the oligonucleotide contains a modified internucleoside linkage.

[0069] The modified oligonucleotides of the present invention preferably contain at least one nucleoside containing a modified sugar. Modified sugars include, for example, substituted sugars or bicyclic sugars. Modified oligonucleotides containing such modified sugars have advantageous properties, such as enhanced nuclease stability and increased binding affinity for target nucleic acids.

[0070] Specific examples of the substituted sugar include 5'-vinyl, 5'-methyl, 4'-S, 2'-F, 2'-OCH ３ (2'-OMe), 2'-OCH ２ CH ３ , 2'-OCH ２ CH ２ F or 2'-O(CH ２ ) ２ OCH ３ The sugar moiety of a nucleoside modified with (2'-MOE) is an example.

[0071] Bicyclic sugars specifically include, for example, sugar moieties of nucleosides that include a bridge between the 4' and 2' ribosyl ring atoms, specifically including, for example, sugar moieties of nucleosides in which the bridge includes one of the following formulas: 4'-(CH ２ )-O-2'(LNA);4'-(CH ２ )-S-2';4'-(CH ２ ) ２ -O-2'(ENA). Other examples of bicyclic sugars include the sugar moiety of the bridged artificial nucleic acid ALNA disclosed in WO 2020 / 100826, specifically the sugar moiety of ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Oxz], or ALNA[Trz], preferably the sugar moiety of ALNA[Ms].

[0072] In certain embodiments (ALNA[mU]), the nucleoside containing a bicyclic sugar (the sugar portion of ALNA[mU]) has the following formula (I): wherein B is a base; １ , R ２ , R ３ and R ４ are each independently a hydrogen atom; ５ and R ６ are each independently a hydrogen atom; m is 1; X is a group represented by the following formula (II-1): The symbol in formula (II-1): indicates the point of attachment to the 2'-amino group; R ７ and R ８ One of the nucleosides is a hydrogen atom, and the other is an unsubstituted methyl group.] (See, for example, WO 2020 / 100826).

[0073] In certain embodiments (ALNA[ipU]), the nucleoside containing a bicyclic sugar (the sugar portion of ALNA[ipU]) is a nucleoside having formula (I) as defined above in ALNA[mU], wherein: B is a base; R １ , R ２ , R ３ and R ４ are each independently a hydrogen atom; ５ and R ６ are each independently a hydrogen atom; m is 1; X is a group represented by the following formula (II-1): R ７ and R ８ is a nucleoside in which one of the nucleosides is a hydrogen atom and the other is an unsubstituted isopropyl group (see, for example, WO 2020 / 100826).

[0074] In certain embodiments (ALNA[Trz]), the bicyclic (sugar portion of ALNA[Trz]) containing nucleosides are nucleosides having formula (I) as defined above in ALNA[mU], wherein: B is a base; R １ , R ２ , R ３ and R４ are each independently a hydrogen atom; ５ and R ６ are each independently a hydrogen atom; m is 1; X is a group represented by the following formula (II-2): A is a 1,5-dimethyl-1,2,4-triazol-3-yl group (see, for example, WO 2020 / 100826).

[0075] In certain embodiments (ALNA[Oxz]), the nucleoside containing a bicyclic sugar (the sugar portion of ALNA[Oxz]) is a nucleoside having formula (I) as defined above in ALNA[mU], wherein: B is a base; R １ , R ２ , R ３ and R ４ are each independently a hydrogen atom; ５ and R ６ are each independently a hydrogen atom; m is 1; X is a group represented by the following formula (II-2): A is a nucleoside represented by the formula:

[0076] In certain embodiments (ALNA[Ms]), the nucleoside containing a bicyclic sugar (ALNA[Ms]) is a nucleoside having formula (I) as defined above in ALNA[mU], wherein: B is a base; R １ , R ２ , R ３ and R ４ are each independently a hydrogen atom; ５ and R ６ are each independently a hydrogen atom; m is 1; X is a group represented by the following formula (II-3): and M is a sulfonyl group substituted with an unsubstituted methyl group (see, for example, WO 2020 / 100826).

[0077] The modified oligonucleotide of the present invention is preferably one in which at least one nucleoside constituting the oligonucleotide contains a modified base. Modified oligonucleotides containing such modified bases have advantageous properties such as enhanced nuclease stability and increased binding affinity to target nucleic acids. Specific examples of modified bases include 5-methylcytosine. 5-methylcytosine refers to cytosine modified with a methyl group attached to the 5-position.

[0078] The modified oligonucleotide of the present invention preferably contains at least one internucleoside bond containing a modified internucleoside bond. Modified oligonucleotides having such modified internucleoside bond have advantageous properties such as enhanced cellular uptake, enhanced affinity for target nucleic acids, and increased stability in the presence of nucleases.

[0079] Specific examples of modified internucleoside linkages include phosphorothioate internucleoside linkages, which refer to linkages between nucleosides in which the phosphodiester bond is modified by replacing one of the non-bridging oxygen atoms with a sulfur atom.

[0080] The modified oligonucleotides of the present invention can have a gapmer structure to achieve increased resistance to nuclease degradation, increased cellular uptake, increased binding affinity for target nucleic acids, and / or increased MRLN expression-suppressing activity. A gapmer structure refers to a structure in which an internal region having multiple nucleosides that supports RNase H cleavage is located between external regions having one or more nucleosides. The internal region can be referred to as a "gap segment." The external region can be referred to as a "wing segment." The wing segment located 5' from the gap segment can be referred to as a "5' wing segment." The wing segment located 3' from the gap segment can be referred to as a "3' wing segment." In a gapmer, the sugar moieties of all nucleosides in the wing segment are modified sugars. The sugar moieties of the nucleosides adjacent to the wing segment (the 5'-most and 3'-most nucleosides of the gap segment) are natural DNA sugar moieties. The sugar moieties of the nucleosides of the gap segment that are not adjacent to the wing segments can consist solely of native DNA sugar moieties, or can contain one or more modified sugars.

[0081] An example of a modified oligonucleotide having such a gapmer structure is a modified oligonucleotide comprising 1) a gap segment, 2) a 5' wing segment, and 3) a 3' wing segment, wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment, and the sugar moieties of the nucleosides constituting the 5' wing segment and the 3' wing segment are all modified sugars. Furthermore, the sugar moiety of ALNA[Ms] is preferably used as the modified sugar.

[0082] The modified oligonucleotides of the present invention can exist in the form of their pharmaceutically acceptable salts. The pharmaceutically acceptable salts may be any salts that retain the desired biological activity of the modified oligonucleotides of the present invention and do not have any undesirable toxicological effects on them, but salts formed with inorganic ions such as metal ions are preferred. Specific examples include sodium salts and potassium salts, with sodium salts being preferred. The modified oligonucleotides of the present invention can also exist in the form of ions.

[0083] The modified oligonucleotide of the present invention or a pharmaceutically acceptable salt thereof can be synthesized by a conventional method, for example, by the phosphoramidite method using amidites that are commercially available or can be synthesized by known methods.

[0084] The modified oligonucleotide or a pharmaceutically acceptable salt thereof of the present invention may be conjugated to a conjugate group at one or more positions. Specific examples of conjugate groups include proteins having affinity for biological molecules such as fatty acids, cholesterol, carbohydrates, phospholipids, and antibodies, as well as biotin, phenazine, vitamins, peptides, folate, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, and dyes. The conjugated modified oligonucleotide can be produced by known methods, and one that enhances its activity, tissue distribution, cellular distribution, or cellular uptake can be selected. The conjugate group may be directly attached to the modified oligonucleotide or via a linker.

[0085] (Method for Evaluating the Compound of the Present Invention) The method for evaluating the compound of the present invention may be any method that can verify the suppression of the expression level of MRLN in cells by the compound of the present invention. Specifically, for example, the following in vitro and in vivo MRLN expression measurement methods can be used.

[0086] In the in vitro MRLN expression measurement method for evaluating the suppression of MRLN expression in cells by the compound of the present invention, any cells expressing MRLN (hereinafter, sometimes referred to as "MRLN-expressing cells") can be used, and examples thereof include RD cells (human rhabdomyosarcoma cells).

[0087] The method for contacting the compound of the present invention with MRLN-expressing cells is not particularly limited, and includes methods generally used for introducing nucleic acids into cells. Specific examples include lipofection, electroporation, and Gymnosis. In the lipofection or Gymnosis method, the compound of the present invention can be used at a final concentration of, for example, 3, 10, 30, or 100 nM. The treatment time can be, for example, 48 hours.

[0088] The intracellular mRNA level of MRLN can be assayed by various methods known in the art, including, for example, Northern blot analysis, competitive polymerase chain reaction (PCR), or quantitative real-time PCR.

[0089] Protein levels in MRLN cells can be assayed by a variety of methods known in the art, including, for example, immunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA), quantitative protein assays, protein activity assays (e.g., caspase activity assays), immunohistochemistry, immunocytochemistry, or fluorescence-activated cell sorting (FACS).

[0090] An in vivo MRLN expression measurement method for evaluating the suppression of MRLN expression in cells by the compound of the present invention includes, for example, a method in which the compound of the present invention is administered to an animal expressing MRLN, and the above-mentioned MRLN expression level in the cells is analyzed.

[0091] (2) Pharmaceutical composition containing a modified oligonucleotide or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable salt thereof The modified oligonucleotide of the present invention or a pharmaceutically acceptable salt thereof can be used as a pharmaceutical composition. Furthermore, the modified oligonucleotide of the present invention conjugated with a conjugate group or a pharmaceutically acceptable salt thereof can be used as a pharmaceutical composition. The pharmaceutical composition may further contain a pharmaceutically acceptable carrier. That is, the pharmaceutical composition may contain the modified oligonucleotide of the present invention or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0092] The pharmaceutically acceptable carrier is appropriately selected depending on the dosage form of the pharmaceutical composition, and examples thereof include excipients, lubricants, binders, and disintegrants in solid preparations, and solvents, solubilizers, suspending agents, isotonicity agents, buffers, and soothing agents in liquid preparations. Furthermore, conventional additives such as preservatives, antioxidants, colorants, sweeteners, adsorbents, and wetting agents can also be used as needed in appropriate amounts.

[0093] Dosage forms for parenteral administration of pharmaceutical compositions include injection preparations (e.g., drip infusion preparations, intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections, intracerebral administration preparations, intraspinal administration preparations), topical preparations (e.g., ointments, poultices, lotions), suppositories, inhalants, eye preparations, eye ointments, nasal drops, ear drops, liposomes, etc. Methods for preparing dosage forms for parenteral administration are well known to those skilled in the art.

[0094] For example, an injectable formulation can be prepared by dissolving the modified oligonucleotide of the present invention or a pharmaceutically acceptable salt thereof in a sterile aqueous solution. Examples of sterile aqueous solutions include sterile saline, sterile water, and sterile phosphate-buffered saline. Furthermore, solubilizers, buffers, pH adjusters, isotonicity agents, soothing agents, preservatives, stabilizers, and the like can be added as needed. Furthermore, the formulation can also be prepared as a lyophilized formulation for preparation immediately before use.

[0095] Dosage forms of pharmaceutical compositions for oral administration include solid or liquid dosage forms, specifically tablets, coated tablets, pills, fine granules, granules, powders, capsules, syrups, emulsions, suspensions, injections, troches, etc. Methods for preparing dosage forms for oral administration are well known to those skilled in the art.

[0096] The modified oligonucleotides of the present invention or pharmaceutically acceptable salts thereof, or pharmaceutical compositions containing them, can be used for the treatment or prevention of muscle diseases. It has been reported that suppressing the expression level of MRLN in muscle diseases increases calcium uptake in the sarcoplasmic reticulum, thereby reducing the amount of calcium in the cytoplasm and increasing muscle contractility. Therefore, the modified oligonucleotides of the present invention or pharmaceutically acceptable salts thereof, or pharmaceutical compositions containing them, are effective in treating or preventing muscle diseases accompanied by muscle damage, muscle cell death, and / or decreased muscle contractility due to abnormal calcium influx into the cytoplasm.

[0097] Specific examples of such muscle diseases include muscular dystrophy, inclusion body myositis, amyotrophic lateral sclerosis, disuse muscular atrophy, and sarcopenia, preferably muscular dystrophy. Specific examples of muscular dystrophy include Duchenne muscular dystrophy, Becker muscular dystrophy, and sarcoglycanopathy, preferably Duchenne muscular dystrophy or sarcoglycanopathy, more preferably Duchenne muscular dystrophy. Sarcoglycanopathy includes LGMD2C, LGMD2D, LGMD2E, and LGMD2F.

[0098] In particular, two mechanisms are known for muscular dystrophies: 1) deficiency of the dystrophin-glycoprotein complex increases membrane permeability, thereby increasing the amount of calcium in the cytoplasm and causing muscle damage and muscle cell death; and 2) deficiency of the dystrophin-glycoprotein complex reduces nNOS (neuronal nitric oxide synthase), increasing iNOS (inducible nitric oxide synthase) in the cytoplasm, nitrosylation of ryanodine receptors, reducing calcium in the sarcoplasmic reticulum, and reducing muscle contractility. The modified oligonucleotides of the present invention or pharmaceutically acceptable salts thereof, or pharmaceutical compositions containing them, can reduce the amount of calcium in the cytoplasm and increase calcium uptake into the sarcoplasmic reticulum, thereby suppressing muscle damage and muscle cell death in muscular dystrophies and / or maintaining or restoring muscle contractility.

[0099] Accordingly, the present invention provides: a pharmaceutical composition for treating or preventing a muscular disease, comprising the modified oligonucleotide of the present invention or a pharmaceutically acceptable salt thereof; a method for treating or preventing a muscular disease, comprising the step of administering to a subject an effective amount of a modified oligonucleotide of the present invention or a pharmaceutically acceptable salt thereof (or a pharmaceutical composition comprising the modified oligonucleotide or a pharmaceutically acceptable salt thereof); use of the modified oligonucleotide of the present invention or a pharmaceutically acceptable salt thereof (or a pharmaceutical composition comprising the modified oligonucleotide or a pharmaceutically acceptable salt thereof) in the manufacture of a pharmaceutical composition for treating or preventing a muscular disease; a modified oligonucleotide of the present invention or a pharmaceutically acceptable salt thereof (or a pharmaceutical composition comprising the modified oligonucleotide or a pharmaceutically acceptable salt thereof) for use in the manufacture of a pharmaceutical composition for treating or preventing a muscular disease; and use of the modified oligonucleotide of the present invention or a pharmaceutically acceptable salt thereof (or a pharmaceutical composition comprising the modified oligonucleotide or a pharmaceutically acceptable salt thereof) for the treatment or prevention of a muscular disease.

[0100] The "subject" to which the compound is administered includes humans and non-human mammals (e.g., one or more of mice, guinea pigs, hamsters, rats, mice, rabbits, pigs, sheep, goats, cows, horses, cats, dogs, marmosets, monkeys, or chimpanzees).

[0101] The dosage form of the modified oligonucleotide of the present invention, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing the same, can be systemic administration, such as oral administration, intravenous administration, or intra-arterial administration. The dosage of the modified oligonucleotide of the present invention, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing the same can be appropriately varied depending on the purpose of use, severity of the disease, age, weight, sex, etc. of the patient, and can be, for example, 0.1 ng to 100 mg / kg / day of the modified oligonucleotide.

[0102] The modified oligonucleotides of the present invention or pharmaceutically acceptable salts thereof, or pharmaceutical compositions containing them, can be used alone or in combination with one or more other drugs.

[0103] NON-LIMITING DISCLOSURE AND INCORPORATION BY REFERENCE While certain compounds, compositions, and methods described herein are specifically described according to certain embodiments, the following examples serve only to illustrate the compounds described herein and are not intended to be limiting thereof. Each of the references described in this application is incorporated herein by reference in its entirety.

[0104] Example 1 Preparation of Modified Oligonucleotides Modified oligonucleotides were synthesized and purified by Nippon Gene Co., Ltd. / Nippon Gene Material Co., Ltd. using ALNA[Ms] amidite (synthesized by the method described in International Publication WO 2020 / 100826) according to a general solid-phase synthesis method for oligonucleotides. The compounds were identified using a high-performance liquid chromatograph mass spectrometer.

[0105] The synthesized modified oligonucleotide compounds are shown in Tables 1-1 to 1-6 (16-residue modified oligonucleotides). Each nucleoside is represented by a single letter, and the internucleoside bond is a phosphorothioate bond. Each nucleoside is represented as follows: A = adenosine containing the sugar moiety of ALNA[Ms] as a modified sugar; T = thymidine containing the sugar moiety of ALNA[Ms] as a modified sugar; G = guanosine containing the sugar moiety of ALNA[Ms] as a modified sugar; C = 5-methylcytidine containing the sugar moiety of ALNA[Ms] as a modified sugar and 5-methylcytosine as a modified base; a = 2'-deoxyribose adenosine; t = 2'-deoxyribose thymidine; g = 2'-deoxyribose guanosine; c = 2'-deoxyribose cytidine. The premature target start position indicates the MRLN pre-mRNA 5' target site of the modified oligonucleotide (the position of SEQ ID NO: 1 in the Sequence Listing corresponding to the 3' end of the modified oligonucleotide), and the premature target end position indicates the MRLN pre-mRNA 3' target site of the modified oligonucleotide (the position of SEQ ID NO: 1 in the Sequence Listing corresponding to the 5' end of the modified oligonucleotide). The mature target start position indicates the MRLN mRNA 5' target site of the modified oligonucleotide (the position of SEQ ID NO: 2 in the Sequence Listing corresponding to the 3' end of the modified oligonucleotide), and the mature target end position indicates the MRLN mRNA 3' target site of the modified oligonucleotide (the position of SEQ ID NO: 2 in the Sequence Listing corresponding to the 5' end of the modified oligonucleotide).

[0106]

[0107]

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[0110]

[0111]

[0112] Example 2 In vitro MRLN expression ratio test (lipofection method) The modified oligonucleotide synthesized in Example 1 was mixed with a lipofection reagent and added to a 384-well plate at 5 μL / well. RD cells suspended in a 10% serum-containing medium were then added to the mixture at 5 × 10 ３ Cells were seeded at 45 μL / well and CO ２ The cells were cultured in an incubator. After 48 hours, the modified oligonucleotide-containing medium was removed from the cells and washed with phosphate-buffered saline (PBS). Subsequently, using the SuperPrep Cell Lysis & RT Kit for qPCR (Toyobo), a cell lysate containing RNA was prepared, and an RT reaction was performed from the lysate to synthesize template cDNA. Real-time PCR was performed using this cDNA to quantify the expression level of MRLN mRNA. The MRLN expression ratio was calculated as a percentage by comparing the MRLN mRNA level in cells to which the modified oligonucleotide was added at a final concentration of 100 nmol / L with the MRLN mRNA level in cells to which the modified oligonucleotide was not added, and is shown in Tables 1-1 to 1-6.

[0113] Example 3 In vitro MRLN expression ratio test (Gymnosis method) 5 × 10 RD cells suspended in a 2% serum-containing medium were ３ Cells were seeded in a 384-well plate at 45 μL / well and incubated with CO ２ After 72 hours, the medium was replaced with the same medium, and the modified oligonucleotide synthesized in Example 1 was added at 5 μL / well. ２The cells were cultured in an incubator. After 72 hours, the modified oligonucleotide-containing medium was removed from the cells and washed with phosphate-buffered saline (PBS). Subsequently, using the SuperPrep Cell Lysis & RT Kit for qPCR (Toyobo), a cell lysate containing RNA was prepared, and an RT reaction was performed from the lysate to synthesize template cDNA. Real-time PCR was performed using this cDNA to quantify the expression level of MRLN mRNA. The MRLN expression ratio was calculated as a percentage by comparing the MRLN mRNA level in cells to which the modified oligonucleotide was added at a final concentration of 1000 nmol / L with the MRLN mRNA level in cells to which the modified oligonucleotide was not added, and is shown in Tables 1-1 to 1-6.

Claims

1. A modified oligonucleotide consisting of 12 to 30 linked nucleosides or a pharmaceutically acceptable salt thereof, which has the activity of inhibiting Myoregulin expression, and which is selected from the base sequence of SEQ ID NO: 1 in the Sequence Listing, and which is located at positions 15 to 41, 62 to 108, 448 to 471, 649 to 666, 848 to 863, 1104 to 1119, 1232 to 1254, 1691 to 1719, 1735 to 1751, 2195 to 2211, 2285 to 2288, and 2289 from the 5' end of the base sequence. 9-2310th, 2479-2512, 2616-2631, 2692-2716, 2751-2766, 2893-2909, 2979-2995, 3431-3447, 3564-3585, 3601-3617 3721-3736, 4074-4089, 4127-4142, 4156-4171, 4277-4292, 4305-4322, 4500-4515, 4613-4628, 5039-5055, 5063 ~5079th, 5103-5119, 5522-5540, 5727-5742, 5772-5803, 5998-6013, 6272-6288, 6695-6711, 6763-6778, 7224-7247 , 8013-8028th, 9844-9864, 14649-14665, 14903-14920, 14951-14995, 16113-16129, 16235-16254, 16256-16273, 1628 A modified oligonucleotide or a pharmaceutically acceptable salt thereof, comprising a base sequence that is 100% complementary to any 8 or more consecutive bases in any of the base sequences at positions 1 to 16306, 16316 to 16336, 16514 to 16536, 16578 to 16593, 16610 to 16637, or 16724 to 16739, and the full-length base sequence of the modified oligonucleotide is 85% or more complementary to an equal-length portion of the base sequence of SEQ ID NO: 1 in the Sequence Listing.

2. The modified oligonucleotide is selected from the group consisting of positions 1232 to 1254, 1691 to 1719, 1735 to 1751, 2195 to 2211, 2289 to 2308, 3431 to 3446, 4127 to 4142, 4156 to 4171, 4500 to 4515, 5522 to 5537, 5727 to 5742, 5772 to 5803, 5998 to 6013, 6272 to 6287, 6763 to 6778, 8013 to 8028, 9846 to 9999, and 10 ...

2. The modified oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 1, comprising a base sequence that is 100% complementary to any 8 or more consecutive bases in any of the base sequences of positions 16235 to 16254, 16256 to 16273, 16281 to 16306, or 16316 to 16331, and the full-length base sequence of the modified oligonucleotide is 85% or more complementary to an equal-length portion of the base sequence of SEQ ID NO: 1 in the Sequence Listing.

3. A modified oligonucleotide or a pharmaceutically acceptable salt thereof, which is composed of 12 to 30 linked nucleosides and has the activity of inhibiting Myoregulin expression, wherein the modified oligonucleotide comprises a base sequence that is 100% complementary to any 8 or more consecutive bases in any of the base sequences at positions 15 to 41, 62 to 91, 100 to 129, 160 to 225, 227 to 244, 252 to 277, 287 to 307, 485 to 507, 549 to 564, 581 to 608, or 695 to 710 from the 5' end of the base sequence of SEQ ID NO: 2 in the Sequence Listing, and the full-length base sequence of the modified oligonucleotide is 85% or more complementary to the equal-length portion of the base sequence of SEQ ID NO: 2 in the Sequence Listing.

4. The modified oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 3, wherein the modified oligonucleotide comprises a base sequence that is 100% complementary to any 8 or more consecutive bases in any of the base sequences at positions 172 to 187, 201 to 225, 227 to 244, 252 to 277, or 287 to 302 from the 5' end of the base sequence of SEQ ID NO: 2 in the Sequence Listing, and the full-length base sequence of the modified oligonucleotide is 85% or more complementary to the equal-length portion of the base sequence of SEQ ID NO: 2 in the Sequence Listing.

5. A modified oligonucleotide or a pharmaceutically acceptable salt thereof having the activity of suppressing Myoregulin expression, wherein the base sequence of the modified oligonucleotide is any base sequence selected from the group consisting of SEQ ID NOs: 275 to 279, 229, and 231 in the sequence listing, or a base sequence consisting of 17 or 18 consecutive bases including said base sequence, and the full-length base sequence of the modified oligonucleotide is 100% complementary to the isolength portion of the base sequence of SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof.

6. A modified oligonucleotide or a pharmaceutically acceptable salt thereof having the activity of suppressing Myoregulin expression, wherein the base sequence of the modified oligonucleotide is either the base sequence of SEQ ID NO: 273 or 280 in the Sequence Listing, or a base sequence consisting of 17 or 18 consecutive bases including said base sequence, and the full-length base sequence of the modified oligonucleotide is a base sequence that is 100% complementary to the equal-length portion of the base sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof.

7. The modified oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6, wherein the modified oligonucleotide is single-stranded.

8. The modified oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6, wherein at least one nucleoside constituting the modified oligonucleotide contains a modified sugar.

9. The modified oligonucleotide or a pharmaceutically acceptable salt thereof of claim 8, wherein the modified sugar is a bicyclic sugar.

10. The modified oligonucleotide or a pharmaceutically acceptable salt thereof of claim 9, wherein the bicyclic sugar is selected from the group consisting of LNA, ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Oxz], and ALNA[Trz] sugar moieties.

11. The modified oligonucleotide or a pharmaceutically acceptable salt thereof of claim 10, wherein the bicyclic sugar is the sugar moiety of ALNA[Ms].

12. The modified oligonucleotide or a pharmaceutically acceptable salt thereof of claim 8, wherein the modified sugar is a substituted sugar.

13. The modified oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6, wherein at least one nucleoside constituting the modified oligonucleotide contains a modified base.

14. The modified oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 13, wherein the modified base is 5-methylcytosine.

15. The modified oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6, wherein at least one internucleoside bond constituting the modified oligonucleotide is a modified internucleoside bond.

16. The modified oligonucleotide or a pharmaceutically acceptable salt thereof of claim 15, wherein the modified internucleoside linkage is a phosphorothioate internucleoside linkage.

17. The modified oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6, wherein the modified oligonucleotide comprises: 1) a gap segment, 2) a 5' wing segment, and 3) a 3' wing segment, wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment, and wherein all of the sugar moieties of the nucleosides comprising the 5' wing segment and the 3' wing segment are modified sugars.

18. A pharmaceutical composition comprising the modified oligonucleotide according to any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof.

19. The pharmaceutical composition according to claim 18 for the treatment or prevention of muscle diseases.

20. The pharmaceutical composition of claim 19, wherein the muscle disease is selected from the group consisting of muscular dystrophy, inclusion body myositis, amyotrophic lateral sclerosis, disuse muscle atrophy, and sarcopenia.

21. The pharmaceutical composition of claim 20, wherein the muscle disease is muscular dystrophy.

22. The pharmaceutical composition of claim 21, wherein the muscular dystrophy is selected from the group consisting of Duchenne muscular dystrophy, Becker muscular dystrophy, and sarcoglycanopathy.

23. The pharmaceutical composition of claim 19, wherein the muscle disease is Duchenne muscular dystrophy.

24. The pharmaceutical composition of claim 19, wherein the muscle disease is Becker muscular dystrophy.

25. The pharmaceutical composition of claim 19, wherein the muscle disease is a sarcoglycanopathy.

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