Nucleic acid medicine expressing splicing variant of myostatin

Abstract

Provided is a method for inhibiting the function of myostatin. Provided are: an antisense oligonucleotide having a base length of 15-30 and a base sequence complementary to the target sequence of exon 3 of myostatin gene, the antisense oligonucleotide being capable of expressing a splicing variant of myostatin; and a salt or solvate thereof. Provided are: a medicine, food, and feed comprising the antisense oligonucleotide and a salt or solvate thereof; and a medicine for promoting the proliferation and / or hypertrophy of muscle cells, a medicine for increasing muscle mass and / or inhibiting muscle weakness, a medicine for switching the splicing of myostatin from myostatin to a splicing variant of myostatin, a medicine for reducing a myostatin signal, and an anticancer agent, said medicine and agent each comprising the antisense oligonucleotide and a salt or solvate thereof.

Description

Nucleic acid medicine that induces expression of splicing variants of myostatin

[0001] The present invention relates to a nucleic acid drug that expresses a splicing variant of myostatin.

[0002] Myostatin is a TGF-beta type cell growth factor and is a protein encoded by the myostatin gene. Myostatin inhibits muscle cell proliferation and hypertrophy. Therefore, inhibiting myostatin function promotes muscle cell proliferation and hypertrophy, making it a promising therapeutic target for muscle atrophy (Non-Patent Document 1). Furthermore, reducing myostatin expression inhibits cancer cell proliferation (Non-Patent Document 2). This suggests that reducing myostatin levels and inhibiting myostatin signaling are effective in cancer treatment. Furthermore, because myostatin expression is elevated in patients with type 2 diabetes, it is believed that there is some relationship between myostatin and diabetes (Non-Patent Document 3). These findings suggest that inhibiting myostatin signaling may be effective in suppressing or slowing the progression of diabetes.

[0003] To date, antibodies against myostatin and antisense oligonucleotides (ASOs) that regulate the splicing of the myostatin gene to produce loss-of-function mRNA have been developed (Non-Patent Documents 4 and 5). However, a clinically useful myostatin inhibitor has yet to be established.

[0004] Bogdanovich et al., Nature, 2002, 420;418-421Han et al., Redox Biol. 2018, 19;412-4128Palsgaard et al. 2009, 4;e6575St Andre et al. Skeletal Muscle 2017,7;25Kang et al. Mol Ther 2011,19;159-64

[0005] The present invention aims to provide a method for inhibiting the function of myostatin.

[0006] The myostatin (MSTN) gene has a simple structure with three exons. It was previously thought that only one type of mRNA was produced from the MSTN gene. However, the inventors previously demonstrated that a cryptic splice site in the 3' untranslated region of the gene is activated, resulting in the production of a novel splice variant mRNA (WO2020 / 179571). Furthermore, they demonstrated that the isoform encoded by this mRNA has myostatin inhibitory activity. In this study, we sought nucleic acid drugs that alter the splicing pattern of the human myostatin gene and promote the production of novel mRNA. We screened ASOs with ENA (2'-O,4'-C-Ethylene-bridged Nucleic Acids)-modified nucleic acids as monomers, with sequences complementary to the exon 3 sequence, and identified the ASO that most effectively promoted the production of the splice variant. Furthermore, we demonstrated that the introduction of this ASO resulted in the production of myostatin isoforms in cells. Inhibition of myostatin function is a therapeutic target for muscle atrophy, and numerous myostatin inhibitors have been developed. However, this ASO is the first in the world to produce splicing variants.

[0007] The gist of the present invention is as follows: (1) An antisense oligonucleotide having a base length of 15 to 30 and having a base sequence complementary to a target sequence in exon 3 of the myostatin gene, said antisense oligonucleotide, a salt, or a solvate thereof, capable of expressing a splicing variant of myostatin. (2) The antisense oligonucleotide, a salt, or a solvate thereof according to (1), wherein the base sequence of exon 3 of the myostatin gene is the base sequence of SEQ ID NO: 1, and the target sequence of exon 3 of the myostatin gene is a sequence of a region of base numbers 10 to 33 of the base sequence of SEQ ID NO: 1, and said base sequence is the base sequence of SEQ ID NO: 2. (3) The antisense oligonucleotide, a salt, or a solvate thereof according to (1) or (2), wherein the base sequence of the antisense oligonucleotide comprises a sequence consisting of at least 15 consecutive bases in any of the base sequences of SEQ ID NOs: 24, 30, 31, 27, 32, 33, or 28 (provided that t in the sequence may be u, and u may be t). (4) The antisense oligonucleotide, salt, or solvate thereof according to any one of (1) to (3), wherein the antisense oligonucleotide has a base length of 18. (5) The antisense oligonucleotide, salt, or solvate thereof according to (4), wherein the base sequence of the antisense oligonucleotide is any one of SEQ ID NOs: 24, 30, 31, 27, 32, 33, or 28 (provided that t in the sequence may be u, and u may be t). (6) The antisense oligonucleotide, salt, or solvate thereof according to any one of (1) to (5), wherein at least one nucleotide is modified. (7) The antisense oligonucleotide, salt, or solvate thereof according to (6), wherein the sugar constituting the modified nucleotide is D-ribofuranose, and the hydroxyl group at the 2'-position of the D-ribofuranose is modified. (8) The antisense oligonucleotide, salt, or solvate thereof according to (7), wherein the D-ribofuranose is 2'-O-alkylated and / or 2'-O,4'-C-alkylenated. (9) A pharmaceutical comprising the antisense oligonucleotide according to any one of (1) to (8), or a pharmaceutically acceptable salt or solvate thereof. (10) The pharmaceutical according to (9), for preventing and / or treating a pathology and / or disease involving myostatin.(11) The pharmaceutical composition according to (10), wherein the myostatin-associated pathology and / or disease is muscular atrophy. (12) The pharmaceutical composition 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 pharmaceutical composition according to (10), wherein the myostatin-associated pathology and / or disease is a pathology and / or disease for which muscle mass restoration brings about a therapeutic effect. (14) The pharmaceutical composition according to (13), wherein the myostatin-associated pathology and / or disease is at least one selected from the group consisting of cancer cachexia, diabetes, cardiovascular disease, kidney disease, and bone disease. (15) The pharmaceutical composition 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 product comprising the antisense oligonucleotide according to any one of (1) to (8) or a salt or solvate thereof that is acceptable for food use. (17) A feed product comprising the antisense oligonucleotide according to any one of (1) to (8) or a salt or solvate thereof that is acceptable for food use. (18) A drug for promoting muscle cell proliferation and / or hypertrophy, comprising the antisense oligonucleotide according to any one of (1) to (8) or a salt or solvate thereof. (19) A drug for increasing muscle mass and / or suppressing muscle wasting, comprising the antisense oligonucleotide according to any one of (1) to (8) or a salt or solvate thereof. (20) A drug for switching myostatin splicing from myostatin to a myostatin splicing variant, comprising the antisense oligonucleotide according to any one of (1) to (8) or a salt or solvate thereof. (21) A drug for reducing myostatin signaling, comprising the antisense oligonucleotide according to any one of (1) to (8) or a salt or solvate thereof. (22) An anticancer agent comprising the antisense oligonucleotide according to any one of (1) to (8), or a salt or solvate thereof. (23) A method for preventing and / or treating a condition and / or disease involving myostatin, comprising administering to a subject an effective amount of the antisense oligonucleotide according to any one of (1) to (8), or a salt or solvate thereof.(24) A method for promoting muscle cell proliferation and / or hypertrophy, comprising administering to a subject an effective amount of the antisense oligonucleotide, salt, or solvate thereof according to any one of (1) to (8). (25) A method for increasing muscle mass and / or suppressing muscle wasting, comprising administering to a subject an effective amount of the antisense oligonucleotide, salt, or solvate thereof according to any one of (1) to (8). (26) A method for switching myostatin splicing from myostatin to a myostatin splicing variant, comprising administering to a subject an effective amount of the antisense oligonucleotide, salt, or solvate thereof according to any one of (1) to (8). (27) A method for reducing myostatin signaling, comprising administering to a subject an effective amount of the antisense oligonucleotide, salt, or solvate thereof according to any one of (1) to (8). (28) A method for preventing and / or treating cancer, comprising administering to a subject an effective amount of the antisense oligonucleotide, salt, or solvate thereof according to any one of (1) to (8). (29) The antisense oligonucleotide, salt or solvate thereof according to any one of (1) to (8) for use in a method for preventing and / or treating a pathology and / or disease involving myostatin. (30) The antisense oligonucleotide, salt or solvate thereof according to any one of (1) to (8) for use in a method for promoting proliferation and / or hypertrophy of muscle cells. (31) The antisense oligonucleotide, salt or solvate thereof according to any one of (1) to (8) for use in a method for increasing muscle mass and / or suppressing muscle wasting. (32) The antisense oligonucleotide, salt or solvate thereof according to any one of (1) to (8) for use in a method for switching myostatin splicing from myostatin to a myostatin splicing variant. (33) The antisense oligonucleotide, salt or solvate thereof according to any one of (1) to (8) for use in a method for reducing myostatin signaling. (34) The antisense oligonucleotide, salt or solvate thereof according to any one of (1) to (8) for use in a method for preventing and / or treating cancer.(35) Use of the antisense oligonucleotide according to any one of (1) to (8), or a salt or solvate thereof, for preventing and / or treating a pathology and / or disease involving myostatin. (36) Use of the antisense oligonucleotide according to any one of (1) to (8), or a salt or solvate thereof, for promoting muscle cell proliferation and / or hypertrophy. (37) Use of the antisense oligonucleotide according to any one of (1) to (8), or a salt or solvate thereof, for increasing muscle mass and / or suppressing muscle wasting. (38) Use of the antisense oligonucleotide according to any one of (1) to (8), or a salt or solvate thereof, for switching myostatin splicing from myostatin to a myostatin splicing variant. (39) Use of the antisense oligonucleotide according to any one of (1) to (8), or a salt or solvate thereof, for reducing myostatin signaling. (40) Use of the antisense oligonucleotide, salt or solvate thereof according to any one of (1) to (8) for preventing and / or treating cancer.

[0008] The ASO of the present invention modifies the splicing of the MSTN gene, promoting the production of splicing variants and inducing the expression of a new myostatin isoform. A patent application has already been filed for this new isoform as a myostatin inhibitory protein (WO2020 / 179571). Therefore, the ASO of the present invention serves as a new myostatin inhibitor. By administering the ASO of the present invention to humans, it inhibits the function of myostatin, enabling the prevention and treatment of muscle atrophy. Specifically, it is believed that the ASO can be applied to the treatment of diseases in which muscle atrophy is the primary symptom, such as muscular dystrophy and myopathy. Furthermore, it can be applied to the prevention of sarcopenia and the treatment and prevention of a wide range of conditions and diseases where muscle mass restoration is expected to have a therapeutic effect, including cancer cachexia and diabetes.

[0009] By promoting the production of myostatin splicing variants and inducing the expression of myostatin isoforms, myostatin signaling can be inhibited. This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2021-029890, which is the priority basis of the present application.

[0010] Search for ASOs that promote alternative splicing. Schematic diagram of an ASO that switches the splicing of MSTN pre-mRNA from producing MSTN to producing MSTN-b. Numbers indicate the exons in MSTN pre-mRNA. MSTN has the full length of exon 3, while MSTN-b has exon 3s, a portion of exon 3. ASOs complementary to the sequences of intron 2 and exon 3 of MSTN pre-mRNA. 54 types of ASOs were created for MSTN pre-mRNA. ■ indicates the recognition position of ASOs complementary to MSTN pre-mRNA. Numbers indicate the exons in MSTN pre-mRNA. Comparison of the effectiveness of ASOs (MSTN_Ex3_BP, MSTN_Ex3_SS, MSTN_Ex3_Fox1, MSTN_Ex3_LESE12, MSTN_Ex3_LESE3, MSTN_Ex3_LESE4). (A) The recognition sites of six ASOs in intron 2 and exon 3 of MSTN pre-mRNA are shown. (B) The results of RT-PCR of MSTN mRNA, MSTN-b mRNA, and GAPDH mRNA in human rhabdomyosarcoma cells (CRL-2061) transfected with each ASO are shown. w / o indicates no ASO treatment. Black arrowheads indicate MSTN, and white arrowheads indicate MSTN-b. The graph shows the ratio of MSTN-b to total MSTN. Results from three independent experiments are summarized. Comparison of the efficacy of ASOs (MSTN_Ex3_Fox1+6, MSTN_Ex3_Fox1+4, MSTN_Ex3_Fox1+2, MSTN_Ex3_Fox1, MSTN_Ex3_Fox1-2, MSTN_Ex3_Fox1-4, MSTN_Ex3_Fox1-6). (A) The recognition sites of seven ASOs in exon 3 of MSTN pre-mRNA are shown. (B) ASOs were introduced into human rhabdomyosarcoma cells (CRL-2061), and the results of RT-PCR for MSTN mRNA, MSTN-b mRNA, and GAPDH mRNA are shown. w / o indicates no ASO treatment. Black arrowheads indicate MSTN, and white arrowheads indicate MSTN-b. The graph shows the ratio of MSTN-b to total MSTN. Results are pooled from three independent experiments. **P<0.01.Comparison of the efficacy of ASOs (MSTN_Ex3_Fox1, MSTN_Ex3_1, MSTN_Ex3_2, MSTN_Ex3_3, MSTN_Ex3_4, MSTN_Ex3_5, MSTN_Ex3_6). (A) The recognition sites of seven ASOs relative to exon 3 of MSTN pre-mRNA are shown. (B) The results of RT-PCR of MSTN mRNA, MSTN-b mRNA, and GAPDH mRNA after transfection of each ASO into human rhabdomyosarcoma cells (CRL-2061) are shown. w / o indicates no ASO treatment. Black arrowheads indicate MSTN, and white arrowheads indicate MSTN-b. The graph shows the ratio of MSTN-b to total MSTN. Results are pooled from three independent experiments. *P<0.05. Comparison of the efficacy of ASOs (MSTN_In2_4, MSTN_In2_3, MSTN_In2_2, MSTN_Ex3_BP, MSTN_Ex3_SS, MSTN_Ex3_7, MSTN_Ex3_Fox1). (A) The recognition sites of seven ASOs in intron 2 and exon 3 of MSTN pre-mRNA are shown. (B) The results of RT-PCR of MSTN mRNA, MSTN-b mRNA, and GAPDH mRNA after transfection of each ASO into human rhabdomyosarcoma cells (CRL-2061) are shown. w / o indicates no ASO treatment. Black arrowheads indicate MSTN, and white arrowheads indicate MSTN-b. The graph shows the ratio of MSTN-b to total MSTN. Results are pooled from three independent experiments. **P<0.01. Comparison of the efficacy of ASOs (MSTN_Ex3_7+6, MSTN_Ex3_7+3, MSTN_Ex3_7, MSTN_Ex3_7-3, MSTN_Ex3_7-6, MSTN_Ex3_7-9). (A) The recognition sites of six ASOs relative to exon 3 of MSTN pre-mRNA are shown. (B) The results of RT-PCR of MSTN mRNA, MSTN-b mRNA, and GAPDH mRNA in human rhabdomyosarcoma cells (CRL-2061) transfected with each ASO are shown. w / o indicates no ASO treatment. Black arrowheads indicate MSTN, and white arrowheads indicate MSTN-b. The graph shows the ratio of MSTN-b to total MSTN. Results are pooled from three independent experiments. *P<0.05, ***P<0.001.Comparison of the efficacy of ASOs (MSTN_Ex3_7, MSTN_Ex3_7-1, MSTN_Ex3_7-2, MSTN_Ex3_7-3, MSTN_Ex3_7-4, MSTN_Ex3_7-5, MSTN_Ex3_7-6, MSTN_Ex3_7-7, MSTN_Ex3_7-8, MSTN_Ex3_7-9). (A) The recognition sites of 10 ASOs in exon 3 of MSTN pre-mRNA are shown. (B) The results of RT-PCR for MSTN mRNA, MSTN-b mRNA, and GAPDH mRNA are shown for human rhabdomyosarcoma cells (CRL-2061) transfected with each ASO. w / o indicates no ASO treatment. Black arrowheads indicate MSTN, and white arrowheads indicate MSTN-b. The graph shows the ratio of MSTN-b to total MSTN. Results from three independent experiments are summarized. *P<0.05, **P<0.01, ***P<0.001. Comparison of the efficacy of ASOs (MSTN_Ex3_8, MSTN_Ex3_9, MSTN_Ex3_10, MSTN_Ex3_11, MSTN_Ex3_12). (A) The recognition sites of five ASOs relative to exon 3 of MSTN pre-mRNA are shown. (B) The results of RT-PCR for MSTN mRNA, MSTN-b mRNA, and GAPDH mRNA are shown for human rhabdomyosarcoma cells (CRL-2061) transfected with each ASO. w / o indicates no ASO treatment. Black arrowheads indicate MSTN, and white arrowheads indicate MSTN-b. The graph shows the ratio of MSTN-b to total MSTN. Results are pooled from three independent experiments. ***P<0.001. Comparison of the efficacy of ASOs (MSTN_Ex3_13, MSTN_Ex3_14, MSTN_Ex3_15). (A) The recognition sites of three ASOs relative to exon 3 of MSTN pre-mRNA are shown. (B) The results of RT-PCR of MSTN mRNA, MSTN-b mRNA, and GAPDH mRNA after each ASO was introduced into human rhabdomyosarcoma cells (CRL-2061) are shown. w / o indicates no ASO treatment. Black arrowheads indicate MSTN, and white arrowheads indicate MSTN-b. The graph shows the ratio of MSTN-b to total MSTN. Results from three independent experiments are summarized. Comparison of the efficacy of ASOs (MSTN_Ex3_16, MSTN_Ex3_17, MSTN_Ex3_18, MSTN_Ex3_19).(A) The recognition sites of four ASOs in exon 3 of MSTN pre-mRNA are shown. (B) The results of RT-PCR for MSTN mRNA, MSTN-b mRNA, and GAPDH mRNA are shown for human rhabdomyosarcoma cells (CRL-2061) transfected with each ASO. w / o indicates no ASO treatment. Black arrowheads indicate MSTN, and white arrowheads indicate MSTN-b. The graph shows the ratio of MSTN-b to total MSTN. Results from three independent experiments are summarized. Comparison of the efficacy of ASOs (MSTN_Ex3_20, MSTN_Ex3_21, MSTN_Ex3_22, MSTN_Ex3_23). (A) The recognition sites of four ASOs in exon 3 of MSTN pre-mRNA are shown. (B) The results of RT-PCR for MSTN mRNA, MSTN-b mRNA, and GAPDH mRNA are shown for human rhabdomyosarcoma cells (CRL-2061) transfected with each ASO. W / o indicates no ASO treatment. Black arrowheads indicate MSTN, and white arrowheads indicate MSTN-b. The graph shows the ratio of MSTN-b to total MSTN. Results from three independent experiments are summarized. Comparison of the efficacy of ASOs (MSTN_Ex3_24, MSTN_Ex3_25, MSTN_Ex3_26, MSTN_Ex3_27, MSTN_Ex3_28). (A) The recognition sites of five ASOs relative to exon 3 of MSTN pre-mRNA are shown. (B) The results of RT-PCR of MSTN mRNA, MSTN-b mRNA, and GAPDH mRNA after ASO transfection into human rhabdomyosarcoma cells (CRL-2061) are shown. W / o indicates no ASO treatment. Black arrowheads indicate MSTN, and white arrowheads indicate MSTN-b. The graph shows the ratio of MSTN-b to total MSTN. Results from three independent experiments are summarized. Myostatin-b expression following ASO administration. A schematic diagram of the promyostatin and myostatin-b proteins is shown (left). Each domain and amino acid number are indicated. Promyostatin consists of 375 amino acids, while myostatin-b consists of 251 amino acids. Promyostatin and myostatin-b share the same sequence from amino acid numbers 1 to 250, with myostatin-b containing a valine at the 251st amino acid.Exon 3 of MSTN encodes amino acids 250-375, while exon 3s of MSTN-b encodes amino acids 250 and 251. Myostatin (mature myostatin) is produced by protease excision of the active domain of promyostatin translated from MSTN mRNA; therefore, it is not produced from MSTN-b mRNA, which does not retain the region encoding the active domain. The expression of myostatin and actin after MSTN_Ex3_7-2 ASO treatment was examined (right panel). ASO treatment reduced promyostatin dimers, and expression of myostatin-b dimers and myostatin-b monomers was confirmed. ASO administration reduces myostatin signaling. A schematic diagram of the effect of myostatin on an in vitro myostatin transcriptional activity assay system is shown (left panel). When myostatin (mature myostatin) binds to its receptor, it activates the transcription factor Samd2 / 3, which activates the transcription of target genes. The in vitro myostatin transcriptional activity assay uses a luciferase reporter gene as the target gene for Samd2 / 3, which is activated by myostatin, allowing myostatin signaling to be evaluated by luciferase activity. MSTN_Ex3_7-2 ASO was administered to rhabdomyosarcoma cells (CRL-2061), and myostatin signaling was evaluated using the in vitro myostatin transcriptional activity assay (right panel). Luciferase activity (luciferase activity / β-galactosidase activity) is expressed relative to the value measured in the extract from cells without ASO administration (w / o), set at 1. The results of two independent experiments are summarized. The ASO target sequence is highly conserved across species. The nucleic acid sequence alignment of MSTN exon 3 from human, dog, mouse, chicken, bovine, porcine, and ovine is shown. The underlined sequences are identical to those of human MSTN. Comparison of the effectiveness of ASOs with different ENA configurations of MSTN_Ex3_7-2 (MSTN_Ex3_7-2a, MSTN_Ex3_7-2b, MSTN_Ex3_7-2c, MSTN_Ex3_7-2d, MSTN_Ex3_7-2e, MSTN_Ex3_7-2f, MSTN_Ex3_7-2g, MSTN_Ex3_7-2h, MSTN_Ex3_7-2i).Human rhabdomyosarcoma cells (CRL-2061) were transfected with ASOs, and the results of RT-PCR for MSTN mRNA, MSTN-b mRNA, and GAPDH mRNA are shown. Con indicates no ASO treatment. Black arrowheads indicate MSTN, and white arrowheads indicate MSTN-b. The graph shows the ratio of MSTN-b to total MSTN. Results are summarized from three independent experiments. *P<0.05, **P<0.01, ***P<0.001. Examination of the concentration dependency of MSTN_Ex3_7-2b. Human rhabdomyosarcoma cells (CRL-2061) were treated with ASOs at final concentrations of 0, 25, 50, 75, 100, and 200 nM, and the results of RT-PCR for MSTN mRNA, MSTN-b mRNA, and GAPDH mRNA are shown. Black arrowheads indicate MSTN, and white arrowheads indicate MSTN-b. The graph shows the ratio of MSTN-b to total MSTN. Results from three independent experiments are pooled. ***P<0.001. MSTN_Ex3_7-2b administration resulted in a concentration-dependent decrease in myostatin signaling. Human rhabdomyosarcoma cells (CRL-2061) were treated with ASO at final concentrations of 0, 25, 50, 75, 100, 200, 300, and 400 nM, and myostatin signaling was assessed using an in vitro myostatin transcription activity assay. The luciferase activity (luciferase activity / β-galactosidase activity) derived from cells treated with ASO at each concentration is shown. The IC50 was 87.4 nM. Results from three independent experiments are pooled. *P<0.05, ***P<0.001. MSTN_Ex3_7-2b was administered to human myoblasts at final concentrations of 0, 1, 5, 10, 25, 50, and 100 nM, and a CCK-8-mediated cell proliferation assay was performed. Results are shown for the third day after administration. Results are from three independent experiments. *P<0.05, ***P<0.001. Human rhabdomyosarcoma cells (CRL-2061) were administered with MSTN_Ex3_7-2b at final concentrations of 0, 1, 5, 10, 25, 50, and 100 nM, and a CCK-8-mediated cell proliferation assay was performed. Results are shown for the third day after administration. Results are from three independent experiments. *P<0.05, **P<0.01, ***P<0.001.

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

[0012] The present invention provides an antisense oligonucleotide, a salt, or solvate thereof, having a base sequence complementary to a target sequence in exon 3 of the myostatin gene and capable of expressing a myostatin splicing variant, and having a length of 15 to 30 bases. The present inventors previously demonstrated that expression of myostatin-b (MSTN-b), an isoform of myostatin (MSTN), reduces myostatin signaling in cultured cells ( WO 2020 / 179571 ). This suggests that myostatin-b inhibits myostatin and promotes muscle formation. The base sequence of MSTN-b is shown in SEQ ID NO: 68. Myostatin is composed of, from the N-terminus, a signal peptide (residues 1-18), a prodomain (residues 19-266), and mature myostatin (residues 267-375). The nucleic acid sequences of myostatin and myostatin-b are identical in exons 1 and 2, and are shared up to amino acid 249. In myostatin-b, the base sequence of exon 3 is different, resulting in an asparagine (N) at the 250th amino acid from the N-terminus, a valine (V) at the 251st amino acid, and a stop codon (*) at the 252nd amino acid. Therefore, myostatin-b does not possess the domains of mature myostatin. The amino acid sequence of myostatin-b protein is shown in SEQ ID NO: 69. The amino acid sequence of myostatin is shown in SEQ ID NO: 70. The antisense oligonucleotides of the present invention can switch splicing from myostatin to myostatin-b. The antisense oligonucleotides of the present invention can decrease myostatin or increase myostatin-b at the protein level in cells expressing myostatin. Furthermore, they can also decrease myostatin signaling.

[0013] The base sequence of exon 3 of the human myostatin gene is shown in SEQ ID NO: 1. In the present invention, when the base sequence of exon 3 of the myostatin gene is the base sequence of SEQ ID NO: 1, the target sequence of exon 3 of the myostatin gene may be present within the region of base numbers 10 to 33 of the base sequence of SEQ ID NO: 1. The base sequence of the region of base numbers 10 to 33 of the base sequence of SEQ ID NO: 1 is shown in SEQ ID NO: 2. In the present invention, a "target sequence" refers to a sequence that promotes the production of a myostatin splicing variant by an ASO having a sequence complementary to all or part of that sequence. An ASO that promotes the production of a myostatin splicing variant may include a sequence complementary to a sequence consisting of at least 15 consecutive bases in the base sequence of SEQ ID NO: 2.

[0014] Furthermore, in the present invention, the base sequence of the antisense oligonucleotide preferably comprises a sequence consisting of at least 15 consecutive bases in any of the base sequences of SEQ ID NOs: 24, 30, 31, 27, 32, 33, or 28 (however, t in the sequence may be u, and u may be t). The base sequences of SEQ ID NOs: 24, 30, 31, 27, 32, 33, and 28 all share a sequence complementary to the region of bases 7 to 18 in the base sequence of SEQ ID NO: 2. The sequences of SEQ ID NOs: 61 to 67 are examples of sequences in which t in the sequence of SEQ ID NO: 31 is changed to u.

[0015] The antisense oligonucleotide may be 18 bases long, and the base sequence of the antisense oligonucleotide may be any of the base sequences of SEQ ID NOs: 24, 30, 31, 27, 32, 33, or 28 (however, t in the sequence may be u, and u may be t).

[0016] The nucleotides constituting the antisense oligonucleotide may be natural DNA, natural RNA, or modified versions thereof, but it is preferable that at least one of them is a modified nucleotide.

[0017] Examples of modified nucleotides include those with modified sugars (e.g., those in which the hydroxyl group at the 2' position of D-ribofuranose is modified, such as those in which D-ribofuranose is 2'-O-alkylated or 2'-O,4'-C-alkylenated), those with modified phosphodiester bonds (e.g., thioated), those with modified bases, and combinations thereof. Antisense oligonucleotides in which at least one D-ribofuranose moiety is 2'-O-alkylated or 2'-O,4'-C-alkylenated have high RNA binding strength and high resistance to nucleases, and are therefore expected to have a higher therapeutic effect than natural nucleotides (i.e., oligo-DNAs and oligo-RNAs). Furthermore, oligonucleotides in which at least one phosphodiester bond is thioated are also expected to have a higher therapeutic effect than natural nucleotides (i.e., oligo-DNAs and oligo-RNAs) due to their high resistance to nucleases. Oligonucleotides containing both the above-described modified sugar and modified phosphate are expected to have even greater therapeutic effects because they are more resistant to nucleases.

[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-propargylation, etc.), 2'-O,4'-C-alkylenation of D-ribofuranose (e.g., Examples include 2'-O,4'-C-ethylenated, 2'-O,4'-C-methylenated, 2'-O,4'-C-propylenated, 2'-O,4'-C-tetramethylenated, 2'-O,4'-C-pentamethyleneated, etc.), 3'-deoxy-3'-amino-2'-deoxy-D-ribofuranose, 3'-deoxy-3'-amino-2'-deoxy-2'-fluoro-D-ribofuranose, etc.

[0019] For antisense oligonucleotides, examples of modifications of phosphodiester bonds 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-fluorolation, 5-bromination, 5-iodination, and N4-methylation of cytosine; 5-demethylation (uracil), 5-fluorolation, 5-bromination, and 5-iodination of thymidine; N6-methylation and 8-bromination of adenine; N2-methylation and 8-bromination of guanine; pseudouridylation and 1-methylpseudourylation of uridine.

[0021] The antisense oligonucleotide of the present invention may be in the form of a salt. When the antisense oligonucleotide of the present invention is used as a medicine, the salt may be a pharmaceutically acceptable salt, and examples of such salts include metal salts such as alkali metal salts (e.g., sodium salt, potassium salt, and lithium salt), alkaline earth metal salts (e.g., calcium salt and magnesium salt), aluminum salt, iron salt, zinc salt, copper salt, nickel salt, and cobalt salt; inorganic salts (e.g., ammonium salt), 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-benzoyl Examples of suitable salts include amine salts, such as organic salts like 4-phenethylamine salts, piperazine salts, tetramethylammonium salts, and tris(hydroxymethyl)aminomethane salts; inorganic acid salts like hydrohalogen salts like hydrofluoride, hydrochloride, hydrobromide, and hydroiodide, nitrate, perchlorate, sulfate, and phosphate; organic acid salts like lower alkane sulfonates like methanesulfonate, trifluoromethanesulfonate, and ethanesulfonate, arylsulfonates like benzenesulfonate and p-toluenesulfonate, acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, and maleate; and amino acid salts like glycine salt, lysine salt, arginine salt, ornithine salt, glutamate, and aspartate. These salts can be prepared by known methods.

[0022] Furthermore, the antisense oligonucleotide may exist as a solvate (for example, a hydrate), and may be such a solvate.

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

[0024] Conventional methods can be used to synthesize antisense oligonucleotides. Examples of the synthesis method include synthesis by genetic engineering techniques and chemical synthesis. Examples of the genetic engineering techniques include in vitro transcription synthesis, vector-based synthesis, and PCR cassette-based synthesis. Examples of the vector include non-viral vectors such as plasmids and viral vectors. Examples of the chemical synthesis method include the phosphoramidite method and the H-phosphonate method. For example, a commercially available automated nucleic acid synthesizer can be used for the chemical synthesis method. Amidites are generally used in the chemical synthesis method. The amidites are not particularly limited; in the examples described below, antisense oligonucleotides were synthesized by the phosphoramidite method using ENA-2CE phosphoramidite and 2'-OMe-2CE phosphoramidite.

[0025] Commercially available phosphoramidite reagents can be used for natural nucleosides and 2'-O-methylnucleosides (i.e., 2'-O-methylguanosine, 2'-O-methyladenosine, 2'-O-methylcytosine, and 2'-O-methyluridine). The following are the phosphoramidite reagents for 2'-O-alkylguanosine, adenosine, cytosine, and uridine, each of which has an alkyl group with 2 to 6 carbon atoms.

[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, EA et al., Biochemistry (1993), 32, 7832-7838).

[0028] Commercially available reagents can be used for 2'-O-allylguanosine, adenosine, cytosine, and uridine.

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

[0030] 2'-O-butylguanosine, adenosine, cytosine, and uridine can be synthesized according to the literature (Lesnik, EA et al., Biochemistry (1993), 32, 7832-7838).

[0031] 2'-O-pentylguanosine, adenosine, cytosine, and uridine can be synthesized according to the literature (Lesnik, EA et al., Biochemistry (1993), 32, 7832-7838).

[0032] Commercially available reagents can be used for 2'-O-propargylguanosine, adenosine, cytosine, and uridine.

[0033] 2'-O,4'-C-methyleneguanosine, adenosine, 5-methylcytosine, and thymidine can be produced according to the method described in WO99 / 14226, and 2'-O,4'-C-alkyleneguanosine, adenosine, 5-methylcytosine, and thymidine having an alkylene group with 2 to 5 carbon atoms can be produced according to the method described in WO00 / 47599.

[0034] Antisense oligonucleotides having phosphorothioate bonds can be synthesized by coupling a phosphoramidite reagent followed by reaction with a reagent such as sulfur, tetraethylthiuram disulfide (TETD, Applied Biosystems), Beaucage reagent (Glen Research), or xanthan hydride (Tetrahedron Letters, 32, 3005 (1991), J. Am. Chem. Soc. 112, 1253 (1990), PCT / WO98 / 54198).

[0035] Commercially available controlled pore glass (CPG) for use in synthesizers can be used to conjugate 2'-O-methylnucleosides. For 2'-O,4'-C-methyleneguanosine, adenosine, 5-methylcytosine, and thymidine, nucleosides containing 2 to 5 carbon atoms in the alkylene group can be conjugated to CPG according to the method described in WO 99 / 14226. For 2'-O,4'-C-alkyleneguanosine, adenosine, 5-methylcytosine, and thymidine, nucleosides prepared according to the method described in WO 00 / 47599 can be conjugated to CPG according to the literature (Oligonucleotide Synthesis, Edited by MJ Gait, Oxford University Press, 1984). Using modified CPG (described in Example 12b of JP-A-7-87982), oligonucleotides bearing a 2-hydroxyethyl phosphate group at the 3' end can be synthesized. In addition, 3'-amino-Modifier C3 CPG, 3'-amino-Modifier C7 CPG, Glyceryl CPG (Glen Research), 3'-specer C3 SynBase CPG 1000, and 3'-specer C9 SynBase CPG 1000 (link technologies) can be used to synthesize oligonucleotides with a hydroxyalkyl phosphate group or an aminoalkyl phosphate group attached to the 3' end.

[0036] The antisense oligonucleotides of the present invention can be used in pharmaceuticals. When used as pharmaceuticals, the antisense oligonucleotides may be in the form of a pharmaceutically acceptable salt, solvate, or prodrug. Thus, the present invention provides pharmaceuticals comprising the above-described antisense oligonucleotides or pharmaceutically acceptable salts or solvates thereof. The pharmaceuticals are preferably intended for the prevention and / or treatment of myostatin-related conditions and / or diseases, including muscle atrophy (e.g., muscle atrophy caused by muscular dystrophy, myopathy, spinal muscular atrophy, sarcopenia, disuse muscle atrophy, etc.), and conditions and / or diseases in which muscle mass restoration provides therapeutic benefits (e.g., cancer cachexia, diabetes, cardiovascular disease (e.g., heart failure, arteriosclerosis, etc.), renal disease (e.g., chronic renal failure, etc.), bone disease (e.g., inflammatory arthritis, etc.)). Because myostatin inhibition increases skeletal muscle mass, it is believed that the pharmaceuticals can be used to treat all diseases that exhibit muscle atrophy, regardless of the cause of the atrophy. Increasing skeletal muscle mass promotes increased exercise and contributes to improved whole-body metabolism. It is also expected to act on the myocardium, restoring its function. Meanwhile, myostatin inhibition is expected to suppress bone destruction by acting on osteoclasts, activate the homeostatic ability of vascular endothelial cells, induce apoptosis, and increase insulin sensitivity. The present invention also provides a method for preventing and / or treating myostatin-associated pathologies and / or diseases, comprising administering to a subject an effective amount of the antisense oligonucleotide, salt, or solvate thereof. The present invention also provides the antisense oligonucleotide, salt, or solvate thereof for use in a method for preventing and / or treating myostatin-associated pathologies and / or diseases. Furthermore, the present invention provides the use of the antisense oligonucleotide, salt, or solvate thereof for preventing and / or treating myostatin-associated pathologies and / or diseases.

[0037] The antisense oligonucleotides of the present invention, or pharmaceutically acceptable salts, solvates, or prodrugs thereof (hereinafter referred to as "active ingredients"), can be administered orally or parenterally to mammals (e.g., humans, rabbits, dogs, cats, rats, and mice) alone or in appropriate dosage forms together with pharmacologically acceptable carriers, diluents, or excipients. The dosage varies depending on the subject, target disease, symptoms, and route of administration. For example, when used to prevent or treat muscle atrophy (e.g., muscular dystrophy), a single dose of the active ingredient is typically about 0.1 to 50 mg / kg body weight, preferably about 0.5 mg / kg body weight, administered orally, intramuscularly, subcutaneously, or intravenously (preferably continuously or every other day) about once a week to once a month, preferably about once a year. Similar amounts can also be administered for other parenteral and oral administrations. In particularly severe cases, the dosage may be increased depending on the symptoms. For other diseases, the above dosage may be used as a reference and the dosage may be increased or decreased as appropriate.

[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] Preparations for parenteral administration include, for example, injections and suppositories, and injections may be in the form of intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip infusion injections, or the like. Such injections are prepared by conventional methods, i.e., by dissolving, suspending, or emulsifying the active ingredient in a sterile aqueous or oily liquid typically used for injections. Aqueous liquids for injection include physiological saline, isotonic liquids containing glucose and other adjuvants, and the like, which may be used in combination with an appropriate solubilizing agent, such as alcohol (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), or nonionic surfactants (e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)). Oily liquids include sesame oil and soybean oil, and may be used in combination with a solubilizing agent such as benzyl benzoate or benzyl alcohol. The prepared injection solution is usually filled into a suitable ampule. Suppositories for rectal administration can be prepared by mixing the active ingredient with a conventional suppository base.

[0040] The oral or parenteral pharmaceutical preparations may be prepared in dosage unit forms suitable for the dosage of the active ingredient. Examples of dosage unit forms include tablets, pills, capsules, injections (ampoules), suppositories, etc., and each dosage unit preferably contains 0.1 to 1,000 mg of the active ingredient.

[0041] The antisense oligonucleotides of the present invention can also be used in foods and feeds. For example, they can be used as food or feed additives or as supplements for humans or animals. The antisense oligonucleotides of the present invention may be in the form of a salt, solvate, or prodrug that is acceptable for use in foods or feeds. Thus, the present invention provides foods containing the above-mentioned antisense oligonucleotides and their salts or solvates that are acceptable for use in foods. The present invention also provides feeds containing the above-mentioned antisense oligonucleotides and their salts or solvates that are acceptable for use in foods. Examples of salts, solvates, or prodrugs that are acceptable for use in foods or feeds include pharmaceutically acceptable salts, solvates, or prodrugs, which are described above.

[0042] The antisense oligonucleotides of the present invention can be used to promote muscle cell proliferation and / or hypertrophy. Muscle cells are contractile cells that form muscle tissue in humans and animals, and include skeletal muscle cells, smooth muscle cells, and cardiac muscle cells. Myostatin inhibition by the antisense oligonucleotides of the present invention is effective in myoblasts, promoting myoblast proliferation and differentiation, which can ultimately lead to muscle cell proliferation / hypertrophy. Muscle cells also include precursor cells such as myoblasts. The antisense oligonucleotides 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 are described above. Any animal that expresses myostatin may be used, and examples include domestic animals used for work or food, such as mammals such as cats, dogs, sheep, pigs, cows, chickens, and turkeys, and fish such as salmon, trout, cod, tuna, and yellowtail. Thus, the present invention provides a drug for promoting muscle cell proliferation and / or hypertrophy, comprising the above-mentioned antisense oligonucleotide, a salt thereof, or a solvate thereof. The present invention also provides a method for promoting muscle cell proliferation and / or hypertrophy, comprising administering an effective amount of the above-mentioned antisense oligonucleotide, a salt thereof, or a solvate thereof to a subject. The present invention also provides the above-mentioned antisense oligonucleotide, a salt thereof, or a solvate thereof for use in the method for promoting muscle cell proliferation and / or hypertrophy. The present invention also provides use of the above-mentioned antisense oligonucleotide, a salt thereof, or a solvate thereof for promoting muscle cell proliferation and / or hypertrophy. The subject may be a human or an animal. The animal is described above. The dosage form, dosage, administration route, administration frequency, etc. of the drug may be similar to those of the above-mentioned pharmaceuticals, and can be appropriately modified to achieve the desired effect.

[0043] The antisense oligonucleotides of the present invention can also be used to increase muscle mass and suppress muscle wasting in humans and animals. The antisense oligonucleotides 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 are described above. The animal may be any animal that expresses myostatin, including mammals such as cats, dogs, sheep, pigs, cows, chickens, and turkeys, and farmed animals used for work or food, such as salmon, trout, cod, tuna, and yellowtail. Thus, the present invention provides a pharmaceutical agent for increasing muscle mass and / or suppressing muscle wasting, comprising the above-described antisense oligonucleotide, a salt thereof, or a solvate thereof. The present invention also provides a method for increasing muscle mass and / or suppressing muscle wasting, comprising administering an effective amount of the above-described antisense oligonucleotide, a salt thereof, or a solvate thereof to a subject. The present invention also provides the above-described antisense oligonucleotide, a salt thereof, or a solvate thereof for use in the method for increasing muscle mass and / or suppressing muscle wasting. The present invention also provides use of the above-mentioned antisense oligonucleotide, its salt, or solvate for increasing muscle mass and / or suppressing muscle wasting. The subject may be a human or an animal. Animals are described above. The dosage form, dosage amount, administration route, administration frequency, etc. of the drug may be similar to those of the above-mentioned pharmaceuticals, and may be appropriately modified to achieve the desired effect.

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

[0045] The food of the present invention may contain general ingredients such as protein, lipids, carbohydrates, 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, galactooligosaccharides, dietary fiber, excipients (e.g., water, carboxymethylcellulose, lactose), sweeteners, flavorings (e.g., malic acid, citric acid, amino acids), and flavorings. When the food of the present invention is prepared as a liquid, water, physiological saline, soup, milk, fruit juice, and the like can be used as a liquid for dispersing or dissolving the food ingredients. The food of the present invention may be in the form of a powder, granules, tablet, liquid, or the like. To facilitate easy intake by sick individuals and the elderly, it is preferable to prepare the food in a gel form such as jelly.

[0046] The feed to which the antisense oligonucleotide of the present invention, or its salt, solvate, or prodrug that is acceptable for the feed is added may be any feed, such as a single feed such as grains, vegetable oil cakes, bran, manufacturing by-products, or animal feed, or a compound feed containing a plurality of feed ingredients or feed additives.

[0047] The feed of the present invention may contain antioxidants (ethoxyquin, dibutylhydroxytoluene, butylhydroxyanisole, etc.), fungicides (propionic acid, calcium propionate, sodium propionate, etc.), binders (sodium alginate, sodium caseinate, sodium carboxymethylcellulose, propylene glycol, sodium polyacrylate, etc.), emulsifiers (glycerin fatty acid esters, sucrose fatty acid esters, sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene glycerin fatty acid esters, etc.). , adjusters (formic acid, etc.), amino acids (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 (L-ascorbic acid, calcium L-ascorbate, sodium L-ascorbate, sodium calcium L-ascorbate 2-phosphate, L-ascorbic acid 2-phosphate ester, sodium calcium L-ascorbate 2-phosphate ester) Magnesium nitrate, 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-hydroxycholecalciferol, menadione sulfite water Ingredients: dimethylpyrimidinol, menadione sodium bisulfite, folic acid, riboflavin, riboflavin butyrate, etc.), minerals (potassium chloride, iron citrate, calcium gluconate, sodium ferrous citrate succinate, 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, zinc peptide, iron peptide, copper peptide, manganese peptide, potassium iodide, potassium iodate,Calcium iodate, zinc sulfate (dried), zinc sulfate (crystalline), zinc methionine sulfate, sodium sulfate (dried), magnesium sulfate (dried), magnesium sulfate (crystalline), cobalt sulfate (dried), cobalt sulfate (crystalline), iron sulfate (dried), copper sulfate (dried), copper sulfate (crystalline), manganese sulfate, potassium monohydrogen phosphate (dried), sodium monohydrogen phosphate (dried), potassium dihydrogen phosphate (dried), sodium dihydrogen phosphate (dried), sodium dihydrogen phosphate (crystalline), etc.), pigments, synthetic antibacterial agents, antibiotics, flavorings, taste-imparting agents, enzymes, probiotics, organic acids, etc. may also be added.

[0048] The food or feed should be ingested in an amount, frequency, and duration that will ensure the desired effect (for example, promotion of muscle formation).

[0049] The antisense oligonucleotides of the present invention can switch myostatin splicing from myostatin to a myostatin splicing variant. Therefore, the present invention provides a drug that switches myostatin splicing from myostatin to a myostatin splicing variant, comprising the antisense oligonucleotide. The drug of the present invention can be used as a human or animal medicine, as an additive or supplement to food or feed, as an animal growth promoter, or as an experimental reagent. The antisense oligonucleotides 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 are described above. The present invention also provides a method for switching myostatin splicing from myostatin to a myostatin splicing variant, comprising administering an effective amount of the antisense oligonucleotide, its salt, or solvate to a subject. The present invention also provides the above-mentioned antisense oligonucleotide, a salt or solvate thereof for use in a method for switching myostatin splicing from myostatin to a myostatin splicing variant.The present invention also provides use of the above-mentioned antisense oligonucleotide, a salt or solvate thereof for switching myostatin splicing from myostatin to a myostatin splicing variant.

[0050] The antisense oligonucleotides of the present invention can reduce myostatin signaling. Therefore, the present invention provides a drug for reducing myostatin signaling, comprising the above-described antisense oligonucleotide. The myostatin signaling reducer of the present invention can be used as a pharmaceutical for humans or animals, as an additive or supplement for food or feed, as an animal growth promoter, or as an experimental reagent. 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 are described above. The present invention also provides a method for reducing myostatin signaling, comprising administering an effective amount of the above-described antisense oligonucleotide, its salt, or solvate to a subject. The present invention also provides the above-described antisense oligonucleotide, its salt, or solvate for use in the method for reducing myostatin signaling. The present invention also provides the use of the above-described antisense oligonucleotide, its salt, or solvate to reduce myostatin signaling. Since inhibition of myostatin signaling is thought to be effective in suppressing diabetes and inhibiting its progression, the above-mentioned antisense oligonucleotides, salts thereof, or solvates thereof may be effective in preventing and / or treating diabetes. Therefore, the present invention also provides a prophylactic and / or therapeutic agent for diabetes, comprising the above-mentioned antisense oligonucleotides, salts thereof, or solvates thereof.

[0051] When used as an experimental reagent, treating myostatin-expressing cells, tissues, or organs with the antisense oligonucleotides, salts, or solvates of the present invention can switch myostatin splicing from myostatin to a myostatin splicing variant, inducing expression of a myostatin isoform, which can inhibit myostatin signaling. The antisense oligonucleotides, salts, and solvates of the present invention can be used in an amount effective to exert the desired function. Examples of myostatin-expressing cells include muscle cells, sarcoma cells, and cancer cells of the digestive tract, lungs, esophagus, etc. Examples of cells that express myostatin include naturally occurring cells and recombinant cells into which the myostatin gene has been introduced. Examples of tissues and organs that express myostatin include skeletal muscle, cardiac muscle, blood vessels, kidneys, digestive tract, uterus, liver, pancreas, lungs, etc. The switch in myostatin splicing from myostatin to a myostatin splice variant can be analyzed by analyzing the mRNA of the myostatin splice variant in a sample by RT-PCR, or by detecting the protein translated from the myostatin splice variant in a sample by Western blotting or mass spectrometry.

[0052] The antisense oligonucleotides of the present invention can inhibit the proliferation of cancer cells. Examples of cancer cells include rhabdomyosarcoma cells, liver cancer cells, cervical cancer cells, and alveolar basal epithelial adenocarcinoma cells. Therefore, the present invention provides an anticancer agent containing the above-mentioned antisense oligonucleotide. Indications for the anticancer agent of the present invention include cancers such as sarcoma, liver cancer, cervical cancer, and lung cancer. The anticancer agent of the present invention can be used as a medicine for humans or animals. 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 are described above. The present invention also provides a method for preventing and / or treating cancer, comprising administering an effective amount of the above-mentioned antisense oligonucleotide, its salt, or solvate to a subject. The present invention also provides the above-mentioned antisense oligonucleotide, its salt, or solvate for use in the method for preventing and / or treating cancer. The present invention also provides the use of the above-mentioned antisense oligonucleotide, its salt, or solvate for preventing and / or treating cancer. Cancer prevention is a concept that includes preventing cancer recurrence.

[0053] The present invention is described in detail below based on the following examples, but is not limited to these examples. [Examples 1-54] Synthesis of Antisense Oligonucleotides (ASOs) To identify ASOs (Figure 1) that promote alternative splicing to produce MSTN-β, the antisense oligonucleotides (ASOs) listed in Table 1 were synthesized. The recognition sites of ASOs complementary to MSTN pre-mRNA are shown in Figure 2. The modified nucleic acid ENA (2'-O,4'-C-Ethylene-bridged Nucleic Acids) was introduced into A (adenine), G (guanine), C (cytosine), and T (thymine) in the ASO sequence to improve affinity and stability.

[0054] Synthesis of gaaCaaTCagTaaTaTCa (MSTN_Ex3_BP) (Example 1) This was performed 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 solvents, reagents, and phosphoramidites in each synthesis cycle were the same as those used in natural oligonucleotide synthesis. Reagents and 2'-O-methylnucleoside phosphoramidites (adenosine product No. 10-3100-10, guanosine product No. 10-3121-10) were manufactured by Glen Research. Solvents were used from Wako Pure Chemical Industries, Ltd. The unnatural phosphoramidites used were those described in 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 A. The compounds shown were synthesized using Universal Control Pore Glass (CPG) (Glen Research, product No. 25-5040) as the solid support. The amidite condensation time was 15 minutes.

[0055] The protected oligonucleotide analogs containing the target sequence were cleaved from the support by heat treatment with concentrated aqueous ammonia (55°C for 8 hours), which removed the cyanoethyl protecting groups on the phosphorus atom and the nucleobases. The resulting ammonia solution was then used in a Glen-Pak DNA Purification Cartridge (Glen Research, product no. 60-5100) to remove DMT within the cartridge according to the protocol recommended by Glen Research. The collected solution was evaporated under reduced pressure, and the residue was purified by reverse-phase HPLC (Shimadzu LC-2a, column (YMC Triart C18, 10 x 150 mm)) using a mixture of 0.1 M triethylamine acetate (TEAA), pH 7.0, and acetonitrile (B), with a linear gradient of 10% to 25% B (30 min, 50°C, 4.7 mL / min, 280 nm). After distilling off the solvent, the residue was dissolved in 10 mM NaOH solution, and the solution was subjected to ultrafiltration using a Microsep centrifugal filtration device (product No. MCP003C, manufactured by Nippon Pall Corporation) to replace the solvent with pure water. After lyophilization, the target compound was obtained.

[0056] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6440.35, measured value: 6433.59).

[0057] The base sequence of this compound is complementary to nucleotides 5058-5075 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0058] Synthesis of aaaCggaTTCTgTTTgaa (MSTN_Ex3_SS) (Example 2) The compound of Example 2 having the target sequence similar to that of the compound of Example 1 was synthesized.

[0059] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6476.33, measured value: 6469.82).

[0060] The base sequence of this compound is complementary to nucleotides 5083-5100 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0061] Synthesis of TCaTCaCagTCaagaCCa (MSTN_Ex3_Fox1) (Example 3) The compound of Example 3 having the target sequence similar to that of the compound of Example 1 was synthesized.

[0062] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6443.39, measured value: 6436.58).

[0063] The base sequence of this compound is complementary to nucleotides 5184-5165 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0064] Synthesis of CCTgCTgaaCCTCTgggg (MSTN_Ex3_LESE12SF2) (Example 4) The compound of Example 4 having the target sequence similar to that of the compound of Example 1 was synthesized.

[0065] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6510.38, measured value: 6503.83).

[0066] The base sequence of this compound is complementary to nucleotides 5337-5354 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0067] Synthesis of aTTgTTgaggggaaaaCC (MSTN_Ex3_LESE3) (Example 5) The compound of Example 5 having the target sequence similar to that of the compound of Example 1 was synthesized.

[0068] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6502.33, measured value: 6495.71).

[0069] The base sequence of this compound is complementary to nucleotides 5512-5529 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0070] Synthesis of TaTagCCTgTggTaCTTa (MSTN_Ex3_LESE4) (Example 6) The compound of Example 6 having the target sequence similar to that of the compound of Example 1 was synthesized.

[0071] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6481.33, measured value: 6474.94).

[0072] The base sequence of this compound is complementary to nucleotides 5546-5563 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0073] Synthesis of CagTCaagaCCaaaaTCC (MSTN_Ex3_Fox1+6) (Example 7) The compound of Example 7 having the target sequence similar to that of the compound of Example 1 was synthesized.

[0074] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6439.64, measured value: 6439.92).

[0075] The base sequence of this compound is complementary to nucleotides 5142-5159 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0076] Synthesis of CaCagTCaagaCCaaaaT (MSTN_Ex3_Fox1+4) (Example 8) The compound of Example 8 having the target sequence similar to that of the compound of Example 1 was synthesized.

[0077] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6437.58, measured value: 6437.93).

[0078] The base sequence of this compound is complementary to nucleotides 5144-5161 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0079] Synthesis of aTCaCagTCaagaCCaaa (MSTN_Ex3_Fox1+2) (Example 9) The compound of Example 9 having the target sequence similar to that of the compound of Example 1 was synthesized.

[0080] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6437.58, measured value: 6437.82).

[0081] The base sequence of this compound is complementary to nucleotides 5146-5163 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0082] Synthesis of gCTCaTCaCagTCaagaC (MSTN_Ex3_Fox1-2) (Example 10) The compound of Example 10 having the target sequence similar to that of the compound of Example 1 was synthesized.

[0083] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6458.60, measured value: 6458.70).

[0084] The base sequence of this compound is complementary to nucleotides 5150-5167 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0085] Synthesis of gTgCTCaTCaCagTCaag (MSTN_Ex3_Fox1-4) (Example 11) The compound of Example 11 having the target sequence similar to that of the compound of Example 1 was synthesized.

[0086] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6475.50, measured value: 6475.67).

[0087] The base sequence of this compound is complementary to nucleotides 5152-5169 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0088] Synthesis of gagTgCTCaTCaCagTCa (MSTN_Ex3_Fox1-6) (Example 12) The compound of Example 12 having the target sequence similar to that of the compound of Example 1 was synthesized.

[0089] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6475.50, measured value: 6475.55).

[0090] The base sequence of this compound is complementary to nucleotides 5154-5171 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0091] Synthesis of TCCagagCagTaaTTggC (MSTN_Ex3_1) (Example 13) The compound of Example 13 having the target sequence similar to that of the compound of Example 1 was synthesized.

[0092] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6489.44, measured value: 6487.83).

[0093] The base sequence of this compound is complementary to nucleotides 5260-5277 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0094] Synthesis of TgagCaCCCaCagCggTC (MSTN_Ex3_2) (Example 14) The compound of Example 14 having the target sequence similar to that of the compound of Example 1 was synthesized.

[0095] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6489.70, measured value: 6488.90).

[0096] The base sequence of this compound is complementary to nucleotides 5452-5469 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0097] Synthesis of CTCgCCgaTgTTgTaaTa (MSTN_Ex3_3) (Example 15) The compound of Example 15 having the target sequence similar to that of the compound of Example 1 was synthesized.

[0098] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6479.36, measured value: 6477.62).

[0099] The base sequence of this compound is complementary to nucleotides 5734-5751 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0100] Synthesis of CaTaTgCTgCaCCaTCCC (MSTN_Ex3_4) (Example 16) The compound of Example 16 having the target sequence similar to that of the compound of Example 1 was synthesized.

[0101] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6449.68, measured value: 6450.12).

[0102] The base sequence of this compound is complementary to nucleotides 5965-5982 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0103] Synthesis of TgTaggaaaaTgCaCCTg (MSTN_Ex3_5) (Example 17) The compound of Example 17 having the target sequence similar to that of the compound of Example 1 was synthesized.

[0104] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6487.38, measured value: 6486.69).

[0105] The base sequence of this compound is complementary to nucleotides 6122-6139 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0106] Synthesis of CaaaggCagTgTTgTaaT (MSTN_Ex3_6) (Example 18) The compound of Example 18 having the target sequence similar to that of the compound of Example 1 was synthesized.

[0107] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6488.28, measured value: 6488.01).

[0108] The base sequence of this compound is complementary to nucleotides 6322-6339 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0109] Synthesis of CCCCTaaggTCagTaCCa (MSTN_In2_4) (Example 19) The compound of Example 19 having the target sequence similar to that of the compound of Example 1 was synthesized.

[0110] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6460.66, measured value: 6460.14).

[0111] The base sequence of this compound is complementary to nucleotides 4866-4883 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0112] Synthesis of gTTgaagTaaTaaaCTaa (MSTN_In2_3) (Example 20) The compound of Example 20 having the target sequence similar to that of the compound of Example 1 was synthesized.

[0113] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6454.22, measured value: 6454.25).

[0114] The base sequence of this compound is complementary to nucleotides 4998-5015 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0115] Synthesis of ggCCTggaaaCaCTTTTC (MSTN_In2_2) (Example 21) The compound of Example 21 having the target sequence similar to that of the compound of Example 1 was synthesized.

[0116] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6478.46, measured value: 6478.47).

[0117] The base sequence of this compound is complementary to nucleotides 5037-5054 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0118] Synthesis of TgTTaCCTTgaCCTCTaa (MSTN_Ex3_7) (Example 22) The compound of Example 22 having the target sequence similar to that of the compound of Example 1 was synthesized.

[0119] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6452.38, measured value: 6452.23).

[0120] The base sequence of this compound is complementary to nucleotides 5101-5118 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0121] Synthesis of CTTgaCCTCTaaaaaCgg (MSTN_Ex3_7+6) (Example 23) The compound of Example 23 having the target sequence similar to that of the compound of Example 1 was synthesized.

[0122] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6459.50, measured value: 6458.24).

[0123] The base sequence of this compound is complementary to nucleotides 5095-5112 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0124] Synthesis of TaCCTTgaCCTCTaaaaa (MSTN_Ex3_7+3) (Example 24) The compound of Example 24 having the target sequence similar to that of the compound of Example 1 was synthesized.

[0125] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6430.46, measured value: 6429.70).

[0126] The base sequence of this compound is complementary to nucleotides 5098-5115 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0127] Synthesis of gTCTgTTaCCTTgaCCTC (MSTN_Ex3_7-3) (Example 25) The compound of Example 25 having the target sequence similar to that of the compound of Example 1 was synthesized.

[0128] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6470.44, measured value: 6469.11).

[0129] The base sequence of this compound is complementary to nucleotides 5104-5121 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0130] Synthesis of TgTgTCTgTTaCCTTgaC (MSTN_Ex3_7-6) (Example 26) The compound of Example 26 having the target sequence similar to that of the compound of Example 1 was synthesized.

[0131] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6485.28, measured value: 6484.44).

[0132] The base sequence of this compound is complementary to nucleotides 5107-5124 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0133] Synthesis of TggTgTgTCTgTTaCCTT (MSTN_Ex3_7-9) (Example 27) The compound of Example 27 having the target sequence similar to that of the compound of Example 1 was synthesized.

[0134] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6502.18, measured value: 6500.63).

[0135] The base sequence of this compound is complementary to nucleotides 5110-5127 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0136] Synthesis of CTgTTaCCTTgaCCTCTa (MSTN_Ex3_7-1) (Example 28) The compound of Example 28 having the target sequence similar to that of the compound of Example 1 was synthesized.

[0137] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6454.44, measured value: 6454.34).

[0138] The base sequence of this compound is complementary to nucleotides 5102-5119 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0139] Synthesis of TCTgTTaCCTTgaCCTCT (MSTN_Ex3_7-2) (Example 29) The compound of Example 29 having the target sequence similar to that of the compound of Example 1 was synthesized.

[0140] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6457.40, measured value: 6457.37).

[0141] The base sequence of this compound is complementary to nucleotides 5103-5120 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0142] Synthesis of TgTCTgTTaCCTTgaCCT (MSTN_Ex3_7-4) (Example 30) The compound of Example 30 having the target sequence similar to that of the compound of Example 1 was synthesized.

[0143] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6471.34, measured value: 6471.68).

[0144] The base sequence of this compound is complementary to nucleotides 5105-5122 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0145] Synthesis of gTgTCTgTTaCCTTgaCC (MSTN_Ex3_7-5) (Example 31) The compound of Example 31 having the target sequence similar to that of the compound of Example 1 was synthesized.

[0146] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6484.38, measured value: 6484.65).

[0147] The base sequence of this compound is complementary to nucleotides 5106-5123 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0148] Synthesis of gTgTgTCTgTTaCCTTga (MSTN_Ex3_7-7) (Example 32) The compound of Example 32 having the target sequence similar to that of the compound of Example 1 was synthesized.

[0149] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6515.22, measured value: 6515.64).

[0150] The base sequence of this compound is complementary to nucleotides 5108-5125 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0151] Synthesis of ggTgTgTCTgTTaCCTTg (MSTN_Ex3_7-8) (Example 33) The compound of Example 33 having the target sequence similar to that of the compound of Example 1 was synthesized.

[0152] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6499.22, measured value: 6499.59).

[0153] The base sequence of this compound is complementary to nucleotides 5109-5126 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0154] Synthesis of gCTTCaaaaTCCaCagTT (MSTN_Ex3_8) (Example 34) The compound of Example 34 having the target sequence similar to that of the compound of Example 1 was synthesized.

[0155] This compound was identified by Q-TOF LC / MS (calculated value: 6446.35, measured value: 6446.24).

[0156] The base sequence of this compound is complementary to nucleotides 5202-5219 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0157] Synthesis of TCTTTTaggagCgaTaaT (MSTN_Ex3_9) (Example 35) The compound of Example 35 having the target sequence similar to that of the compound of Example 1 was synthesized.

[0158] This compound was identified by Q-TOF LC / MS (calculated value: 6478.31, measured value: 6478.52).

[0159] The base sequence of this compound is complementary to nucleotides 5236-5253 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0160] Synthesis of ggaTaTTTTTgTaaaaaT (MSTN_Ex3_10) (Example 36) The compound of Example 36 having the target sequence similar to that of the compound of Example 1 was synthesized.

[0161] This compound was identified by Q-TOF LC / MS (calculated value: 6461.28, measured value: 6461.43).

[0162] The base sequence of this compound is complementary to nucleotides 5292-5309 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0163] Synthesis of agaCaTCTTTgTgggagT (MSTN_Ex3_11) (Example 37) The compound of Example 37 having the target sequence similar to that of the compound of Example 1 was synthesized.

[0164] This compound was identified by Q-TOF LC / MS (calculated value: 6507.31, measured value: 6507.26).

[0165] The base sequence of this compound is complementary to nucleotides 5365-5382 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0166] Synthesis of aTaTaTTaTTTgTTCTTT (MSTN_Ex3_12) (Example 38) The compound of Example 38 having the target sequence similar to that of the compound of Example 1 was synthesized.

[0167] This compound was identified by Q-TOF LC / MS (calculated value: 6443.28, measured value: 6443.39).

[0168] The base sequence of this compound is complementary to nucleotides 5410-5427 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0169] Synthesis of aCTCTggaaaTCaTaaaa (MSTN_Ex3_13) (Example 39) The compound of Example 39 having the target sequence similar to that of the compound of Example 1 was synthesized.

[0170] This compound was identified by Q-TOF LC / MS (calculated value: 6439.33, measured value: 6439.67).

[0171] The base sequence of this compound is complementary to nucleotides 5674-5691 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0172] Synthesis of TaaTTCaTCagaaCTCaa (MSTN_Ex3_14) (Example 40) The compound of Example 40 having the target sequence similar to that of the compound of Example 1 was synthesized.

[0173] This compound was identified by Q-TOF LC / MS (calculated value: 6428.34, measured value: 6428.49).

[0174] The base sequence of this compound is complementary to nucleotides 5771-5788 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0175] Synthesis of TgTaaaTaTTaaaCaaaa (MSTN_Ex3_15) (Example 41) The compound of Example 41 having the target sequence similar to that of the compound of Example 1 was synthesized.

[0176] This compound was identified by Q-TOF LC / MS (calculated value: 6422.31, measured value: 6422.37).

[0177] The base sequence of this compound is complementary to nucleotides 5822-5839 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0178] Synthesis of CagCCTaTCgTaTTagCa (MSTN_Ex3_16) (Example 42) The compound of Example 42 having the target sequence similar to that of the compound of Example 1 was synthesized.

[0179] This compound was identified by Q-TOF LC / MS (calculated value: 6462.35, measured value: 6462.34).

[0180] The base sequence of this compound is complementary to nucleotides 6037-6054 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0181] Synthesis of CTggTagCCTCagaCaTT (MSTN_Ex3_17) (Example 43) The compound of Example 43 having the target sequence similar to that of the compound of Example 1 was synthesized.

[0182] This compound was identified by Q-TOF LC / MS (calculated value: 6478.35, measured value: 6478.26).

[0183] The base sequence of this compound is complementary to nucleotides 6055-6072 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0184] Synthesis of CCgTTggCaTggaTTgTT (MSTN_Ex3_18) (Example 44) The compound of Example 44 having the target sequence similar to that of the compound of Example 1 was synthesized.

[0185] This compound was identified by Q-TOF LC / MS (calculated value: 6512.32, measured value: 6512.38).

[0186] The base sequence of this compound is complementary to nucleotides 6019-6036 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0187] Synthesis of gTTTTTTaTgTgaTaaaC (MSTN_Ex3_19) (Example 45) The compound of Example 45 having the target sequence similar to that of the compound of Example 1 was synthesized.

[0188] This compound was identified by Q-TOF LC / MS (calculated value: 6466.29, measured value: 6466.19).

[0189] The base sequence of this compound is complementary to nucleotides 6073-6090 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0190] Synthesis of agagaaaCTTaCTaTTTT (MSTN_Ex3_20) (Example 46) The compound of Example 46 having the target sequence similar to that of the compound of Example 1 was synthesized.

[0191] This compound was identified by Q-TOF LC / MS (calculated value: 6446.31, measured value: 6446.34).

[0192] The base sequence of this compound is complementary to nucleotides 6098-6115 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0193] Synthesis of TCTTaCaTTaagaaaaT (MSTN_Ex3_21) (Example 47) The compound of Example 47 having the target sequence similar to that of the compound of Example 1 was synthesized.

[0194] This compound was identified by Q-TOF LC / MS (calculated value: 6427.32, measured value: 6427.14).

[0195] The base sequence of this compound is complementary to nucleotides 6158-6175 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0196] Synthesis of TgaaagCCaaCCTCTaga (MSTN_Ex3_22) (Example 48) The compound of Example 48 having the target sequence similar to that of the compound of Example 1 was synthesized.

[0197] This compound was identified by Q-TOF LC / MS (calculated value: 6456.36, measured value: 6456.24).

[0198] The base sequence of this compound is complementary to nucleotides 6187-6204 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0199] Synthesis of aTaaaaaCaaaCaCCaTT (MSTN_Ex3_23) (Example 49) The compound of Example 49 having the target sequence similar to that of the compound of Example 1 was synthesized.

[0200] This compound was identified by Q-TOF LC / MS (calculated value: 6406.35, measured value: 6406.36).

[0201] The base sequence of this compound is complementary to nucleotides 6252-6269 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0202] Synthesis of TTTCTaTTaTTTTaCCaT (MSTN_Ex3_24) (Example 50) The compound of Example 50 having the target sequence similar to that of the compound of Example 1 was synthesized.

[0203] This compound was identified by Q-TOF LC / MS (calculated value: 6428.31, measured value: 6428.34).

[0204] The base sequence of this compound is complementary to nucleotides 6356-6373 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0205] Synthesis of aaagTaaaTaaaaaagga (MSTN_Ex3_25) (Example 51) The compound of Example 51 having the target sequence similar to that of the compound of Example 1 was synthesized.

[0206] This compound was identified by Q-TOF LC / MS (calculated value: 6515.37, measured value: 6515.22).

[0207] The base sequence of this compound is complementary to nucleotides 6460-6477 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0208] Synthesis of TgTCCTTagTgTaaaaaT (MSTN_Ex3_26) (Example 52) The compound of Example 52 having the target sequence similar to that of the compound of Example 1 was synthesized.

[0209] This compound was identified by Q-TOF LC / MS (calculated value: 6462.31, measured value: 6462.37).

[0210] The base sequence of this compound is complementary to nucleotides 6483-6500 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0211] Synthesis of TTCTgTgaTgCaTgaCaT (MSTN_Ex3_27) (Example 53) The compound of Example 53 having the target sequence similar to that of the compound of Example 1 was synthesized.

[0212] This compound was identified by Q-TOF LC / MS (calculated value: 6480.32, measured value: 6480.14).

[0213] The base sequence of this compound is complementary to nucleotides 6530-6547 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0214] Synthesis of TCaTgTaaaaaaaTaTaa (MSTN_Ex3_28) (Example 54) The compound of Example 54 having the target sequence similar to that of the compound of Example 1 was synthesized.

[0215] This compound was identified by Q-TOF LC / MS (calculated value: 6422.31, measured value: 6422.49).

[0216] The base sequence of this compound is complementary to nucleotides 6642-6659 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0217] (Table 1) Sequences of the ASOs synthesized in this example. Uppercase letters indicate ENA nucleic acids, and lowercase letters indicate 2'OMe.

[0218] Examples 55-63: Synthesis of Antisense Oligonucleotides (ASOs) Antisense oligonucleotides (ASOs) shown in Table 2 were synthesized by changing the ENA configuration of MSTN_Ex3_7-2 (Example 29). Synthesis of TcTguTaCcuTgaCcTcT (MSTN_Ex3_7-2a) (Example 55) The compound of Example 55, 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: 6301.1, measured value: 6301.8). The base sequence of this compound is complementary to nucleotides 5103-5120 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession no. NG_009800.1). Synthesis of TcTguTACCTTgacCTCT (MSTN_Ex3_7-2b) (Example 56) The compound of Example 56, 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: 6391.3, measured value: 6398.1). The base sequence of this compound is complementary to nucleotides 5103-5120 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1). Synthesis of TcTguuACcTugaccTcT (MSTN_Ex3_7-2c) (Example 57) The compound of Example 57, 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: 6261.1, measured value: 6262.4). The base sequence of this compound is complementary to nucleotides 5103-5120 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession no. NG_009800.1).Synthesis of TcTguuaCcTugaccTcT (MSTN_Ex3_7-2d) (Example 58) The compound of Example 58, 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: 6249.1, measured value: 6249.7). The base sequence of this compound is complementary to nucleotides 5103-5120 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1). Synthesis of TcTguuAccTugaccTcT (MSTN_Ex3_7-2e) (Example 59) The compound of Example 59, 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: 6235.0, measured value: 6237.5). The base sequence of this compound is complementary to nucleotides 5103-5120 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1). Synthesis of TcTguTaCcTugaccTcT (MSTN_Ex3_7-2f) (Example 60) The compound of Example 60, 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: 6275.1, measured value: 6275.4). The base sequence of this compound is complementary to nucleotides 5103-5120 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession number NG_009800.1). Synthesis of TcTguTaCcTugacCucT (MSTN_Ex3_7-2g) (Example 61) The compound of Example 61, 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: 6275.1, measured value: 6275.0).The base sequence of this compound is complementary to nucleotides 5103-5120 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession number NG_009800.1). Synthesis of TcTguuAcCuTgaccTcT (MSTN_Ex3_7-2h) (Example 62) The compound of Example 62, 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: 6261.1, measured value: 6262.4). The base sequence of this compound is complementary to nucleotides 5103-5120 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession number NG_009800.1). Synthesis of TcTguuAcCuTgacCucT (MSTN_Ex3_7-2i) (Example 63) The compound of Example 63, 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: 6261.1, measured value: 6263.0). The base sequence of this compound is complementary to nucleotides 5103-5120 of Homo sapiens myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank accession No. NG_009800.1).

[0219] (Table 2)

[0220] [Test Example] Experimental Method Evaluation of MSTN mRNA Changes in the splicing pattern of MSTN pre-mRNA after ASO introduction were evaluated by RT-PCR in human rhabdomyosarcoma cells (CRL-2061, ATCC).

[0221] Cell culture: Human rhabdomyosarcoma cells (CRL-2061, ATCC) were cultured in RPMI 1640 medium (#30263-95, Nacalai Tesque) supplemented with 10% FBS (10270-106, Gibco). Human myoblasts (Wada et al. Development 2002, 129;2987-2995) were cultured in DMEM medium (08458-16, Nacalai Tesque) supplemented with 20% FBS (10270-106, Gibco) and 2% Ultroser G (15950-017, Pall Life Sciences).

[0222] ASO transfection: 1) 100 μl of Opti-MEM medium (#31985070, Thermo Fisher Scientific) was mixed with 2 μl of each ASO (prepared at 50 pmol / μl in Nuclease-Free Water (#AM9932, Thermo Fisher Scientific)). For non-ASO treatment, 2 μl of Nuclease-Free Water was added. 2) In a separate tube, 100 μl of Opti-MEM medium was mixed with 4 μl of Lipofectamine 3000 Transfection Reagent (#L3000015, Thermo Fisher Scientific). 3) Solutions 1 and 2 were mixed and left at room temperature for 15 minutes. 4) Human rhabdomyosarcoma cells (CRL-2061, ATCC) cultured in a 12-well plate were washed once with PBS, and 800 μl of Opti-MEM medium was added to each well. 5) Solution 3) was added to solution 4) (final ASO concentration: 100 nM), and the cells were cultured at 37°C under 5% CO2 for 3 hours. The medium was then replaced with RPMI 1640 medium containing 10% FBS, and the cells were further cultured.

[0223] RNA Preparation: 1) Cells transfected with each ASO were cultured for 24 hours, then washed once with PBS. 300 μl of RNA extraction reagent from the High Pure RNA Isolation Kit (#11828665001, Roche Life Science) was added to the cells. 2) After leaving the cells at room temperature for 10 minutes, the RNA extraction reagent in the wells was collected into a tube. 3) RNA was extracted according to the High Pure RNA Isolation Kit protocol, and 50 μl of RNA solution was finally obtained.

[0224] Reverse transcription reaction: 1) Random primers (#48190011, Thermo Fisher Scientific) and dNTP mixture (2.5 mM each) (#4030, Takara) were added to 500 ng of RNA and incubated at 65°C for 5 minutes and at 25°C for 10 minutes. 2) M-MLV Reverse Transcriptase (#28025013, Thermo Fisher Scientific), RNaseOUT Recombinant Ribonuclease Inhibitor (#10777-019, Thermo Fisher Scientific), DTT (provided with M-MLV), and 5x First Strand Buffer (provided with M-MLV) were added to solution 1), and the mixture was incubated at 37°C for 55 minutes and at 70°C for 10 minutes to obtain cDNA.

[0225] PCR Reaction and Product Verification: 1) To 2 μl of the resulting cDNA, 1 μl of primer MSTN_Ex1F1 (5'-agattcactggtgtggcaag-3': SEQ ID NO: 57), 1 μl of primer MSTN_R2 (5'-tgcatgacatgtctttgtgc-3': SEQ ID NO: 58), 0.1 μl of Takara Ex Taq DNA Polymerase (#RR001A, Takara), 1.6 μl of dNTP Mixture (2.5 mM each), 2 μl of 10x Ex Taq Buffer, and 12.3 μl of nuclease-free water were added. 2) The mixture was heated to 94°C for 3 minutes. 3) 30 cycles of 94°C for 0.5 minutes, 60°C for 0.5 minutes, and 72°C for 1.5 minutes were performed. 4) The mixture was heated to 72°C for 3 minutes. 5) The PCR reaction products were electrophoresed on a 2% agarose gel (#318-01195, Nippon Gene) and quantified using Image J (NIH, http: / / imagej.nih.gov / ij / ). The PCR reaction products were also electrophoresed and quantified using an Agilent 2100 Bioanalyzer Electrophoresis System (Agilent Technologies, Inc.). 6) For GAPDH, steps 1) to 5) above were performed using primers GAPDH H_F (5'-cccttcattgacctcaac-3': SEQ ID NO: 59) and GAPDH H_R (5'-ttcacacccatgacgaac-3': SEQ ID NO: 60). Step 3) was performed for 18 cycles.

[0226] Myostatin protein expression after ASO administration was confirmed by Western blotting. ASOs were transfected into human rhabdomyosarcoma cells (CRL-2061, ATCC) using Lipofectamine 3000 Transfection Reagent (#L3000015, Thermo Fisher Scientific). 24 hours after ASO administration, cells were lysed using Cell Lysis Buffer (#9803, Cell Signaling) supplemented with 1 mM PMSF (#8553, Cell Signaling), and the soluble fraction was isolated. Protein quantification of the resulting sample was performed using the Qubit Protein Assay Kit (#Q33211, Thermo Fisher Scientific). Samples for SDS-PAGE were prepared by mixing the sample with 4x Laemmli Sample Buffer (#1610747, Bio-Rad) and then heat-treating. SDS-PAGE was performed using Mini-PROTEAN TGX Precast Gels 4-20% (#456-1094, BIO-RAD). Protein Ladder One Plus, Triple-color for SDS-PAGE (#19593-25, Nacalai Tesque) was used as a molecular weight marker. Proteins were transferred to the membrane using the Trans-Blot Turbo Transfer System (Bio-Rad). After protein transfer, the membrane was blocked with 2% ECL Prime Blocking Agent (#RPN418, Amersham) for 1 hour at room temperature and then treated overnight at 4°C with either an antibody recognizing the N-terminus of myostatin (Anti-GDF8 / Myostatin antibody, #ab71808, Abcam) or an antibody recognizing actin (β-Actin antibody (C4), #sc-47778, Santa Cruz Biotechnology).The secondary antibodies were HRP-conjugated anti-rabbit IgG (#NA934, GE) and HRP-conjugated anti-mouse IgG (#NA931, GE), and the sections were incubated at room temperature for 1 hour. Detection was performed using Amersham ECL Select Western Blotting Detection Reagent (#RPN2235, GE) on a ChemiDoc XRS+ system (Bio-Rad).

[0227] Analysis of myostatin signaling. Inhibition of myostatin signaling by ASO administration was evaluated using an in vitro myostatin transcription activity assay. In this assay, a reporter gene (SBE4-Luc plasmid, #16495, Addgene) containing a luciferase gene downstream of an Smad binding site was transfected into cells, and myostatin signaling was assessed by measuring the luminescence of induced luciferase. A β-galactosidase expression vector (pSV-β-Galactosidase Control Vector, #E1081, Promega) was also transfected as a control for luciferase reporter gene transfection.

[0228] ASOs were transfected into human rhabdomyosarcoma cells (CRL-2061, ATCC) using Lipofectamine 3000 Transfection Reagent (#L3000015, Thermo Fisher Scientific). Twenty-four hours after ASO administration, cells were lysed using Reporter Lysis Buffer from the Luciferase Assay System with Reporter Lysis Buffer (E4030, Promega), and the soluble fraction was isolated. Protein quantification of the resulting sample was performed using the Qubit Protein Assay Kit (#Q33211, Thermo Fisher Scientific). Luciferase activity was assessed using the Luciferase Assay System with Reporter Lysis Buffer (#E4030, Promega) as a substrate, measuring the luciferase luminescence signal using an ARVO X3 multilabel plate reader (PerkinElmer). β-Galactosidase activity was assessed by incubation at 37°C for 1 hour using the β-Gal Assay System with Reporter Lysis Buffer (E2000, Promega) as the substrate, followed by the addition of 1 M sodium carbonate to terminate the reaction. After incubation at room temperature for 15 minutes, the plate was assayed by measuring absorbance at 420 nm using an ARVO X3 multilabel plate reader (PerkinElmer). Luciferase activity was normalized by β-galactosidase activity (luciferase activity / β-galactosidase activity), and the value measured in the extract from cells not treated with ASO was set to 1, and the relative values ​​were expressed.

[0229] Cell proliferation assay Cell proliferation was assessed using human myoblasts (Wada et al. Development 2002,129;2987-2995) and human rhabdomyosarcoma cells (CRL-2061, ATCC) using Cell Counting Kit-8 (CCK-8) (347-07621, Dojindo Laboratories).

[0230] Experimental Results: To switch the splicing of MSTN exon 3 from MSTN to MSTN-b, we constructed 54 18-nt ASOs complementary to exon 3 and intron 2 of MSTN pre-mRNA (Fig. 2). Each ASO was designed based on predicted binding of splicing factors to MSTN pre-mRNA. Human rhabdomyosarcoma cells (CRL-2061) were treated with each ASO for 24 hours, and MSTN mRNA was analyzed by RT-PCR (Figs. 3–13). Treatment with MSTN_Ex3_7, MSTN_Ex3_7-1, MSTN_Ex3_7-2, MSTN_Ex3_7-3, MSTN_Ex3_7-4, MSTN_Ex3_7-5, and MSTN_Ex3_7-6 significantly increased the proportion of MSTN-b in total MSTN (Fig. 8). The results of these ASOs indicate that the sequence important for the splicing switch is 5'-TTAGAGGTCAAGGTAACAGACACA-3' (SEQ ID NO: 2).

[0231] To examine the expression of myostatin-b, human rhabdomyosarcoma cells (CRL-2061) were treated with MSTN_Ex3_7-2 ASO for 24 hours and examined by Western blotting. The expression of myostatin-b was confirmed by ASO treatment.

[0232] Myostatin signaling was examined by treating human rhabdomyosarcoma cells (CRL-2061) with MSTN_Ex3_7-2 ASO for 24 hours and measuring myostatin transcription activity in vitro. ASO treatment resulted in a decrease in specific luciferase activity (Figure 15). Luciferase activity in this experimental system correlated with myostatin signaling, with increased luciferase activity indicating enhanced myostatin signaling and decreased luciferase activity indicating suppression of myostatin signaling. These findings demonstrate that ASO treatment inhibits myostatin signaling.

[0233] MSTN_Ex3_7-2b, which has a different ENA configuration from MSTN_Ex3_7-2, showed a stronger effect on MSTN-b production than MSTN_Ex3_7-2 (Figure 17). This effect was concentration-dependent (Figure 18) and also caused a decrease in myostatin signaling (Figure 19). Furthermore, MSTN_Ex3_7-2b promoted the growth of human myoblasts (Figure 20) and inhibited the growth of human rhabdomyosarcoma cells (Figure 21).

[0234] Discussion We previously showed that expression of myostatin-b, a myostatin isoform, attenuates myostatin signaling in cultured cells (WO2020 / 179571), suggesting that myostatin-b inhibits myostatin and promotes muscle formation.

[0235] The ASO of the present invention can switch splicing from MSTN to MSTN-b, and we have identified the target sequence of the ASO that is important for this splicing switch. Analysis of the splicing factors that bind to this sequence is expected to reveal the factors that control the splicing switch.

[0236] Administration of the ASO (MSTN_Ex3_7-2) of the present invention, which induces the expression of MSTN-b, resulted in a decrease in myostatin protein levels and an increase in myostatin-b. Furthermore, a decrease in myostatin signaling was observed. The decrease in myostatin signaling is thought to be due to a decrease in myostatin levels and myostatin inhibition by myostatin-b.

[0237] MSTN_Ex3_7-2b, which has a different ENA configuration than MSTN_Ex3_7-2, showed a stronger effect on MSTN-b production than MSTN_Ex3_7-2. The promotion of human myoblast proliferation shown by MSTN_Ex3_7-2b administration leads to the promotion of myogenesis. Furthermore, the results of the inhibition of human rhabdomyosarcoma cell proliferation by MSTN_Ex3_7-2b administration suggest that MSTN_Ex3_7-2b may also be used as an anticancer agent.

[0238] The greatest advantage of the ASO of the present invention is its dual-action mechanism, which reduces myostatin and increases myostatin-b by switching endogenous MSTN splicing to one that produces MSTN-b (Figure 1). In addition, the production of myostatin-b by ASO administration utilizes the myostatin-b produced by the individual, making it non-immunogenic and safe. At the same time, it utilizes the function of the patient's own gene, eliminating the need to introduce a large gene from outside the body.

[0239] Furthermore, by using ENA, a modified nucleic acid whose transfer to skeletal muscle has been confirmed, as a synthetic monomer for ASO, the barrier to muscle delivery is eliminated. The development of ASO using ENA has a proven track record, and Daiichi Sankyo is currently conducting Phase I / II clinical trials of DS5141b as a treatment for Duchenne muscular dystrophy (DMD). This was conducted after preclinical trials, and the ASO created this time uses the same monomer, so it is expected to be used clinically without any problems.

[0240] The target sequence of the ASO of the present invention is highly conserved in cattle and pigs, making it possible to use it to promote the growth of these livestock (Figure 16). Suppression of MSTN expression by administering ASO to livestock is easy to use because it does not involve the creation of genetically modified organisms. It is also believed to be similarly effective in dogs, and could be used to treat muscle wasting in pet dogs (Figure 16). All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

[0241] The present invention can be used as a nucleic acid drug that can express a splicing variant of myostatin.

[0242] <SEQ ID NO: 1> This shows the base sequence of exon 3 of the myostatin gene. MSTN Ex3 1939 nt (NM_005259.3) The ASO target sequence is shown in square. <Sequence number 2> Shows the base sequence of the ASO target sequence of exon 3 of the myostatin gene. 5'-TTAGAGGTCAAGGTAACAGACACA-3' <Sequence numbers 3 to 56> Shows the sequences of the ASO synthesized in Examples 1 to 54. <Sequence numbers 57 to 60> Shows the sequences of the primers used in the test examples. <Sequence numbers 61 to 67> Shows the sequences of the ASO synthesized in Examples 55 to 63. <Sequence number 68> Shows the base sequence of MSTN-b (1,860 bases in total. The start codon (atg) and stop codon (tga) are shown in square). <SEQ ID NO: 69> This shows the amino acid sequence of MSTN-b protein (total 251 amino acids). <SEQ ID NO: 70> Shows the amino acid sequence of MSTN protein (total 375 amino acids). 1 MQKLQLCVYIYLFMLIVAGPVDLNENSEQKENVEKEGLCNACTWRQNTKSSRIEAIKIQI 60 LSKLRLETAPNISKDVIRQLLPKAPPLRELIDQYDVQRDDSSDGSLEDDDYHATTETIIT 120 MPTESDFLMQVDGKPKCCFFKFSSKIQYNKVVKAQLWIYLRPVETPTTVFVQILRLIKPM 180 KDGTRYTGIRSLKLDMNPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHDLAVT 240 FPGPGEDGLNPFLEVKVTDTPKRSRRDFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIA 300 PKRYKANYCSGECEFVFLQKYPHTHLVHQANPRGSAGPCCTPTKMSPINMLYFNGKEQII 360 YGKIPAMVVDRCGCS 375

Claims

1. An antisense oligonucleotide having a base length of 15 to 30 bases and having a base sequence complementary to a target sequence of exon 3 of the myostatin gene, comprising any of the base sequences of SEQ ID NOs: 24, 30, 31, 27, 32, 33 or 28 (wherein t in the sequence may be u, and u may be t), and capable of expressing a splicing variant of myostatin, a salt or solvate thereof.

2. The antisense oligonucleotide, salt or solvate thereof described in claim 1, wherein the base sequence of exon 3 of the myostatin gene is the base sequence of SEQ ID NO: 1, the target sequence of exon 3 of the myostatin gene is the region of base numbers 10 to 33 of the base sequence of SEQ ID NO: 1, and the base sequence is the base sequence of SEQ ID NO:

2.

3. The antisense oligonucleotide, its salt or solvate according to claim 1 or 2, wherein the base sequence of the antisense oligonucleotide comprises the base sequence of SEQ ID NO: 31 (wherein t in the sequence may be u, and u may be t).

4. The antisense oligonucleotide, its salt or solvate described in claim 3, wherein the base sequence of the antisense oligonucleotide comprises any of the base sequences of SEQ ID NOs: 61 to 67.

5. The antisense oligonucleotide, its salt or solvate according to any one of claims 1 to 4, wherein the antisense oligonucleotide has a base length of 18.

6. The antisense oligonucleotide, its salt or solvate according to claim 5, wherein the base sequence of the antisense oligonucleotide is any one of the base sequences of SEQ ID NOs: 24, 30, 31, 27, 32, 33 or 28 (wherein t in the sequence may be u, and u may be t).

7. The antisense oligonucleotide, its salt or solvate described in claim 6, wherein the base sequence of the antisense oligonucleotide is any of the base sequences of SEQ ID NOs: 61 to 67.

8. The antisense oligonucleotide, salt or solvate thereof according to any one of claims 1 to 7, wherein at least one nucleotide is modified.

9. The antisense oligonucleotide, its salt or solvate according to claim 8, wherein the sugar constituting the modified nucleotide is D-ribofuranose, and the hydroxyl group at the 2'-position of D-ribofuranose is modified.

10. The antisense oligonucleotide, its salt or solvate according to claim 9, wherein D-ribofuranose is 2'-O-alkylated and / or 2'-O,4'-C-alkylenated.

11. A pharmaceutical comprising the antisense oligonucleotide according to claim 3 or a pharma- ceutically acceptable salt or solvate thereof.

12. The pharmaceutical composition according to claim 11, for preventing and / or treating pathologies and / or diseases in which myostatin is involved.

13. The pharmaceutical composition according to claim 12, wherein the pathology and / or disease in which myostatin is involved is muscle atrophy.

14. The pharmaceutical composition according to claim 13, 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.

15. The pharmaceutical composition according to claim 12, wherein the pathological condition and / or disease in which myostatin is involved is a pathological condition and / or disease in which the therapeutic effect is brought about by muscle mass recovery.

16. The pharmaceutical composition according to claim 15, wherein the pathological condition and / or disease for 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, renal disease and bone disease.

17. The pharmaceutical composition according to claim 16, wherein the cardiovascular disease is heart failure and / or arteriosclerosis, the renal disease is chronic renal failure, and the bone disease is inflammatory arthritis.

18. A food product comprising the antisense oligonucleotide according to claim 3 or a food-acceptable salt or solvate thereof.

19. A feed comprising the antisense oligonucleotide according to claim 3 or a feed-acceptable salt or solvate thereof.

20. A drug for promoting proliferation and / or hypertrophy of muscle cells, comprising the antisense oligonucleotide, salt or solvate thereof according to claim 3.

21. A drug for increasing muscle mass and / or suppressing muscle loss, comprising the antisense oligonucleotide, salt or solvate thereof according to claim 3.

22. A drug that switches myostatin splicing from myostatin to a myostatin splicing variant, comprising the antisense oligonucleotide, salt or solvate thereof according to any one of claims 1 to 10.

23. A drug that reduces myostatin signaling, comprising the antisense oligonucleotide, its salt, or solvate according to any one of claims 1 to 10.

24. An anticancer agent comprising the antisense oligonucleotide according to claim 3, or a salt or solvate thereof.