Antisense oligonucleotide targeting hic-5

Abstract

The present invention relates to an antisense oligonucleotide targeting a base sequence selected from SEQ ID NOs: 157-312.

Description

Antisense oligonucleotides targeting Hic-5

[0001] The present disclosure relates to antisense oligonucleotides that target Hic-5, methods for treating diseases using the same, and the like.

[0002] Hydrogen peroxide-inducible clone-5 (Hic-5) is a component of focal adhesions belonging to the paxillin family. Focal adhesions are cellular mechanisms that connect the extracellular matrix (ECM) with the intracellular actin skeleton. These cellular mechanisms are composed of integrins and multiple focal adhesion proteins, including Hic-5. Focal adhesions play important roles in cell adhesion, cell motility, morphological changes, and the transmission of stimuli from the cell's surrounding environment. Studies using Hic-5 knockout mice and other methods have suggested that Hic-5 may be involved in various diseases (Non-Patent Documents 1-7). Furthermore, attempts have been made to treat various diseases by inhibiting Hic-5 (Patent Document 1).

[0003] WO2021 / 149329

[0004] Kim-Kaneyama et al., J Atheroscler Thromb. 2012;19(7):601-7Omoto et al., Oncogene. 2018;37(9):1205-1219Petropoulos et al., J Cell Biol. 2016;213(5):585-99Lei et al., J Hepatol. 2016;64(1):110-7Jamba et al., PLoS One. 2015;10(4):e0122773Arita-Okubo et al., Cardiovasc Res. 2015;105(3):361-71Yund et al., J Mol Cell Cardiol. 2009;47(4):520-7

[0005] Additional therapeutic agents that target Hic-5 are needed.

[0006] Some aspects of the present disclosure relate to the following: [1] An antisense oligonucleotide targeting a nucleotide sequence selected from SEQ ID NOs: 157 to 312. [2] The antisense oligonucleotide according to [1], comprising a nucleotide sequence selected from SEQ ID NOs: 1 to 156. [3] The antisense oligonucleotide according to [1] or [2], having modifications in the 5'-terminal and 3'-terminal nucleotides. [4] The antisense oligonucleotide according to any one of [1] to [3], consisting of a sequence set forth in SEQ ID NOs: 313 to 485. [5] A composition comprising the antisense oligonucleotide according to any one of [1] to [4]. [6] The antisense oligonucleotide according to any one of [1] to [4] or the composition according to [5], for treating a disease involving Hic-5. [7] The antisense oligonucleotide or composition according to [6], wherein the disease involving Hic-5 is selected from vascular disease, fibrotic disease, cardiac hypertrophy, osteoarthritis, and tumor. [8] A composition for treating sclerosing cholangitis, comprising a Hic-5 inhibitor.

[0007] The present disclosure provides antisense oligonucleotides against Hic-5 that have excellent properties.

[0008] Figure 1 shows the dose-response curves of each ASO in KMST-6 cells. Figure 2 shows the dose-response curves of each ASO in JTC-19 cells. Figure 3 shows the dose-response curves of each ASO in human HSCs. Figure 4 shows the Hic-5 mRNA expression level (left) and the Hic-5 mRNA retention rate (right) in each group (mean ± SEM). **P < 0.01 (one-way ANOVA followed by Dunnett's multiple comparison test). Figure 5 shows the Hic-5 protein expression level in each group (mean ± SEM). *P < 0.05, **P < 0.01 (one-way ANOVA followed by Dunnett's multiple comparison test). Figure 6 shows the results of biochemical tests (mean ± SEM). Figure 7 shows representative photographs of liver tissue in each experimental group. Figure 8 shows the fibrotic area (MT-positive area) in each group (mean ± SEM). *P<0.05, **P<0.01 (one-way ANOVA followed by Dunnett's multiple comparison test). Figure 9 shows the liver weight / body weight ratio (A), plasma AST (B), plasma ALT (C), plasma total bilirubin (D), plasma direct bilirubin (E), and Hic-5 mRNA expression levels (F) for each group (mean ± SEM). *P<0.05, **P<0.01 (unpaired two-tailed t-test). Figure 10 shows representative HE-stained, SR-stained, ORO (Oil Red)-stained, and Hic-5 immunostained liver tissues for each group. The scale bar indicates 200 μm. Figure 11 shows the degree of liver fibrosis, steatosis, and mRNA expression levels of Hic-5, Col1a1, Col1a2, Lox, Loxl1, and Loxl2 (mean ± SEM). *P<0.05, **P<0.01 (Dunnett's multiple comparison test after one-way ANOVA). Figure 12 is a graph showing the OARSI score results. Figure 13 is a photograph showing representative images of Safranin-O staining from each group.

[0009] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. All patents, applications, and other publications (including online information) referenced herein are incorporated herein by reference in their entirety. This specification also incorporates the contents of the specification and drawings of Japanese Patent Application No. 2023-104574, filed on June 26, 2023, from which this application claims priority.

[0010] In one aspect, the present disclosure relates to an antisense oligonucleotide (ASO) targeting Hic-5 (hereinafter, sometimes referred to as the "ASO of the present disclosure"). As is well known in the art, ASOs are oligonucleotides that hybridize in a sequence-specific manner to RNA molecules (e.g., mRNA (mature mRNA), pre-mRNA, ncRNA, miRNA, etc., associated with a target gene) and affect the function of the target gene. Examples of effects on the function of a target gene include suppression of target gene expression by cleavage of the RNA molecule via RNase H.

[0011] The ASO of the present disclosure targets a specific base sequence in Hic-5 mRNA or pre-mRNA. Therefore, the ASO of the present disclosure can hybridize to that base sequence and affect the function of the Hic-5 gene. Furthermore, the ASO of the present disclosure typically contains a base sequence that is complementary to that base sequence. The degree of complementarity may be, for example, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% of the entire length of the target base sequence.

[0012] In some embodiments, the ASO of the present disclosure targets a nucleotide sequence selected from SEQ ID NOs: 157 to 312. In one embodiment, the ASO of the present disclosure targets a nucleotide sequence for mouse Hic-5 selected from SEQ ID NOs: 157 to 224. In another embodiment, the ASO of the present disclosure targets a nucleotide sequence for human Hic-5 selected from SEQ ID NOs: 163 to 171 and 225 to 312. In a preferred embodiment, the ASO of the present disclosure targets a nucleotide sequence selected from SEQ ID NOs: 163 to 165, 237, and 250.

[0013] In some preferred embodiments, the ASOs of the present disclosure target an ASO target sequence selected from ASO numbers H10-H29, H31, H35, H36, H38, H39, H41, H42, and H44-H53 (any of SEQ ID NOs: 234-253, 255, 259, 260, 262, 263, 265, 266, and 268-277). In other preferred embodiments, the ASOs of the present disclosure target an ASO target sequence selected from ASO numbers H11-H15, H19, H25-H31, H36, H38, and H44-H53 (any of SEQ ID NOs: 235-239, 243, 249-255, 260, 262, and 268-277). In another preferred embodiment, the ASO of the present disclosure targets an ASO target sequence selected from ASO Nos. H8, H13, H16-H18, H21, and H23-H28 (any of SEQ ID NOs: 232, 237, 240-242, 245, and 247-252). In another preferred embodiment, the ASO of the present disclosure targets an ASO target sequence selected from ASO Nos. M1-M10, M12-M20, M22, M23, M25, M26, M29, M30, M33-M48, M50-M56, and M67 (any of SEQ ID NOs: 157-166, 168-176, 178, 179, 181, 182, 185, 186, 189-204, 206-212, and 223). In another preferred embodiment, the ASO of the present disclosure targets an ASO target sequence selected from ASO Nos. M2 to M5, M7 to M16, M23, M28, M29, M33 to M35, M46, and M48 to M50 (any of SEQ ID NOs: 158-161, 163-172, 179, 184, 185, 189-191, 202, and 204-206). In another preferred embodiment, the ASO of the present disclosure targets an ASO target sequence selected from ASO Nos. H1 to H3, H8, H10-H18, H20-H29, H36, H38 to H42, and H44 to H53 (any of SEQ ID NOs: 225-227, 232, 234-242, 244-253, 260, 262-266, and 268-277).In another preferred embodiment, the ASOs of the disclosure are ASO numbers H1687, H1790, H1790_18, H1791, H1791_18, H1994_18, H1995_18, H1996_18, H2002_18, H2003_18, H2004_18, H2005_18, H2007_18, H2008_18, H2010_18, H2576_18, H2577, H2577_17, H2578_18, H26, H26_1, H26_2, H26_3, H26_4, H26_5, H26_6, H26_7, H26_8, H26_9, H26_10, H26_11, H26_12, H26_13, H26_14, H26_15, H26_16, H26_17, H26_18, H26_19, H26_20, H26_21, H26_22, H26_23, H26_24, H26_25, H26_26, H26_27, H26_28, H26_39, H26_40, H26_41, H26_42, H26_43, H26_44, H26_45, H26_46, H26_47, H26_48, H26_49, H26_51, H26_52, H26_53, H26_54, H26_55, H26_56, H26_57, H26_68, H26_69, H26_7 The ASO targets a target sequence selected from H26_3, H26A4_1, H26A4_2, H26A4_3, H26A5_1, H26A5_2, H26A5_3, H3974_1, H3974_2, H3975, H3977_18, H3978_17, H3979_18, H3980_17, H4745_18, H4801_17, and H4802_18 (any of SEQ ID NOs: 250, 278-299, 301, 305-309, 311, and 312). In another preferred embodiment, the ASO of the present disclosure targets an ASO target sequence selected from ASO numbers H1687, H2577_17, H26, H26 cho, H26 toco, H26_1, H26_2, H26_3, H26A4_1, H26A4_2, H26A4_3, H26A5_1, H26A5_2, H26A5_3, H3979_18, H3980_17, H4745_18, H4801_17, and H4802_18 (any of SEQ ID NOs: 250, 278, 294, 297, 298, 307-309, 311, and 312). In another preferred embodiment, the ASO of the present disclosure targets an ASO target sequence selected from ASO numbers H26, H3979_18_43_2, H3979_18_43_3 and H4745_18 (any of SEQ ID NOs: 250, 307 and 309). In another preferred embodiment, the ASO of the present disclosure targets an ASO target sequence selected from ASO numbers H1687, H1687_16_32_1, H1687_16_32_2, H26, H26A4_3, H26A5_3_, H3979_18, H3979_18_33_1, H3979_18_33_2, H3979_18_43_1, H3979_18_43_2, H3979_18_43_3 and H4745_18 (any of SEQ ID NOs: 250, 278, 297, 298, 307 and 309).In another preferred embodiment, the ASO of the present disclosure targets an ASO target sequence selected from ASO numbers H26, H26A4_3, H26A5_3, H3979_18, H3979_18_33_1, H3979_18_43_1, H3979_18_43_2, H3979_18_43_3 and H4745_18 (any of SEQ ID NOs: 250, 297, 298, 307 and 309).

[0014] In some embodiments, the ASO of the present disclosure comprises or consists of a nucleotide sequence selected from SEQ ID NOs: 1 to 156. In one embodiment, the ASO of the present disclosure comprises or consists of a nucleotide sequence selected from SEQ ID NOs: 1 to 68. In another embodiment, the ASO of the present disclosure comprises or consists of a nucleotide sequence selected from SEQ ID NOs: 7 to 15 and 67 to 156. In a preferred embodiment, the ASO of the present disclosure comprises or consists of a nucleotide sequence selected from SEQ ID NOs: 7 to 9, 81, and 94. The ASO of the present disclosure may also comprise or consist of a nucleotide sequence having 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to the full length of the above nucleotide sequences.

[0015] In some preferred embodiments, the ASO of the present disclosure comprises the base sequence of an ASO selected from ASO numbers H10 to H29, H31, H35, H36, H38, H39, H41, H42, and H44 to H53 (any of SEQ ID NOs: 78 to 97, 99, 103, 104, 106, 107, 109, 110, and 112 to 121). In another preferred embodiment, the ASO of the present disclosure comprises the base sequence of an ASO selected from ASO numbers H11 to H15, H19, H25 to H31, H36, H38, and H44 to H53 (any of SEQ ID NOs: 79 to 83, 87, 93 to 99, 104, 106, and 112 to 121). In another preferred embodiment, the ASO of the present disclosure comprises the base sequence of an ASO selected from ASO numbers H8, H13, H16 to H18, H21, and H23 to H28 (any of SEQ ID NOs: 76, 81, 84 to 86, 89, and 91 to 96). In another preferred embodiment, the ASO of the present disclosure comprises the base sequence of an ASO selected from ASO numbers M1 to M10, M12 to M20, M22, M23, M25, M26, M29, M30, M33 to M48, M50 to M56, and M67 (SEQ ID NOs: 1 to 10, 12 to 20, 22, 23, 25, 26, 29, 30, 33 to 48, 50 to 56, and 67). In another preferred embodiment, the ASO of the present disclosure comprises the base sequence of an ASO selected from ASO numbers M2 to M5, M7 to M16, M23, M28, M29, M33 to M35, M46, and M48 to M50 (SEQ ID NOs: 2 to 5, 7 to 16, 23, 28, 29, 33 to 35, 46, and 48 to 50). In another preferred embodiment, the ASO of the present disclosure comprises the base sequence of an ASO selected from ASO numbers H1 to H3, H8, H10 to H18, H20 to H29, H36, H38 to H42, and H44 to H53 (any of SEQ ID NOs: 69 to 71, 76, 78 to 86, 88 to 97, 104, 106 to 110, and 112 to 121).In another preferred embodiment, the ASOs of the present disclosure are ASO numbers H1687, H1790, H1790_18, H1791, H1791_18, H1994_18, H1995_18, H1996_18, H2002_18, H2003_18, H2004_18, H2005_18, H2007_18, H2008_18, H2010_18, H2576_18, H2577, H2577_17, H2578_18, H26, H26_1, H26_2 , H26_3, H26A4_1, H26A4_2, H26A4_3, H26A5_1, H26A5_2, H26A5_3, H3974_1, H3974_2, H3975, H3977_18, H3978_17, H3979_18, H3980_17, H4745_18, H4801_17, and H4802_18 (any of sequence numbers 94, 122 to 143, 145, 149 to 153, 155, and 156). In another preferred embodiment, the ASO of the present disclosure comprises the base sequence of an ASO selected from ASO numbers H1687, H2577_17, H26, H26 cho, H26 toco, H26_1, H26_2, H26_3, H26A4_1, H26A4_2, H26A4_3, H26A5_1, H26A5_2, H26A5_3, H3979_18, H3980_17, H4745_18, H4801_17 and H4802_18 (any of SEQ ID NOs: 94, 122, 138, 141, 142, 151 to 153, 155 and 156). In another preferred aspect, the ASO of the present disclosure comprises the base sequence of an ASO selected from ASO numbers H26, H3979_18_43_2, H3979_18_43_3 and H4745_18 (any of SEQ ID NOs: 94, 151 and 153). In another preferred aspect, the ASO of the present disclosure comprises the base sequence of an ASO selected from ASO numbers H1687, H1687_16_32_1, H1687_16_32_2, H26, H26A4_3, H26A5_3, H3979_18, H3979_18_33_1, H3979_18_33_2, H3979_18_43_1, H3979_18_43_2, H3979_18_43_3 and H4745_18 (any of SEQ ID NOs: 94, 122, 141, 142, 151 and 153).In another preferred aspect, the ASO of the present disclosure comprises the base sequence of an ASO selected from ASO numbers H26, H26A4_3, H26A5_3, H3979_18, H3979_18_33_1, H3979_18_43_1, H3979_18_43_2, H3979_18_43_3 and H4745_18 (any of SEQ ID NOs: 94, 141, 142, 151 and 153).

[0016] Table 1 shows the base sequences and target sequences contained in the ASO of the present disclosure.

[0017]

[0018]

[0019]

[0020] In some embodiments, the ASO of the present disclosure targets a nucleotide sequence contained in one exon of Hic-5 mRNA. In another embodiment, the ASO of the present disclosure targets a nucleotide sequence spanning two exons of Hic-5 mRNA. In yet another embodiment, the ASO of the present disclosure targets a nucleotide sequence spanning an intron and an exon of Hic-5 pre-mRNA. In some embodiments, the ASO of the present disclosure targets human Hic-5. In another embodiment, the ASO of the present disclosure targets mouse Hic-5. In a specific embodiment, the ASO of the present disclosure targets a nucleotide sequence common to human Hic-5 and mouse Hic-5. The nucleotide sequences of human Hic-5 mRNA (NM_001042454.3) and unspliced ​​transcript (ENST00000394863.8) are shown in SEQ ID NOs: 503 and 504, and the nucleotide sequences of mouse Hic-5 mRNA (NM_001289553.1) and unspliced ​​transcript (ENSMUST00000164710.8) are shown in SEQ ID NOs: 505 and 506, respectively.

[0021] The length of the ASO of the present disclosure is not particularly limited as long as it can function as an ASO, and may be, for example, 10 to 50 nucleotides, 11 to 40 nucleotides, 12 to 35 nucleotides, 13 to 30 nucleotides, or 14 to 28 nucleotides. In certain embodiments, the length of the ASO of the present disclosure may be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides.

[0022] ASOs may contain unmodified nucleotides and / or modified nucleotides. In this specification, unmodified nucleotides and modified nucleotides may be collectively referred to simply as "nucleotides." Unmodified nucleotides refer to nucleotides that constitute naturally occurring DNA and RNA, i.e., those composed of a nucleic acid base (adenine, guanine, uracil, thymine, cytosine), a sugar (ribose, deoxyribose), and a phosphate group. In unmodified nucleic acid molecules composed of unmodified nucleotides, two adjacent unmodified nucleotides are typically linked by a phosphodiester bond between the 3' position of one unmodified nucleotide and the 5' position of the other unmodified nucleotide. In one embodiment, the unmodified nucleotides are unmodified deoxyribonucleotides, and the unmodified nucleic acid molecule is composed of unmodified deoxyribonucleotides.

[0023] A modified nucleotide refers to a nucleotide that contains a chemical modification compared to an unmodified nucleotide. Modified nucleotides may be artificially synthesized or naturally occurring. Modified nucleotides include those whose nucleic acid base, sugar, backbone (internucleotide bond), 5'-end, and / or 3'-end are modified. Modified nucleotides include those whose any one of the above positions is modified, as well as those whose two or more of the above positions are modified.

[0024] Modified nucleobases include, but are not limited to, xanthine, hypoxanthine, 2-aminoadenine, 6-thioguanine, 6-methyl and other alkyl derivatives of adenine and guanine, universal bases, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and 5-halocytosine, 5-propynyluracil and 5-propynylcytosine, 5-bromouracil and 5-bromocytosine, 5-iodouracil, and the like. 2-thiouracil, 2-thiothymine, 5-methyl-2-thiocytosine and 2-thiocytosine, dihydrouracil, dihydrocytosine and dihydrothymine, 6-azouracil, 6-azocytosine and 6-azothymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracil cytosine and 5-substituted cytosines, 7-methylguanine, deazapurines (e.g., 7-deazaguanine, 7-deaza-8-azaguanine, 7-substituted-7-deazaguanines, 7-substituted-7-deaza-8-azaguanines and the corresponding deazaadenines), heterocyclic substituted analogs of purines and pyrimidines, such as aminoethyloxyphenoxazines, derivatives of purines and pyrimidines (e.g., 1-alkyl derivatives, 1-alkenyl derivatives, heteroaromatic derivatives and 1-methyl-2-pyrimidinyl derivatives), -alkynyl derivatives) and their tautomers, 8-oxo-N6-methyladenine, 7-diazaxanthine, 5-alkylcytosines such as 5-methylcytosine, 5-alkyluracils such as 5-ethyluracil, 3,N4-ethenocytosine, non-purine bases and non-pyrimidine bases such as 2-aminopyridine and triazine, abasic nucleotides, deoxy abasic nucleotides, inverted abasic nucleotides, inverted deoxy abasic nucleotides, and the like.

[0025] Modifications to sugars include, but are not limited to, modifications at the 2'-position, such as 2'-O-alkyl modifications (e.g., 2'-O-methyl modifications, 2'-O-ethyl modifications, 2'-O-propyl modifications, etc.), 2'-methoxyethoxy modifications, 2'-deoxy modifications, 2'-halogen modifications (e.g., 2'-fluoro modifications, 2'-chloro modifications, 2'-bromo modifications, etc.), 2'-O-allyl modifications, 2'-O-carbamoylethyl modifications, 2'-amino modifications, 2'-S-alkyl modifications, 2' -O-[2(methylamino)-2-oxoethyl] modification, 2'-propargyloxy, 2'-O-(N-methylcarbamate) modification, 2'-O-(2,4-dinitrophenyl) modification, 2'-deoxy-2'-fluoro-β-D-arabino modification, etc.; 4'-position modifications, for example, 4'-thio modification, 4'-C-hydroxymethyl modification, etc.; and others, ethynyl, ethenyl, propenyl, CF, cyano, imidazole, carboxylate, thioate, C1-C 10 Examples of modified sugars include lower alkyl, substituted lower alkyl, alkaryl or aralkyl, OCF3, OCN, O-, S-, or N-alkyl, O-, S-, or N-alkenyl, SOCH3, SO2CH3, ONO2, NO2, N3, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, and substituted silyl. Examples of modified sugars other than those mentioned above include locked nucleic acids (LNA), oxetane-LNA (OXE), unlocked nucleic acids (UNA), ethylene-bridged nucleic acids (ENA), altritol nucleic acids (ANA), hexitol nucleic acids (HNA), methylated LNA (cEt), amide-bridged nucleic acids (AmNA), guanidine-bridged nucleic acids (GuNA), tricyclic bridged nucleic acids (TriCyclo-DNA), spirocyclopropylene-bridged nucleic acids (scpBNA), and 5'-cyclopropylene-modified nucleic acids (see WO2020158910).

[0026] In this disclosure, alkyl groups include saturated aliphatic groups, including straight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.), branched-chain alkyl groups (e.g., isopropyl, tert-butyl, isobutyl, etc.), cycloalkyl (alicyclic) groups (cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl), and alkyl-substituted cycloalkyl groups. In some embodiments, straight-chain or branched-chain alkyls have 6 or fewer carbon atoms in their backbone (e.g., C1-C6 for straight chain, C3-C6 for branched chain), and more preferably 4 or fewer carbon atoms. Likewise, preferred cycloalkyls can have 3-8 carbon atoms in their ring structure, and more preferably 5 or 6 carbons in the ring structure. The term C1-C6 includes alkyl groups containing 1-6 carbon atoms. Alkyl groups can also be substituted alkyl groups, for example, alkyl moieties having substituents replacing hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents may include, for example, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl, and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfate, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

[0027] In this disclosure, alkoxy groups include substituted and unsubstituted alkyl, alkenyl, and alkynyl groups covalently linked to an oxygen atom. Examples of alkoxy groups include methoxy, ethoxy, isopropyloxy, propoxy, butoxy, and pentoxy groups. Examples of substituted alkoxy groups include halogenated alkoxy groups. Alkoxy groups may be substituted with, for example, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl, and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfate, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Examples of halogen substituted alkoxy groups include, but are not limited to, fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloromethoxy, dichloromethoxy, trichloromethoxy, etc. In this disclosure, halogen includes fluorine, bromine, chlorine, and iodine.

[0028] Modified backbones include, but are not limited to, for example, phosphorothioate, thiophosphate, phosphoroselenoate, selenophosphate, carbophosphonate, diphosphate diester, triazole, dialkyl sulfide, boronate, piperazine, guanidine, amide, urea, morpholinophosphoramidate, morpholinophosphorodiamidate, S-methylthiourea, thiophosphate-D-ribose derivative, 2'-5' linkage (also referred to as 5'-2' or 2'5' nucleotide or 2'5' ribonucleotide), PACE, PNA, 3'-(or -5')deoxy-3'-(or -5')thio-phosphorothioate, phosphorodithioate, 3'-(or -5')deoxyphosphinate, boranophosphate, 3'-(or - 5')deoxy-3'- (or 5'-)amino phosphoramidate, hydrogen phosphonate, phosphonate, boranophosphate, phosphoramidate, N-sulfonyl phosphoramidate, alkyl or aryl phosphonate and phosphotriester modifications, alkyl phosphotriester, phosphotriester phosphorus linkage, 5'-ethoxy phosphodiester, P-alkyloxy phosphotriester, methyl phosphonate, morpholino, etc., and non-phosphorus containing linkages such as carbonate, carbamate, silyl, sulfur, sulfonate, sulfonamide, formacetal, thioformacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo, and methyleneoxymethylimino.

[0029] Examples of 5'-end and / or 3'-end modifications include addition of a capping moiety to the 5'-end and / or 3'-end, and modification of the phosphate group at the 5'-end and / or 3'-end, such as [3-3']-inverted deoxyribose, deoxyribonucleotide, [5'-3']-3'-deoxyribonucleotide, [5'-3']-ribonucleotide, [5'-3']-3'-O-methylribonucleotide, 3'-glyceryl, [3'-5']-3'-deoxyribonucleotide, [3'-3']-deoxyribonucleotide, [5'-2']-deoxyribonucleotide, and [5-3']-dideoxyribonucleotide. Non-limiting examples of capping moieties include, for example, abasic nucleotides, deoxy abasic nucleotides, inverted (deoxy) abasic nucleotides, hydrocarbon (alkyl) moieties and derivatives thereof, mirror nucleotides (L-DNA or L-RNA), bridged nucleic acids including LNA and ethylene-bridged nucleic acids, linkage-modified nucleotides (e.g., PACE) and base-modified nucleotides, glyceryl, dinucleotides, acyclic nucleotides, amino, fluoro, chloro, bromo, CN, CF, methoxy, imidazole, carboxylate, thioate, C1-C 10 Examples include lower alkyl, substituted lower alkyl, alkaryl or aralkyl, OCF, OCN, O-, S- or N-alkyl, O-, S- or N-alkenyl, SOCH, SOCH, ONO, NO, N, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino or substituted silyl, polyethylene glycol, etc. The capping moiety can also function as a non-nucleotide overhang.

[0030] The ASO of the present disclosure may have a functional moiety attached thereto. Examples of such functional moieties include a cell membrane permeation promoting moiety, an expression suppression enhancing moiety, and a targeting moiety. Examples of cell membrane permeation promoting moieties include various cell membrane penetrating peptides (CPPs). Non-limiting examples of CPPs include TAT, polyarginine (R5, R8, R9, etc.), Penetratin, Pep-1, proline-rich peptide (Pro), TAT-HA2, Hph-1, HP4, LAH4, LAH4-L1, Vectofusin-1, low molecular weight protamine (LMWP), LL-37, Pep-7, Pept1, Pept2, IVV-14, Transportant, Ig(v), pVEC, HRSV, TGN, Derived Ku-70, RW(n), RRRRRRGGRRRRG (SEQ ID NO: 486), SVS-1, L-CPP, RLW, K16ApoE, Angiopep-2, ACPP, KAFAK, hCT(9-32), VP22, and the like (Vanova et al., Materials (Basel). 2019;12(17). pii: E2671, Silva et al. al., Biomolecules. 2019;9(1). pii: E22, Derakhshankhah and Jafari, Biomed Pharmacother. 2018;108:1090-1096, etc.).

[0031] Examples of expression suppression enhancing moieties include RNA molecules (Nishina et al., Nat Commun. 2015;6:7969) and DNA molecules (Asami et al., Mol Ther. 2021;29(2):838-847) complementary to ASO. Furthermore, these RNA or DNA molecules may be integrated with ASO and form a single strand as a whole (WO2019 / 022196), or may hybridize to ASO as a separate molecule.

[0032] Targeting moieties include various targeting molecules, such as GalNAc, mannose, GLP-1, neurotensin, antibodies, cholesterol, tocopherol (e.g., α-tocopherol), and derivatives thereof (e.g., eGLP1). In some embodiments, the targeting molecule may target cells expressing Hic-5, such as fibroblasts of various organs, such as cardiac fibroblasts and pulmonary fibroblasts, hepatic stellate cells, pancreatic stellate cells, myofibroblasts, CAFs, and tumor cells. Examples of such targeting molecules include cyclic RGD peptide, vitamin A, cholesterol, hyaluronic acid, mannose 6-phosphate, and anti-synaptophysin antibodies (see, e.g., Chen et al., J Pharmacol Exp Ther. 2019;370(3):695-702; Ansboro et al., Eur Cell Mater. 2012;23:310-8).

[0033] Non-limiting examples of modified nucleotides are also described, for example, in Deleavy and Damha, Chem Biol. 2012;19(8):937-54, Freier and Altmann, Nucleic Acids Res, 1997;25(22)4429-43, Wan and Seth J Med Chem 2016;59, 9645-67, Hall RNA 2023;29 423-33, Clave et al., RSC Chem Biol. 2020;2(1):94-150, Ochoa and Milam, Molecules. 2020;25(20):4659, etc.

[0034] In some embodiments, the ASOs of the present disclosure have modifications in nucleotides (preferably in their sugar moieties) at the 5'- and / or 3'-terminal portions. In preferred embodiments, the ASOs of the present disclosure have modifications in nucleotides (preferably in their sugar moieties) at the 5'- and 3'-terminal portions. In certain embodiments, the ASOs of the present disclosure have modifications in at least one, preferably two to four, of the 1st to 6th nucleotides from the 5'-terminus and / or at least one, preferably two to four, of the 1st to 6th nucleotides from the 3'-terminus. The position and number of modifications at the 5'-terminus and 3'-terminus may be the same or different. In certain embodiments, the ASOs of the present disclosure have modifications at the first and second, first and third, first and fourth, first and fifth, first and third and fourth, first to second and fourth, first and fourth and fifth, first, third and fifth, first and fifth and sixth, first and third and fifth, first to second and fourth and fifth, first to third and fifth, or first and sixth nucleotides (preferably at their sugar moieties) from the 5'-end. In certain embodiments, the ASOs of the present disclosure have modifications at the first and second, first and third, first and third, or first to second and fourth nucleotides (preferably at their sugar moieties) from the 3'-end. In preferred embodiments, the sugar moiety modifications of the 5'- and / or 3'-end nucleotides are selected from LNA, 2'-MOE, and 2'-OMe.

[0035] In certain preferred embodiments, the ASOs of the disclosure include the first and second from the 5' end and the first and second from the 3' end, the first and third from the 5' end and the first and third from the 3' end, the first and fourth from the 5' end and the first and second from the 3' end, the first and third and fourth from the 5' end and the first and second from the 3' end, the first and third and fourth from the 5' end and the first and second from the 3' end, the first and second and fourth from the 5' end and the first and second from the 3' end, the first and second and fourth from the 5' end and the first and third from the 3' end, the first and second and fourth from the 5' end and the first and third from the 3' end, the first and second and fourth from the 5' end and the first and second from the 3' end, the first and second and fourth from the 5' end and the first and third from the 3' end, the first and second and fourth from the 5' end and the first and second and fourth from the 3' end, the 1 and 3-4 positions from the 5' end and 1-3 positions from the 3' end, 1 and 4 positions from the 5' end and 1-2 and 4 positions from the 3' end, 1 and 3-4 positions from the 5' end and 1-2 and 4 positions from the 3' end, 1 and 4-5 positions from the 5' end and 1-2 positions from the 3' end, 1, 3 and 5 positions from the 5' end and 1-3 positions from the 3' end, 1 and 4-5 positions from the 5' end and 1-3 positions from the 3' end, 1 and 5-6 positions from the 5' end and 1-2 positions from the 3' end, 1 and 5 and 3' from the 5' end 1st to 3rd from the end, 1st and 3rd to 5th from the 5' end and 1st and 3rd from the 3' end, 1st and 3rd from the 5' end and 1st to 2nd and 4th from the 3' end, 1st to 2nd from the 5' end and 1st to 2nd and 4th from the 3' end, 1st and 3rd from the 5' end and 1st to 3rd from the 3' end, 1st and 4th from the 5' end and 1st to 3rd from the 3' end, 1st, 3rd and 5th from the 5' end and 1st to 2nd from the 3' end, 1st to 3rd from the 5' end and 1st to 2nd and 4th from the 3' end, 1st and 4th from the 5' end The nucleotides have modifications at the 5th and the 1st and 3rd from the 3' end, the 1st and 3rd to 4th from the 5' end and the 1st and 3rd from the 3' end, the 1st to 2nd and 4th from the 5' end and the 1st and 3rd from the 3' end, the 1st and 3rd to 5th from the 5' end and the 1st to 3rd from the 3' end, the 1st to 2nd and 4th to 5th from the 5' end and the 1st to 3rd from the 3' end, the 1st to 3rd and 5th from the 5' end and the 1st to 3rd from the 3' end, or the 1st and 5th from the 5' end and the 1st and 2nd from the 3' end (preferably in their sugar moieties).

[0036] Exemplary structures of ASOs of the present disclosure that include modified nucleotides are shown in Table 2.

[0037]

[0038]

[0039] In the table, H26_cho is H26 modified at the 5' end with TEG-cholesterol, and H26_toco is H26 modified at the 5' end with tocopherol. In each ASO, all nucleotides except for the LNA position are deoxyribonucleotides, all internucleotide bonds are phosphorothioate, and all Cs are 5-methylcytosine.

[0040] Additional structures of ASOs of the disclosure that include modified nucleotides are illustrated in Table 3. In the table, all nucleotides in each ASO are deoxyribonucleotides except for the MOE positions, all internucleotide bonds are phosphorothioate bonds, and all Cs are 5-methylcytosine.

[0041] In some embodiments, the ASO of the present disclosure is selected from the ASOs set forth in Table 2. In some preferred embodiments, the ASO of the present disclosure is selected from ASO numbers H10-H29, H31, H35, H36, H38, H39, H41, H42, and H44-H53 (SEQ ID NOs: 390-409, 411, 415, 416, 418, 419, 421, 422, and 424-433). In other preferred embodiments, the ASO of the present disclosure is selected from ASO numbers H11-H15, H19, H25-H31, H36, H38, and H44-H53 (SEQ ID NOs: 391-395, 399, 405-411, 416, 418, and 424-433). In another preferred embodiment, the ASO of the disclosure is selected from ASO Nos. H8, H13, H16-H18, H21, and H23-H28 (SEQ ID NOs: 388, 393, 396-398, 401, and 403-408). In another preferred embodiment, the ASO of the disclosure is selected from ASO Nos. M1-M10, M12-M20, M22, M23, M25, M26, M29, M30, M33-M48, M50-M56, and M67 (SEQ ID NOs: 313-322, 324-332, 334, 335, 337, 338, 341, 342, 345-360, 362-368, and 379). In another preferred embodiment, the ASO of the disclosure is selected from ASO Nos. M2-M5, M7-M16, M23, M28, M29, M33-M35, M46, and M48-M50 (SEQ ID NOs: 314-317, 319-328, 335, 340, 341, 345-347, 358, and 360-362). In another preferred embodiment, the ASO of the disclosure is selected from ASO Nos. H1-H3, H8, H10-H18, H20-H29, H36, H38-H42, and H44-H53 (SEQ ID NOs: 381-383, 388, 390-398, 400-409, 416, 418-422, and 424-433).In another preferred embodiment, the ASOs of the disclosure are selected from the group consisting of ASO numbers H1687, H1790, H1790_18, H1791, H1791_18, H1994_18, H1995_18, H1996_18, H2002_18, H2003_18, H2004_18, H2005_18, H2007_18, H2008_18, H2010_18, H2576_18, H2577, H2577_17, H2578_18, H26, H26_1, H26_2, H26_3, H26_4, H26_5, H26_6, H26_7, H26_8, H26_9, H26_10, H26_11, H26_12, H26_13, H26_14, H26_15, H26_16, H26_17, H26_18, H26_19, H26_20, H26_21, H26_22, H26_23, H26_24, H26_25, H26_26, H26_27, H26_28, H26_39, H26_40, H26_41, H26_42, H26_43, H26_44, H26_45, H26_46, H26_47, H26_48, H26_49, H26_50, H26_51, H26_52, H26_53, H26_54, H26_55, H26_56, H26_57, H26_58 3, H26A4_1, H26A4_2, H26A4_3, H26A5_1, H26A5_2, H26A5_3, H3974_1, H3974_2, H3975, H3977_18, H3978_17, H3979_18, H3980_17, H4745_18, H4801_17, and H4802_18 (SEQ ID NOs: 406, 434, 437-457, 460-466, 468, 469, 473-475, 481, 482, 484, and 485). In another preferred embodiment, the ASO of the disclosure is selected from ASO numbers H1687, H2577_17, H26, H26 cho, H26 toco, H26_1, H26_2, H26_3, H26A4_1, H26A4_2, H26A4_3, H26A5_1, H26A5_2, H26A5_3, H3979_18, H3980_17, H4745_18, H4801_17, and H4802_18 (sequence numbers 406, 434, 453, 455-465, 475, 481, 482, 484, and 485). In another preferred embodiment, the ASO of the present disclosure is selected from ASO numbers H26, H3979_18_43_2, H3979_18_43_3 and H4745_18 (sequence numbers 406, 479, 480 and 482). In another preferred embodiment, the ASO of the disclosure is selected from ASO numbers H1687, H1687_16_32_1, H1687_16_32_2, H26, H26A4_3, H26A5_3, H3979_18, H3979_18_33_1, H3979_18_33_2, H3979_18_43_1, H3979_18_43_2, H3979_18_43_3 and H4745_18 (sequence numbers 406, 434-436, 462, 465, 475-480 and 482).In another preferred embodiment, the ASO of the present disclosure is selected from ASO Nos. H26, H26A4_3, H26A5_3, H3979_18, H3979_18_33_1, H3979_18_43_1, H3979_18_43_2, H3979_18_43_3, and H4745_18 (SEQ ID NOs: 406, 462, 465, 475, 476, 478-480, and 482). In another preferred embodiment, the ASO of the present disclosure is selected from ASO Nos. M7-M9, H13, and H26 (SEQ ID NOs: 319-321, 393, and 406). In another embodiment, the ASO of the present disclosure is selected from the ASOs set forth in Table 3. In some preferred embodiments, the ASO of the present disclosure is selected from ASO Nos. H26M2, H26M3, and H26M4.

[0042] Suppression of Hic-5 gene expression can be assessed, for example, by comparing the expression levels of the Hic-5 gene or protein in cells treated with and untreated with the ASO of the present disclosure. The expression level of the Hic-5 gene can be determined by detecting the nucleic acid molecule by any known method, such as various hybridization methods, Northern blotting, Southern blotting, and various PCR methods, using a nucleic acid that specifically hybridizes to a nucleic acid molecule encoding the gene or a unique fragment thereof. Furthermore, the expression level of the protein can be determined by known protein detection methods, such as, but not limited to, antibody-based immunoprecipitation, enzyme immunoassay (EIA) (e.g., enzyme-linked immunosorbent assay (ELISA)), radioimmunoassay (RIA) (e.g., immunoradiometric assay (IRMA), radioallergosorbent test (RAST), radioimmunosorbent test (RIST)), Western blotting, immunohistochemistry, immunocytochemistry, and flow cytometry.

[0043] The ASOs of the present disclosure may be delivered or administered with any known delivery vehicle that aids, enhances, or facilitates delivery to the site of action, or may be delivered or administered directly without such a delivery vehicle, which may be a viral or non-viral vector.

[0044] Viral vectors include, but are not limited to, vectors based on adenovirus, adeno-associated virus (AAV), retrovirus, vaccinia virus, poxvirus, lentivirus, herpes virus, etc. Viral vectors may be oncolytic.

[0045] Non-viral vectors include, but are not limited to, particulate carriers such as polymer particles, lipid particles, and inorganic particles, as well as bacterial vectors. Nanoparticles with nanometer-sized particles can be used as particulate carriers. Polymer particles include, but are not limited to, those containing polymers such as cationic polymers, polyamidoamine (PAMAM), chitosan, glycol chitosan, cyclodextrin, poly(lactic-co-glycolic acid) (PLGA), poly(lactic-co-caprolactonic acid) (PLCA), poly(β-amino ester), atelocollagen, and polyethyleneimine (PEI). Lipid particles include liposomes and non-liposomal lipid particles. Liposomes are vesicles with an internal lumen surrounded by a lipid bilayer membrane, while non-liposomal lipid particles are lipid particles that do not have such a structure. Examples of inorganic particles include gold nanoparticles, quantum dots, silica nanoparticles, iron oxide nanoparticles (e.g., superparamagnetic iron oxide nanoparticles (SPIONs)), nanotubes (e.g., carbon nanotubes (CNTs)), nanodiamonds, and fullerenes. Examples of bacterial vectors include, but are not limited to, those based on Listeria monocytogenes, Bifidobacterium, and Salmonella. Non-viral vectors can be used to deliver not only nucleic acid molecules but also polypeptides such as antibodies and Cas. The particle carriers can be modified in various ways. Examples include stealth with PEG, targeting with targeting ligands, and modification with CPPs.

[0046] The ASOs of the present disclosure can be administered systemically or locally to the relevant tissue, ex vivo or in vivo, via dermal application, transdermal application, or injection (e.g., intravenous, intradermal, subcutaneous, intramuscular, arterial, or infusion injection).

[0047] The ASOs of the present disclosure can be delivered using a delivery system suitable for the purpose. Delivery systems may include, for example, aqueous and non-aqueous gels, creams, multiple emulsions, microemulsions, liposomes, ointments, aqueous and non-aqueous solutions, lotions, aerosols, hydrocarbon bases, and powders, and may contain excipients such as solubilizers, penetration enhancers (e.g., fatty acids, fatty acid esters, fatty alcohols, and amino acids), and hydrophilic polymers (e.g., polycarbophil and polyvinylpyrrolidone). In one embodiment, the pharmaceutically acceptable carrier is a liposome or a transdermal enhancer.

[0048] Delivery systems may include patches, tablets, suppositories, pessaries, gels, and creams, and may include excipients such as solubilizers and enhancers (e.g., propylene glycol, bile salts, and amino acids), and other vehicles (e.g., polyethylene glycol, fatty acid esters and derivatives, and hydrophilic polymers such as hydroxypropylmethylcellulose and hyaluronic acid). Techniques and systems useful for delivery of the ASOs of the present disclosure are described, for example, in Huang et al., Biomater Res. 2022;26(1):49.

[0049] Some aspects of the present disclosure relate to compositions containing the ASO of the present disclosure (hereinafter, sometimes referred to as the composition of the present disclosure). The composition of the present disclosure may contain, in addition to the ASO of the present disclosure, any of the carriers, diluents, delivery vehicles, delivery systems, etc. described above. The composition of the present disclosure can be used to treat diseases involving Hic-5. Thus, the composition of the present disclosure can be a pharmaceutical composition for treating diseases involving Hic-5 (hereinafter, sometimes referred to as the pharmaceutical composition of the present disclosure). The pharmaceutical composition of the present disclosure may contain one or more pharmaceutically acceptable additives (e.g., surfactants, carriers, diluents, excipients, etc.). Pharmaceutically acceptable additives are well known in the pharmaceutical field and are described, for example, in Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, PA (1990), the entire contents of which are incorporated herein by reference.

[0050] In some embodiments, the ASOs and pharmaceutical compositions of the present disclosure can be used to treat diseases involving Hic-5. Examples of diseases involving Hic-5 include vascular diseases, fibrotic diseases, NASH, cardiac hypertrophy, osteoarthritis, tumors, and the like. Examples of vascular diseases include arteriosclerosis, arteriovenous aneurysms, and thromboembolism. Examples of fibrotic diseases include liver fibrosis, cirrhosis, vocal cord scarring, vocal cord mucosal fibrosis, laryngeal fibrosis, pulmonary fibrosis, pancreatic fibrosis, myelofibrosis, myocardial infarction, myocardial fibrosis after myocardial infarction, myocardial fibrosis, endomyocardial fibrosis, splenic fibrosis, mediastinal fibrosis, tongue submucosal fibrosis, intestinal fibrosis (e.g., associated with inflammatory bowel disease), retroperitoneal fibrosis, uterine fibrosis, scleroderma, and various organ fibroses, such as mammary fibrosis, scars, and keloids. Cardiac hypertrophy includes concentric and eccentric cardiac hypertrophy. Osteoarthritis includes primary (idiopathic) and secondary osteoarthritis. Secondary osteoarthritis can be caused by conditions that alter the cartilage microenvironment, such as severe trauma, congenital joint abnormalities, metabolic disorders (e.g., hemochromatosis, Wilson's disease), infections, endocrine disorders, neuropathic diseases, and diseases that alter the normal structure and function of hyaline cartilage (e.g., rheumatoid arthritis, gout, chondrocalcinosis). Another example of a disease involving HIC-5 is sclerosing cholangitis (e.g., primary sclerosing cholangitis (PSC)). Nonalcoholic steatohepatitis (NASH) has recently been referred to as metabolic dysfunction-associated steatohepatitis (MASH).

[0051] Tumors include benign tumors and malignant tumors (cancers). Cancers include epithelial malignant tumors and non-epithelial malignant tumors. Tumors also include solid tumors. Examples of solid tumors include, but are not limited to, brain tumors, head and neck tumors, breast tumors, lung tumors, esophageal tumors, thyroid tumors, gastric tumors, small intestine tumors, colon tumors, rectal tumors, liver tumors, pancreatic tumors, gallbladder tumors, bile duct tumors, anal tumors, kidney tumors, renal pelvis and ureter tumors, bladder tumors, prostate tumors, penile tumors, testicular tumors, uterine tumors, ovarian tumors, vulvar tumors, vaginal tumors, skin tumors, fibroma, fibrosarcoma, fibrous histiocytoma, lipoma, liposarcoma, rhabdomyoma, rhabdomyosarcoma, leiomyoma, leiomyosarcoma, hemangioma, angiosarcoma, Kaposi's sarcoma, lymphangioma, lymphangiosarcoma, synovium, synovial sarcoma, chondroma, chondrosarcoma, osteoma, osteosarcoma, myeloma, lymphoma, GIST, and the like. In a particular embodiment, the tumor is a colon tumor. The tumor can be present in any part of the body, for example, the brain, head and neck, chest, limbs, lung, heart, thymus, esophagus, stomach, small intestine (duodenum, jejunum, ileum), large intestine (colon, cecum, appendix, rectum), liver, pancreas, gallbladder, anus, kidney, ureter, bladder, prostate, penis, testicle, uterus, ovary, vulva, vagina, skin, striated muscle, smooth muscle, synovium, cartilage, bone, thyroid, adrenal gland, peritoneum, mesentery, bone marrow, vasculature, lymphatic system, such as lymph nodes.

[0052] Cancers include solid cancers, including, but not limited to, brain tumors, head and neck cancer, breast cancer, lung cancer, esophageal cancer, thyroid cancer, gastric cancer, small intestine cancer, colorectal cancer (CRC), rectal cancer, liver cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, anal cancer, kidney cancer, renal pelvis and ureter cancer, bladder cancer, prostate cancer, penile cancer, testicular cancer, uterine cancer, ovarian cancer, vulvar cancer, vaginal cancer, skin cancer, fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, rhabdomyosarcoma, leiomyosarcoma, angiosarcoma, Kaposi's sarcoma, lymphangiosarcoma, synovial sarcoma, chondrosarcoma, osteosarcoma, myeloma, lymphoma, GIST, and the like. Cancer can be present in any part of the body, for example, the brain, head and neck, chest, limbs, lung, heart, thymus, esophagus, stomach, small intestine (duodenum, jejunum, ileum), large intestine (colon, cecum, appendix, rectum), liver, pancreas, gallbladder, anus, kidney, ureter, bladder, prostate, penis, testicles, uterus, ovaries, vulva, vagina, skin, striated muscle, smooth muscle, synovium, cartilage, bone, thyroid, adrenal gland, peritoneum, mesentery, bone marrow, vasculature, lymphatic system, including lymph nodes. Cancer can involve cancer-associated fibroblasts (CAFs). In some embodiments, the cancer is a primary cancer. In other embodiments, the cancer is a metastatic cancer. In certain embodiments, the cancer is colon cancer, particularly primary colon cancer. Colon cancer includes adenocarcinoma, squamous cell carcinoma, and adenosquamous carcinoma. Colorectal adenocarcinoma includes papillary adenocarcinoma, tubular adenocarcinoma, poorly differentiated adenocarcinoma, mucinous carcinoma, signet ring cell carcinoma, and medullary carcinoma.

[0053] In the present disclosure, "treatment" encompasses all types of medically acceptable prophylactic and / or therapeutic interventions aimed at curing, temporarily ameliorating, or preventing a disease. For example, "treatment" encompasses medically acceptable interventions for various purposes, including delaying or halting the progression of a disease, regressing or eliminating lesions, preventing the onset of the disease, or preventing its recurrence. Thus, the ASOs and pharmaceutical compositions can be used for the treatment and / or prevention of a disease.

[0054] The ASO or pharmaceutical composition of the present disclosure may be administered by various routes, including both oral and parenteral, such as, but not limited to, oral, buccal, intraoral, intravenous, intramuscular, subcutaneous, intradermal, topical, rectal, intraarterial, intraportal, intraventricular, transmucosal, transdermal, intranasal, intraperitoneal, intratracheal, intrapulmonary, and intrauterine, and may be formulated into a dosage form suitable for each administration route. Such dosage forms and formulation methods may be any known ones, as appropriate (see, for example, Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, PA (1990)).

[0055] For example, dosage forms suitable for oral administration include, but are not limited to, powders, granules, tablets, capsules, liquids, suspensions, emulsions, gels, syrups, etc., and dosage forms suitable for parenteral administration include injections such as solution injections, suspension injections, emulsion injections, ready-to-use injections, etc. Preparations for parenteral administration can be in the form of aqueous or non-aqueous isotonic sterile solutions or suspensions.

[0056] A further aspect of the present disclosure relates to a kit or pack for preparing the ASO or composition, or for treating a disease involving Hic-5, comprising the ASO or composition or components thereof according to the present disclosure, as well as the ASO or composition, or its necessary components, provided in the form of such a kit or pack. The components of the ASO or composition contained in such a kit or pack are as described above for the ASO or composition. In addition to the above, the kit may further include instructions regarding the preparation and use (e.g., administration method) of the ASO or composition, such as instructions for use, website information (e.g., URL, two-dimensional code) containing information on the use, or media recording information on the use, such as a flexible disk, CD, DVD, Blu-ray disk, memory card, USB memory, etc. Furthermore, such a kit or pack may contain all of the components necessary to complete the ASO or composition, but it does not necessarily have to contain all of the components. Therefore, the kit or pack may not contain reagents or solvents that are commonly available in medical settings, laboratory facilities, etc., such as sterile water, saline, glucose solution, etc.

[0057] Another aspect of the present disclosure relates to a method for treating a disease involving Hic-5, the method comprising administering an effective amount of an ASO or pharmaceutical composition according to the present disclosure to a subject in need thereof (hereinafter, this may be referred to as the "treatment method of the present disclosure"). Here, the effective amount is, for example, an amount that prevents the onset and recurrence of the disease, or cures the disease. Furthermore, the terms "disease involving Hic-5" and "treatment" in the treatment method of the present disclosure are as defined above for the ASO of the present disclosure.

[0058] The specific dose of ASO or pharmaceutical composition administered to a subject in the treatment method can be determined taking into consideration various conditions related to the subject requiring administration, such as the type of target, the purpose of the method, the type of treatment, the type of disease, the severity of symptoms, the subject's general health condition, age, body weight, sex of the subject, diet, timing and frequency of administration, concomitant medications, responsiveness to treatment, and compliance with treatment. The total daily dose of the ASO or pharmaceutical composition is not limited, and may be, for example, about 1 μg to about 1000 mg / kg body weight, about 10 μg to about 100 mg / kg body weight, or about 100 μg to about 10 mg / kg body weight in terms of the amount of ASO. Alternatively, the dose may be calculated based on the patient's surface area.

[0059] The route of administration includes various routes, including both oral and parenteral, such as oral, buccal, intraoral, intravenous, intramuscular, subcutaneous, intradermal, topical, rectal, intra-arterial, intraportal, intraventricular, transmucosal, transdermal, intranasal, intraperitoneal, intratracheal, intrapulmonary, intrathecal, and intrauterine. The frequency of administration varies depending on the properties of the formulation or composition used and the condition of the subject as described above, but may be, for example, multiple times a day (i.e., 2, 3, 4, or 5 or more times a day), once a day, every few days (i.e., every 2, 3, 4, 5, 6, or 7 days, etc.), several times a week (e.g., 2, 3, or 4 times a week, etc.), weekly, or every few weeks (i.e., every 2, 3, or 4 weeks, etc.).

[0060] In the present disclosure, the term "subject" refers to any individual organism, preferably an animal, more preferably a mammal, and even more preferably a human. A subject may be healthy (e.g., not having a specific or any disease) or may be suffering from some disease, but when treatment of a disease associated with a target nucleic acid molecule is intended, the subject typically refers to a subject suffering from or at risk of suffering from the disease.

[0061] Another aspect of the present disclosure relates to the use of an ASO of the present disclosure in the manufacture of a medicament for treating a disease involving Hic-5 (hereinafter, sometimes referred to as the "use of the present disclosure"). The terms "disease involving Hic-5" and "treatment" in the use of the present disclosure are as defined above for the ASO of the present disclosure.

[0062] Another aspect of the present disclosure relates to a composition for treating sclerosing cholangitis (hereinafter, sometimes referred to as Composition A of the present disclosure) containing a Hic-5 inhibitor. In the present disclosure, a Hic-5 inhibitor refers to a substance that inhibits the expression and / or activity of Hic-5. Substances that inhibit Hic-5 expression include, but are not limited to, substances that modify the Hic-5 gene, substances that inhibit Hic-5 gene transcription, substances that degrade Hic-5 gene transcripts, substances that inhibit Hic-5 gene translation, and substances that inhibit normal folding of the Hic-5 gene. Preferred examples of substances that inhibit Hic-5 expression include inhibitory nucleic acids and genome editing molecules, particularly inhibitory nucleic acids and genome editing molecules that target Hic-5. Substances that inhibit Hic-5 activity include, but are not limited to, substances that inhibit the interaction of Hic-5 with other molecules, such as substances that bind to Hic-5, particularly antibodies that target Hic-5 as an antigen, their antigen-binding derivatives, and Hic-5-binding antibody mimics. Another example of a substance that inhibits the activity of Hic-5 includes a dominant-negative mutant of Hic-5 (Shibanuma et al., Mol Biol Cell. 2003;14(3):1158-71). Hic-5 inhibitors in the present disclosure also include nucleic acid molecules that encode the above-mentioned substances that inhibit the expression and / or activity of Hic-5. The Hic-5 inhibitors in the present disclosure may be nucleic acid molecules or polypeptides.

[0063] Inhibitory nucleic acids refer to nucleic acids that inhibit the expression of target molecules, including RNAi molecules, microRNAs, microRNA mimics, Piwi-interacting RNAs (piRNAs), ribozymes (see, e.g., Jimenez et al., Trends Biochem Sci. 2015;40(11):648-661), ASOs, and Bonac nucleic acids (see, e.g., WO 2012 / 005368). RNAi (RNA interference) molecules refer to any molecule with RNAi activity, including, but not limited to, small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs). RNAi interference typically refers to the sequence-specific degradation of target RNAs induced by double-stranded nucleic acid molecules. siRNA typically refers to a small nucleic acid molecule that has an antisense strand complementary to the target sequence and a sense strand complementary to the antisense strand, with both strands at least partially forming a duplex. MicroRNA mimics are nucleic acid molecules that mimic the function of endogenous microRNAs and are well known in the art (e.g., van Rooij and Kauppinen, EMBO Mol Med. 2014;6(7):851-64; Chorn et al., RNA. 2012;18(10):1796-804). MicroRNA mimics may contain chemical modifications, and their nucleotide sequences may be altered relative to endogenous microRNAs. shRNAs are single-stranded RNA molecules that contain complementary antisense and sense regions and a loop region interposed between them. Pairing between the antisense and sense regions forms a duplex region, resulting in a hairpin-like three-dimensional structure.

[0064] Inhibitory nucleic acids can be prepared based on information such as the gene sequence of Hic-5 (also known as transforming growth factor beta-1-induced transcript 1 protein (TGFB1I1), androgen receptor coactivator 55 kDa protein, androgen receptor-associated protein of 55 kDa, or ARA55). For example, the gene sequences of human Hic-5 are registered under accession numbers NM_001042454 (transcript variant 1), NM_015927 (transcript variant 2), and NM_001164719 (transcript variant 3). Known gene sequences of Hic-5 from non-human animals include, for example, the following: dog: accession numbers XM_022419832, XM_005621231, XM_014114355, and XM_005621229. Cat: accession numbers XM_019820649, XM_019820647, XM_003998629, XM_006942108, and XM_019820648. Horse: accession numbers XM_023655571 and XM_023655570. Cattle: accession number BC104510. Mouse: accession number NM_001289550. Examples of the nucleotide sequences of human Hic-5 mRNA and unspliced ​​transcript are shown in SEQ ID NOs: 503 and 504, and examples of the nucleotide sequences of mouse Hic-5 mRNA and unspliced ​​transcript are shown in SEQ ID NOs: 505 and 506, respectively. Methods for designing siRNAs are described, for example, in Naito and Ui-Tei, Front Genet. 2012;3:102, and methods for designing miRNAs are described, for example, in Mickiewicz et al., Acta Biochim Pol. 2016;63(1):71-77.

[0065] Genome editing molecules are molecules or sets of molecules that can site-specifically modify genome sequences, and include, but are not limited to, those based on the CRISPR / Cas system, TALEN, ZFN, meganuclease (MN), etc. Genome editing molecules based on the CRISPR / Cas system function as a set of guide RNA and Cas (CRISPR-associated protein). Examples of Cas included in the CRISPR / Cas system include, but are not limited to, Cas9, Cas12a (Cpf1), Cas12b, Cas13, xCas9, VQR, VRER, spCas9-NG, spCas9-HF1, Cas nickase (e.g., Cas9 nickase), eSpCas9, evoCas9, HypaCas9, CjCas9, Split-Cas (e.g., Split-Cas9), dCas-BE (e.g., dCas9-BE), and the like (Broeders et al., iScience. 2019;23(1):100789). Because CRISPR / Cas13 can edit RNA, it can be used for gene silencing similar to RNAi.

[0066] In the present disclosure, antibodies include full-length antibodies and antigen-binding fragments thereof. The antibody class is not particularly limited and includes IgA, IgD, IgE, IgG, and IgM, with subclasses including IgA1, IgA2, IgG1, IgG2a, IgG2b, IgG3, and IgG4. Full-length antibodies also include IgNAR and heavy chain antibodies. Antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fcab, vNAR, and VHH (nanobody). Antibodies that react with Hic-5 as an antigen (anti-Hic-5 antibodies) are commercially available or can be obtained by well-known techniques, such as immunizing animals (e.g., mice, rats, rabbits, goats, sheep, etc.) with Hic-5 or an immunogenic fragment thereof and isolating the serum, or isolating anti-Hic-5 antibody-producing cells from the immunized animal, immortalizing them as needed, and culturing them. The antibody may be polyclonal or monoclonal, and may be a chimeric antibody, a humanized antibody, or a human antibody. The Hic-5 antigen is preferably human Hic-5, but Hic-5 from other animal species may be used as the antigen, as long as the resulting antibody can recognize human Hic-5. In another embodiment, the antibody may be an equine antibody that recognizes equine Hic-5, a canine antibody that recognizes canine Hic-5, a feline antibody that recognizes feline Hic-5, or a bovine antibody that recognizes bovine Hic-5.

[0067] Antigen-binding derivatives of antibodies include antibody derivatives that combine an antigen-binding portion of an antibody (e.g., a VH region, a VL region, an Fv region, etc.) with other portions of the antibody (e.g., a CH1 region, a CH2 region, a CH3 region, a CL region, an Fc region, etc.), such as scFv, minibody, scFv-Fc, scFv2 (diabody), scFv3 (triabody), scFv4 (tetrabody), Fv-clasp (Arimori et al., Structure. 2017;25(10):1611-1622), BIf (bispecific scFv immunofusion, Kuo et al., Protein Eng Des Sel. 2012;25(10):561-9), and bispecific antibodies. Examples of Hic-5-binding antibody mimics include monobodies (adnectins), affibodies, affimers, affitins, anticalins, atrimers, finomers, armadillo repeat proteins, Kunitz domains, knottins, avimers, DARPins, alpha bodies, O bodies, and repebodies (Simeon and Chen, Protein Cell. 2018;9(1):3-14; Yu et al., Annu Rev Anal Chem (Palo Alto Calif). 2017;10(1):293-320; Wuo and Arora, Curr Opin Chem Biol. 2018 Jun;44:16-22).

[0068] When the Hic-5 inhibitor is a polypeptide (e.g., a polypeptide-type Hic-5 inhibitor, such as an antibody, an antigen-binding derivative of an antibody, an antibody mimetic, or a dominant-negative mutant), Composition A of the present disclosure may contain a nucleic acid molecule encoding the polypeptide-type Hic-5 inhibitor instead of the polypeptide-type Hic-5 inhibitor, and such nucleic acid molecules are also included in the Hic-5 inhibitors of the present disclosure. By expressing a nucleic acid molecule encoding a polypeptide-type Hic-5 inhibitor in a cell, the Hic-5 inhibitor can be allowed to act intracellularly to achieve the desired effect. The use of a nucleic acid molecule encoding a polypeptide-type Hic-5 inhibitor is particularly useful when nucleic acid molecules are easier to introduce into cells than polypeptides.

[0069] Sclerosing cholangitis (SCH) is a disease characterized by sclerosing changes in the bile duct, resulting in bile duct stricture and cholestasis. Sclerosing cholangitis includes primary sclerosing cholangitis (PSC), IgG4-related sclerosing cholangitis (IgG4-SC), and secondary sclerosing cholangitis. Secondary sclerosing cholangitis can occur secondary to diseases such as cholangitis, bile duct stones, bile duct cancer, and ischemia.

[0070] Because Hic-5 is normally present intracellularly, in Composition A of the present disclosure, the Hic-5 inhibitor is preferably delivered intracellularly. The Hic-5 inhibitor can be delivered intracellularly using, but not limited to, electroporation, calcium phosphate, dextran, microinjection, iTOP (D'Astolfo et al., Cell. 2015;161(3):674-690), cell membrane-permeable peptide (CPP), viral vectors, or non-viral vectors (see, for example, Bruce and McNaughton, Cell Chem Biol. 2017;24(8):924-934; Lee et al., J Control Release. 2019;313:80-95). Viral and non-viral vectors can be used as exemplified for ASOs of the present disclosure. Furthermore, the Hic-5 inhibitor can be delivered using a delivery system such as that exemplified for ASOs of the present disclosure. The "treatment" in Composition A of the present disclosure is as described above for the ASO of the present disclosure.

[0071] Another aspect of the present disclosure relates to a method for treating sclerosing cholangitis, comprising administering a therapeutically effective amount of a Hic-5 inhibitor or a composition containing the same to a subject in need thereof (hereinafter, this may be referred to as "Treatment Method A of the present disclosure"). Here, an effective amount refers, for example, to an amount that prevents the onset and recurrence of sclerosing cholangitis or cures the disease. Subjects in this method include, but are not limited to, subjects diagnosed with sclerosing cholangitis and subjects at risk of developing sclerosing cholangitis. The type of disease, Hic-5 inhibitor, and treatment in Treatment Method A of the present disclosure are as described above for Composition A of the present disclosure.

[0072] Another embodiment of the present disclosure relates to a Hic-5 inhibitor for use in the treatment of sclerosing cholangitis. A further embodiment of the present disclosure relates to the use of a Hic-5 inhibitor in the treatment of sclerosing cholangitis. Yet another embodiment of the present disclosure relates to the use of a Hic-5 inhibitor in the manufacture of a medicament for treating sclerosing cholangitis. The type of sclerosing cholangitis, the Hic-5 inhibitor, and the treatment in these embodiments are as described above for Composition A of the present disclosure.

[0073] The specific dose of Composition A, a Hic-5 inhibitor, or a composition comprising a Hic-5 inhibitor of the present disclosure to be administered to a subject can be determined taking into consideration various conditions related to the subject requiring administration, such as the purpose of the method, the type of treatment, the type of disease, the severity of symptoms, the subject's general health condition, age, body weight, the subject's sex, diet, the timing and frequency of administration, concomitant medications, responsiveness to treatment, and compliance with treatment. The total daily dose of Composition A, a Hic-5 inhibitor, or a composition comprising a Hic-5 inhibitor of the present disclosure may be, but is not limited to, about 1 μg to about 1000 mg / kg body weight, about 10 μg to about 100 mg / kg body weight, or about 100 μg to about 10 mg / kg body weight in terms of the amount of Hic-5 inhibitor. Alternatively, the dose may be calculated based on the patient's surface area.

[0074] Administration routes for Composition A, a Hic-5 inhibitor, or a composition comprising a Hic-5 inhibitor of the present disclosure include various routes, including both oral and parenteral, such as oral, buccal, intraoral, intravenous, intramuscular, subcutaneous, intradermal, topical, rectal, intra-arterial, intraportal, intraventricular, intramyocardial, intra-articular, transmucosal, transdermal, intranasal, intraperitoneal, intratracheal, intrabronchial, intraalveolar, intrapulmonary, intrahepatic, and intrauterine. The frequency of administration varies depending on the properties of the formulation or composition used and the condition of the subject, as described above, and may be, for example, multiple times per day (i.e., 2, 3, 4, or 5 or more times per day), once per day, every few days (i.e., every 2, 3, 4, 5, 6, or 7 days, etc.), several times per week (e.g., 2, 3, or 4 times per week, etc.), weekly, or every few weeks (i.e., every 2, 3, or 4 weeks, etc.).

[0075] The present invention will be described in detail below with reference to examples, but the content of the present invention is not limited thereto. For the sake of brevity, product numbers and manufacturers of reagents and the like are listed only where they first appear.

[0076] [Example 1] Evaluation of on-target activity (1) ASOs with the structures shown in Table 2 were designed. Synthesis was outsourced to Gene Design Inc. The on-target activity of the ASOs was evaluated under the following various conditions. <Condition 1> KMST-6 cells (RIKEN BRC, RCB1955) were cultured at 1.0 × 10 4 After incubating for 24 hours, ASO (200 nM) or siRNA (20 nM) was transfected using a transfection reagent (Lipofectamine TM After 24 hours of incubation, the cells were transfected using RNAiMAX Transfection Reagent (Thermo Fisher Scientific). Proteins were extracted with a cell lysis solution containing 2% SDS and analyzed by anti-mouse Hic-5 antibody (BD Transduction Laboratories). TMThe expression level of Hic-5 was quantified by Western blotting using a transfection reagent (Cat#: 611164). The results are based on the ratio of Hic-5 expression to GAPDH expression, and are shown as a ratio with the control (transfection reagent only) set to 1 (n=1). <Condition 2> Same as condition 1, except that the ASO concentration was 100 nM. <Condition 3> Same as condition 1, except that the ASO concentration was 20 nM. <Condition 4> VSMC cells (isolated and cultured from mouse aorta according to the method described by Kim-Kaneyama et al., J Mol Cell Cardiol. 2011;50(1):77-86) were cultured at 0.7 x 10 4 After incubation for 24 hours, ASO (100 nM) or siRNA (20 nM) was transfected using a transfection reagent (Lipofectamine TM After 24 hours of incubation, transfect the cells with SuperPrep. (R) RNA was extracted using the II Cell Lysis & RT Kit for qPCR2 (Toyobo), cDNA was synthesized, and the resultant was analyzed using KOD SYBR (R) Quantify the expression level of Hic-5 by RT-PCR using qPCR Mix (Toyobo) (StepOnePlus TM , Thermo Fisher Scientific). The results are expressed as a ratio (mean ± SEM) with the control (transfection reagent only) set to 1. <Condition 5> Human HSCs (ScienCell Research Laboratories, Cat#: 5300) were transfected at 1.0 × 10 4 Same as condition 4, except that human HSCs were seeded at a density of 2.0 x 10 cells / well. 4 Condition 7: Same as condition 4, except that VSMC cells were seeded at a density of 1.5 x 10 cells / well. 4 Same as condition 4, except that human HSCs were seeded at a density of 0.3 x 10 cells / well. 4After incubation for 24 hours, ASO (5 μM) was transfected without a transfection reagent (for siRNA (20 nM), Lipofectamine was used). TM <Condition 9> Same as condition 8, except that the siRNA concentration was 5 μM and the results were expressed as a ratio (mean ± SEM) when the untreated value was set to 1.

[0077] When human-derived cells (KMST-6, human HSC) were used, the following siRNAs were used: Sense strand: 5'-GGACCAGUCUGAAGAUAAG -3' (SEQ ID NO: 487) Antisense strand: 5'-CUUAUCUUCAGACUGGUCC -3' (SEQ ID NO: 488) When mouse-derived cells were used, the following siRNAs were used: Sense strand: 5'-GGACCAGUCUGAAGAUAAG -3' (SEQ ID NO: 489) Antisense strand: 5'-CUUAUCUUCAGACUGGUCC -3' (SEQ ID NO: 490)

[0078] The primers used in RT-PCR are as follows:

[0079] The results are shown below.

[0080]

[0081]

[0082]

[0083]

[0084] [Example 2] EC 50 In a 24-well plate, 1.0 × 10 KMST-6 cells or JTC-19 cells (JCRB Cell Bank, JCRB0608) were cultured. 4 pcs / well or 2.0 x 10 4After incubation for 24 hours, ASOs (1, 3, 5, 10, 20, 40, or 100 nM) were transfected using a transfection reagent (Lipofectamine). TM After 24 hours of incubation, the expression level of Hic-5 was quantified by RT-PCR in the same manner as in Condition 4 of Example 1, and the EC 50 The results are shown in Figures 1 and 2.

[0085] In a separate experiment, 1.0 × 10 human HSCs were plated onto poly-L-lysine-coated 24-well plates. 4 After incubation for 24 hours, ASOs (1, 3, 10, 30, or 100 nM) were transfected using a transfection reagent (Lipofectamine). TM After 24 hours of incubation, the expression level of Hic-5 was quantified by RT-PCR in the same manner as in Condition 4 of Example 1, and the EC 50 The results are shown in Figure 3.

[0086] [Example 3] Toxicity Evaluation The cytotoxicity of ASO was evaluated by ATP assay and caspase 3 / 7 assay. (1) ATP Assay VSMC cells or NIH3T3 cells (JCRB Cell Bank, JCRM0615) were placed in a 48-well plate at 0.7 × 10 4 After incubating for 24 hours, ASO (100 nM) was transfected into the cells using a transfection reagent (Lipofectamine). TM The cells were transfected using RNAiMAX Transfection Reagent and then incubated for an additional 24 hours. (R)After adding 2.0 Reagent (Promega) and incubating at room temperature for 10 minutes, the luminescence intensity was measured. The results are shown in Table 10 as a ratio (mean ± SEM) of the control (transfection reagent only) set at 100 (VSMC: n = 8, NIH3T3: n = 6).

[0087]

[0088] (2) Caspase 3 / 7 Assay: VSMC cells or NIH3T3 cells were cultured in a 96-well plate at 0.6 × 10 3 After incubating for 24 hours, ASO (100 nM) was transfected into the cells using a transfection reagent (Lipofectamine). (R) The cells were transfected using Caspase-Glo 3000 Reagent (Thermo Fisher Scientific) and then incubated for an additional 24 hours. (R) After adding 3 / 7 Reagent (Promega) and incubating at room temperature for 30 minutes, the luminescence intensity was measured. The results are shown in Table 11 as a ratio (mean ± SEM) of the control (transfection reagent only) set at 100 (n = 2).

[0089] [Example 4] In vivo evaluation (1) ASO administration Mice (C57BL / 6, 9-week-old, male, Sankyo Labo Service) were subjected to the following treatments. (i) Normal group (n = 4): On day 8, saline was administered subcutaneously to the back under isoflurane anesthesia. On day 15, the mice were euthanized, and liver tissue and blood were collected. (ii) Liver fibrosis induction (CCl4) group (n = 4): CCl4 was administered intraperitoneally at a dose of 0.8 mL / kg on days 0, 2, 4, 7, 9, 11, and 14. On day 8, saline was administered subcutaneously to the back under isoflurane anesthesia. On day 15, the mice were euthanized, and liver tissue and blood were collected. (iii) ASO-treated group (n = 3): CCl4 was administered intraperitoneally at a dose of 0.8 mL / kg on days 0, 2, 4, 7, 9, 11, and 14. ASO (M7, M8, M9, H13, or H26) was administered subcutaneously to the back at a dose of 30 mg / kg under isoflurane anesthesia on day 8. Mice were euthanized on day 15, and liver tissue and blood were collected.

[0090] (2) Hic-5 mRNA expression level. RNA was extracted from a portion of the liver tissue collected from each mouse using QuickGene-810 (Fujifilm) and QuickGene RNAtissue kit SII (Kurabo). (R) cDNA was synthesized using qPCR RT Master Mix (Toyobo) and purified using KOD SYBR (R) The expression level of Hic-5 was quantified by RT-PCR using qPCR Mix (Toyobo). The results are shown in Table 12 and Figure 4. The mRNA expression level was analyzed using the △△CT method and normalized based on the expression level of the housekeeping gene GAPDH. The mRNA expression fold is the ratio of the mRNA expression level in each ASO-treated group to the mRNA expression level in the normal group, which was set to 1. The mRNA residual rate is the ratio of the mRNA expression level in each ASO-treated group to the mRNA expression level in the liver fibrosis-induced group, which was set to 100. Each value represents the mean ± SEM.

[0091] (3) Hic-5 protein expression level. A portion of the liver tissue collected from each mouse was homogenized, and proteins were extracted using a tissue lysis solution containing 0.5% SDS. TM Protein concentration was quantified using a BCA Protein Assay Kit (Thermo Fisher Scientific), and Hic-5 protein expression was quantified by Western blotting. The results are shown in Table 13 and Figure 5. The values ​​are based on the ratio of Hic-5 expression to GAPDH expression, with normal set to 1.

[0092] (4) Blood Biochemistry Test Blood samples from each mouse were subjected to biochemistry tests (TP, ALB, BUN, ALT, AST). The results are shown in Figure 6.

[0093] (5) Quantification of fibrotic area A portion of liver tissue collected from each mouse was subjected to Masson's Trichrome (MT) staining, and images of the perivenous area were obtained using a 20x objective lens (10 fields / mouse). (R) Image analysis using imaging software (Molecular Devices) was performed to calculate the percentage of MT-positive areas (fibrotic areas) in the entire field of view. The results are shown in Table 14 and Figures 7 and 8. Statistical analysis was performed using GraphPad Prism 8.0 (GraphPad Software). Although CCl4 administration induced fibrosis and liver parenchymal damage, a significant reduction in the fibrotic area was observed after a single administration of all ASOs.

[0094] [Example 5] Evaluation of on-target activity (2) ASOs with the structures shown in Table 3 were designed. Synthesis was outsourced to Gene Design Inc. The on-target activity of the ASOs was evaluated under the following conditions. H26 as shown in Table 2 was used. <Condition 10> 0.3 x 10 human HSCs (same as in Example 1) were placed in a 24-well plate. 4 After incubating for 24 hours, ASO (5 μM) was transfected into the cells using a transfection reagent (Lipofectamine). TMAfter 72 hours of incubation, the cells were transfected using RNAiMAX Transfection Reagent. (R) RNA was extracted using the II Cell Lysis & RT Kit for qPCR2 (Toyobo), cDNA was synthesized, and the resultant was analyzed using KOD SYBR (R) Quantify the expression level of Hic-5 by RT-PCR using qPCR Mix (Toyobo) (StepOnePlus TM , Thermo Fisher Scientific). The results are expressed as a ratio (mean ± SEM) with the control (transfection reagent only) set to 1. The primers used for RT-PCR were the same as in Example 1. <Condition 11> Same as Condition 10, except that VSMC cells (same as in Example 1) were used.

[0095] The results are shown below.

[0096] Example 6: In Vivo Evaluation (PSC Model) The effect of ASO on primary sclerosing cholangitis (PSC) was examined. (1) ASO Administration Mice (C57BL / 6, 9-week-old, male, Sankyo Labo Service) were treated as follows: (i) Control group (n = 7): From day 0, mice were fed ad libitum with a diet containing 0.1% DDC (3,5-diethoxycarbonyl-1,4-dihydrocollidine, Sigma-Aldrich, #137030) to create a PSC model. Saline (7.5 mL / kg) was administered subcutaneously to the back on days 3, 6, and 9. Mice were euthanized on day 10, and specimens were collected. (ii) ASO-treated group (n = 6): From day 0, mice were fed ad libitum with a diet containing 0.1% DDC to create a PSC model. ASO (H26 in Table 2) was administered subcutaneously to the back at a dose of 3 mg / kg (7.5 mL / kg) on ​​days 3, 6, and 9. The ASO was diluted to 40 mg / mL with nuclease-free water and then diluted 100-fold with saline.

[0097] (2) Liver weight to body weight ratio The body weight of each euthanized mouse and the weight of the liver collected as a sample were measured, and the liver weight to body weight ratio was calculated. The results are shown in Figure 9A. (3) Blood biochemistry tests Blood collected from each euthanized mouse was subjected to biochemistry tests (plasma AST, plasma ALT, plasma total bilirubin, plasma direct bilirubin). The results are shown in Figures 9B to 9E. (4) Hic-5 mRNA expression level The expression level of Hic-5 mRNA was quantified using the same method as in Example 4. The results are shown in Figure 9F.

[0098] Example 7: In Vivo Evaluation (MASH Model) The effect of ASO on MASH was investigated. C57BL / 6 mice (wild-type, 11 weeks old, male) were maintained on a choline-deficient diet (A06071302, Research Diet Co., Ltd.) containing 60 kcal% fat and 0.1% methionine. Starting 12 weeks after the start of feeding, ASO (H26 in Table 2) was administered subcutaneously at 30 mg / kg body weight / wk for 3 weeks. The ASO was diluted to 40 mg / mL with nuclease-free water and then diluted 10-fold with saline. The control group received the same volume of saline. After administration, the mice were euthanized and their livers were removed. To evaluate lesions at the start of administration, some mice were euthanized and their livers were removed 12 weeks after the start of feeding (starter group). Portions of the collected liver were subjected to HE staining, Sirius Red (SR) staining, Oil Red O (ORO) staining, and Hic-5 immunostaining. For Hic-5 immunostaining, anti-Hic-5 antibody (BD Biosciences, 611165, 1:100) was used as the primary antibody, and anti-mouse IgG Alexa Fluor 100 (Abcam) was used as the secondary antibody. (R) 647 (Abcam, ab150115, 1:500), respectively. The results are shown in Figure 10. The SR-stained and ORO-stained images were scanned at 40x magnification using a virtual slide scanner (NanoZoomer S60, Hamamatsu Photonics) and analyzed using QuPath. The degree of fibrosis was quantified based on the area of ​​SR-stained and the degree of steatosis was quantified based on the area of ​​ORO-stained. The results are shown in Figure 11.

[0099] RNA was extracted from a portion of the collected liver tissue using QuickGene-810 (Fujifilm) and QuickGene RNAtissue kit SII (Kurabo), and then purified using ReverTra Ace (R) cDNA was synthesized using qPCR RT Master Mix (Toyobo) and purified using KOD SYBR (R) The mRNA expression levels of Hic-5, Col1a1, Col1a2, Lox, Loxl1, and Loxl2 were quantified by RT-PCR using qPCR Mix (Toyobo). Expression levels were analyzed using the △△CT method and normalized to the expression level of the housekeeping gene GAPDH. The following primers were used. The results are shown in Figure 11.

[0100]

[0101] [Example 8] In vivo evaluation (OA model) The effect of ASO on osteoarthritis (OA) was examined. The ASO administration solution was H13 and AteloGene (R) Local Use "Quick Gelation" (Koken, hereinafter AteloGene (R) It was prepared by mixing Atelogene (R) A 180 μL aliquot of QG was placed in a 2 mL tube provided with the kit, to which 180 μL of the QG buffer provided with the kit and 40 μL of ASO solution (100 μM in nuclease-free water) were added. The mixture was stirred at 12 rpm for 10 minutes at approximately 4°C, taking care to avoid foaming, and then centrifuged at 10,000 rpm for 1 minute at approximately 4°C. The resulting supernatant was used as the ASO dosing solution and stored on ice until use. The vehicle dosing solution was prepared in the same manner as the ASO dosing solution, except that the same volume of nuclease-free water was used instead of the ASO solution.

[0102] Under general anesthesia, the medial collateral ligament and anterior cruciate ligament of the right hind limb of Wistar rats (7 weeks old, male, Japan SLC) were transected, and the meniscus was dissected from the femur and tibia and removed (day 0). After surgery, each rat was maintained under the same conditions. On days 10, 13, and 16, 40 μL of ASO or vehicle solution was administered into the right knee joint using a syringe (Myjector 29G, 0.5 mL syringe, Terumo Corporation). On day 19, rats in the ASO and vehicle control groups were euthanized, and the tibiae were collected. The collected tissues were immersed in 10% neutral buffered formalin, fixed, embedded in paraffin, and thinly sliced ​​to prepare paraffin sections. Each section was stained with Safranin-O according to the standard method, and the severity of OA was scored based on the following scale: Grade 0 to 6 (depth) and Stage 0 to 4 (extension) (Pritzker et al., Osteoarthritis Cartilage. 2006;14(1):13-29).

[0103] Grade 0: Normal articular cartilage Grade 1: Fibrillation or necrosis / proliferation in the superficial layer. Normal in the middle and deep layers. Grade 2: Focal fuzzing, necrosis / cellular proliferation, and increased / decreased matrix staining from the superficial to middle layers. Grade 3: Cracks and matrix fuzzing extend to the middle layers. Cracks branch deep, with marked necrosis / proliferation near the cracks. Grade 4: Loss of the joint matrix. Deep erosion. Grade 5: The condition extends from the calcified layer to the bone. Repair by fibrocartilage. Grade 6: Bone remodeling progresses Stage 0: No change Stage 1: <10% Stage 2: 10-25% Stage 3: 25-50% Stage 4: >50%

[0104] The above Grade and Stage numbers were multiplied to calculate an OARSI score of 0 to 24. For example, if a site was judged to be Grade 3 and Stage 2, the score would be 3 x 2 = 6. The OARSI score results are shown in Figure 12, and representative Safranin-O stained images for each group are shown in Figure 13.

[0105] Those skilled in the art will appreciate that numerous and various modifications can be made without departing from the spirit of the present invention. Accordingly, it should be understood that the forms of the present invention described herein are illustrative only and are not intended to limit the scope of the present invention.

Claims

1. An antisense oligonucleotide targeting a base sequence selected from SEQ ID NOs: 157 to 312.

2. The antisense oligonucleotide of claim 1, comprising a base sequence selected from SEQ ID NOs: 1 to 156.

3. The antisense oligonucleotide according to claim 1 or 2, having modifications in the nucleotides at the 5'-terminal and 3'-terminal portions.

4. The antisense oligonucleotide according to any one of claims 1 to 3, which consists of the sequence shown in SEQ ID NO: 313 to 485.

5. A composition comprising an antisense oligonucleotide according to any one of claims 1 to 4.

6. An antisense oligonucleotide according to any one of claims 1 to 4 or a composition according to claim 5 for treating a disease in which Hic-5 is involved.

7. The antisense oligonucleotide or composition according to claim 6, wherein the disease in which Hic-5 is involved is selected from vascular diseases, fibrotic diseases, cardiac hypertrophy, osteoarthritis and tumors.

8. A composition for the treatment of sclerosing cholangitis comprising a Hic-5 inhibitor.