Antisense compounds that regulate WFDC2 expression

Abstract

The present invention relates to an antisense compound that regulates the expression of WFDC2. The antisense compound according to the present invention that regulates the expression of WFDC2 can exhibit anticancer effects in various carcinomas.

Description

[Technical Field]

[0001] This invention relates to an antisense compound that modulates WFDC2 expression. [Background technology]

[0002] WFDC2 is a glycated protein first observed in human epididymal tissue and has been reported to be overexpressed in a variety of cancers, including ovarian cancer. The WFDC2 gene product is a stable 4-disulfide core protein family member. Research on the WFDC2 protein and the gene that encodes it has included studies such as human epididymal-specific cDNA encoding proteins with sequence homology to extracellular protease inhibitors, comparative hybridization of ovarian cDNA arrays to discover genes overexpressed in ovarian carcinomas, analysis of the molecular characteristics of epididymal proteins, and cloning and analysis of mRNA specifically expressed in the human epididymis. Such research suggests that WFDC2 overexpression indicates that the protein can be used as a biomarker for cancer, particularly ovarian cancer.

[0003] U.S. Patent Registration No. 7,811,778 relates to a method for diagnosing gastrointestinal cancer, and discloses that WFDC2 is one of the genes whose expression is significantly increased and upregulated during the conversion and differentiation of chief cells into SPEM after oxidative atrophy.

[0004] Furthermore, Korean Patent No. 10-2055305 relates to a marker for the diagnosis and targeted therapy of gastroesophageal junction adenocarcinoma, and discloses that the expression level of WFDC2, one of a variety of genes whose expression levels increase, is a gene that measures the BCCP (Bayesian Compound Covariate Predictor) score and has potential as a biomarker for diagnosing gastric or esophageal cancer.

[0005] Thus, while there are prior studies suggesting that WFDC2 is one of the many genes whose expression increases during cancer development and that it may be useful as a biomarker for ovarian cancer, gastric cancer, etc., there are currently very few studies that have confirmed the therapeutic effect of antisense compounds that suppress or inhibit its expression on cancer. [Overview of the project] [Problems that the invention aims to solve]

[0006] One aspect of the present invention provides an antisense compound comprising a modified oligonucleotide consisting of 10 to 30 sequentially linked nucleosides that bind complementarily to the nucleic acid base sequence within the transcription body of the gene encoding WFDC2 (WAP Four-Disulfide Core Domain 2).

[0007] Another aspect of the present invention provides a conjugate in which the antisense compound is covalently bonded to one or more non-nucleotide moieties.

[0008] A further aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of cancer, comprising the antisense compound or the conjugate as an active ingredient. [Means for solving the problem]

[0009] One aspect of the present invention provides an antisense compound comprising a modified oligonucleotide consisting of 10 to 30 sequentially linked nucleosides that bind complementarily to the nucleic acid base sequence within the transcription body of the gene encoding WFDC2 (WAP Four-Disulfide Core Domain 2).

[0010] In one specific example, the nucleic acid base sequence of the transcription of the gene that encodes WFDC2 may be SEQ ID NO: 1 or SEQ ID NO: 2.

[0011] In one specific example, the antisense compound may include a modified oligonucleotide consisting of 16 to 20 sequentially linked nucleosides.

[0012] In one specific example, the modified oligonucleotide may include one or more modifications selected from one or more modified nucleosides containing a linking group between one or more modified nucleosides, one or more modified nucleosides containing a modified sugar moiety, and one or more modified nucleosides containing a modified nuclear base.

[0013] In one specific example, the modified nucleoside may contain one or more modified sugar molecules selected from the group consisting of sugar molecules substituted with 2'-O-methyl, 2'-O-methoxyethyl, 2'-amino, 2'-fluoro, 2'-arabino-fluoro, 2'-O-benzyl, or 2'-O-methyl-4-pyridine.

[0014] In one specific example, the modified nucleoside may be one or more modified nucleosides selected from the group consisting of locked nucleic acid (LNA), constrained ethyl bicyclic nucleic acid (cEt), 2'-O,4'-C-ethylene-bridged nucleic acid (ENA), and tricyclo-DNA.

[0015] In one specific example, the modified nucleoside may be a modified nucleoside containing a sugar substitute having a six-membered ring or an acyclic moiety.

[0016] To give one specific example, the modified nucleosides are pseudouridine, 2'-thiouridine, N6'-methyladenosine, 5'-methylcytidine, 5'-fluoro-2-deoxyuridine, N-ethylpiperidine 7'-EAA triazol modified adenine, and N-ethylpiperidine 6'-triazol modified adenine. The modified nucleoside may also contain one or more modified nucleobases selected from the group consisting of adenine, 6'-phenylpyrrolocytosine, 2',4'-difluorotoluylribonuleoside, and 5'-nitroindole.

[0017] In one specific example, the linking group between the modified nucleosides may be one or more modified linking groups between nucleosides selected from the group consisting of phosphotriester, phosphoramidate, mesyl phosphoramidate, phosphorothioate, phosphorodithioate, methylphosphonate, and methoxypropyl-phosphonate.

[0018] In one specific example, the modified oligonucleotide comprises a gap segment consisting of linked deoxynucleosides, a 5' wing segment consisting of linked nucleosides, and a 3' wing segment consisting of linked nucleosides, wherein the gap segment is located between the 5' wing segment and the 3' wing segment, and the nucleosides of each wing segment may contain modified sugar moieties or sugar substitutes.

[0019] In one specific example, the modified oligonucleotide is a gap segment consisting of 8 to 10 linked deoxynucleosides; A 5' wing segment consisting of 3 to 5 linked nucleosides; and The molecule includes a 3'-wing segment consisting of 3 to 5 linked nucleosides, the gap segment being located between the 5'-wing segment and the 3'-wing segment, and each nucleoside in each wing segment may contain a modified sugar moiety.

[0020] According to one specific example, the antisense compound has a base sequence that is at least 70%, at least 80%, at least 90%, or completely complementary to any sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and the antisense compound has a base sequence from the start site 25 to the middle site 46, from the start site 284 to the middle site 305, from the start site 520 to the middle site 545, from the start site 2222 to the middle site 2344, from the start site 7334 to the middle site 9301, from the start site 9506 to the middle site 9551, from the start site 9733 to the middle site 10143, and from the start site 10271. The modified oligonucleotide comprises a nucleotide sequence having a nucleotide sequence containing at least eight consecutive adjacent nuclear nucleotides that are completely complementary to any oligonucleotide nucleotide sequence selected from the group consisting of middle site 10302, start site 10360 to middle site 10905, start site 10977 to middle site 11292, start site 11448 to middle site 11563, and start site 11633 to middle site 11773, wherein the modified oligonucleotide may reduce one or more of the mRNA level and / or protein level of WFDC.

[0021] In one specific example, the antisense compound is a modified oligonucleotide that binds complementaryly to SEQ ID NO: 1 or SEQ ID NO: 2, and includes SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 20, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 65, SEQ ID NO: 83, SEQ ID NO: 86, SEQ ID NO: 89, SEQ ID NO: 109, SEQ ID NO: 113, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 148, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 165, SEQ ID NO: 169, SEQ ID NO: 176, SEQ ID NO: 191, SEQ ID NO: 205, SEQ ID NO: 210, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 218, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 2 49, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252, SEQ ID NO: 253, SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 264, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 285, SEQ ID NO: 286, SEQ ID NO: 287, SEQ ID NO: 289, SEQ ID NO: 290, SEQ ID NO: 291, SEQ ID NO: 291, SEQ ID NO: 292, SEQ ID NO: 293, SEQ ID NO: 295, SEQ ID NO: 300, SEQ ID NO: 310, SEQ ID NO: 313, SEQ ID NO: 314, SEQ ID NO: 316, SEQ ID NO: 317, SEQ ID NO: 320, SEQ ID NO: 3 The modified oligonucleotide comprises a nucleotide sequence having at least eight consecutive adjacent nuclear bases that perfectly match the oligonucleotide nucleotide sequence selected from the group consisting of 30, SEQ ID NO: 331, SEQ ID NO: 336, SEQ ID NO: 343, SEQ ID NO: 376, SEQ ID NO: 377, SEQ ID NO: 378, SEQ ID NO: 379, SEQ ID NO: 380, SEQ ID NO: 381, SEQ ID NO: 382, ​​and SEQ ID NO: 383, wherein the modified oligonucleotide may reduce one or more of the mRNA and protein levels of WFDC.

[0022] According to one specific example, the antisense compound may be a modified oligonucleotide having any one base sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 20, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 65, SEQ ID NO: 83, SEQ ID NO: 86, SEQ ID NO: 89, SEQ ID NO: 109, SEQ ID NO: 113, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 148, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 165, SEQ ID NO: 169, SEQ ID NO: 176, SEQ ID NO: 191, SEQ ID NO: 205, SEQ ID NO: 210, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 218, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252, SEQ ID NO: 253, SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 264, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 285, SEQ ID NO: 286, SEQ ID NO: 287, SEQ ID NO: 289, SEQ ID NO: 290, SEQ ID NO: 2, SEQ ID NO: 292, SEQ ID NO: 293, SEQ ID NO: 295, SEQ ID NO: 300, SEQ ID NO: 310, SEQ ID NO: 313, SEQ ID NO: 314, SEQ ID NO: 316, SEQ ID NO: 317, SEQ ID NO: 320, SEQ ID NO: 330, SEQ ID NO: 331, SEQ ID NO: 336, SEQ ID NO: 343, SEQ ID NO: 376, SEQ ID NO: 377, SEQ ID NO: 378, SEQ ID NO: 379, SEQ ID NO: 380, SEQ ID NO: 381, SEQ ID NO: 382 and SEQ ID NO: 383.

[0023] Another aspect of the present invention provides a conjugate in which the antisense compound is covalently bonded to one or more non-nucleotide moieties.

[0024] According to one specific example, the non-nucleotide moiety may include a protein, a fatty acid chain, a sugar residue, a glycoprotein, a polymer, or a combination thereof.

[0025] Still another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer, which contains the antisense compound or the conjugate as an active ingredient.

[0026] According to one specific example, the cancer may be selected from the group consisting of gastric cancer, esophageal cancer, cholangiocarcinoma, ovarian cancer, cervical cancer, head and neck cancer, brain tumor, lung cancer, liver cancer, thyroid cancer, prostate cancer, bladder cancer, kidney cancer, gallbladder cancer, colorectal cancer, and pancreatic cancer.

Advantages of the Invention

[0027] The antisense compound that regulates the expression of WFDC2 according to the present invention can exhibit an anti-cancer effect in various cancer tumors.

Brief Description of the Drawings

[0028] [Figure 1] It is a graph showing the cancer growth inhibitory effect (cancer cell size) against subcutaneous administration of an antisense compound according to one specific example in a SNU638 cell line Xenograft mouse model. [Figure 2] It is a graph showing the cancer growth inhibitory effect (cancer cell size) against intravenous administration of an antisense compound according to one specific example in a SNU638 cell line Xenograft mouse model. [Figure 3] It is a graph showing the cancer growth inhibitory effect (cancer cell weight) against subcutaneous or intravenous administration of an antisense compound according to one specific example in a SNU638 cell line Xenograft mouse model. [Figure 4] It is a photograph showing the cancer growth inhibitory effect against subcutaneous or intravenous administration of an antisense compound according to one specific example in a SNU638 cell line Xenograft mouse model. [Figure 5]This graph shows the effect of intravenous administration of an antisense compound on cancer growth inhibition (cancer cell size) in a specific example in the SF268 cell line Xenograft mouse model. [Figure 6] This photograph shows a specific example of the cancer growth inhibitory effect of intravenous administration of an antisense compound in the SF268 cell line Xenograft mouse model. [Modes for carrying out the invention]

[0029] One aspect of the present invention provides an antisense compound comprising a modified oligonucleotide consisting of 10 to 30 sequentially linked nucleosides that bind complementarily to the nucleic acid base sequence within the transcription body of the gene encoding WFDC2 (WAP Four-Disulfide Core Domain 2).

[0030] WFDC2(WAP Four-Disulfide Core Domain2) The WFDC2 gene product is a family member of the WAP4-disulfide core protein. WFDC2 is the first secreted and glycated protein observed in human epididymal tissue and is known to be overexpressed in certain cancers, including ovarian cancer. Overexpression of WFDC2 in cancer cells suggests that this protein and its various isoforms could serve as biomarkers for detecting cancer or diagnosing patients at high risk of developing cancer.

[0031] Antisense compounds In this specification, “nucleotide” means a constituent molecule of a nucleic acid, which consists of a nucleobase, a sugar moiety, and a phosphate group. The term “nucleotide” can be interpreted as encompassing all non-modified or modified nucleobases, sugar moieties, and / or phosphate groups, such as nucleotide analogs, modified nucleotides, non-natural nucleotides, and non-standard nucleotides.

[0032] In this specification, "nucleoside" refers to a glycosylamine, which is considered to be the portion of a nucleotide excluding the phosphate group, and means a unit molecule composed of a nuclear base and a sugar moiety. The nucleoside can be interpreted as a concept that includes all nucleosides in which the nuclear base G / or sugar moiety is modified or unmodified, just like a nucleotide.

[0033] In this specification, "oligonucleotide" means ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), or low polymers or polymers of analogs thereof. Such oligonucleotides include not only oligonucleotides that are commonly found in living organisms and consist of covalent bonds between nuclear bases, sugars, and nucleosides (skeletons), but also modified or substituted oligonucleotides consisting of nucleotide analogs that act similarly, modified nucleotides, non-natural nucleotides, and non-standard nucleotides. Such modified or substituted oligonucleotides have properties such as enhanced cell absorption, increased nucleic acid target affinity, and increased stability compared to unmodified or substituted oligonucleotides in the presence of nucleases.

[0034] In this specification, “antisense compound” is interpreted to include oligonucleotides that can hybridize with a target nucleic acid sequence via hydrogen bonding. Antisense compounds include, but are not limited to, oligonucleotides, oligonucleotide analogs, oligonucleotide pseudos, antisense oligonucleotides, siRNA, single-stranded siRNA (sssiRNA), short hairpin RNA (shRNA), microRNA mimetic, ribozymes, external guide sequence oligonucleotides, and other oligonucleotides, and the term “antisense compound” is interpreted to include single-stranded and double-stranded oligonucleotides.

[0035] In one specific example, when the antisense compound is described in the 5' to 3' direction, it has a nucleic acid base sequence that includes the reverse complement of the target portion of the targeted nucleic acid sequence to be targeted. Preferably, the antisense compound can bind complementarily to the nucleic acid base sequence in the transcript of the gene encoding WFDC2. The transcript of the gene encoding WFDC2 is a nucleic acid targeted by the antisense compound and may be selected from mRNA and pre-mRNA including introns, exons, and untranslated regions.

[0036] In one specific example, the base sequence of the transcript of the gene encoding WFDC2 is Sequence ID No. 1 or Sequence ID No. 2, where the base sequence of Sequence ID No. 1 is the human WFDC2 genome sequence (a complement of GenBank accession number NC_000020.11 (nucleotides 45469753~45481532), a pre-mRNA sequence), and Sequence ID No. 2 is the human WFDC2 mRNA sequence (RefSeq or GenBank accession number NM_006103.4).

[0037] In one specific example, the antisense compound is at least 70%, at least 80%, at least 90%, or completely complementary to any sequence of Sequence ID No. 1 or Sequence ID No. 2, which is a nucleic acid base sequence within the transcription body of the gene encoding WFDC2, and contains at least 8 or more adjacent nuclear bases that are completely complementary to any sequence of Sequence ID No. 1 or Sequence ID No. 2, and may include a modified oligonucleotide consisting of 10 to 30, preferably 12 to 25, more preferably 14 to 23, and most preferably 16 to 20 consecutively linked nucleosides.

[0038] In one specific example, the antisense compound has a base sequence that is at least 70%, at least 80%, at least 90%, or completely complementary to any sequence of SEQ ID NO: 1 or SEQ ID NO: 2, which is a nucleic acid base sequence within the transcription body of the gene encoding WFDC2, and contains a portion of any one of the nucleic acid base sequences of SEQ ID NOs: 7 to 386, and may include a modified oligonucleotide consisting of 10 to 30 consecutively linked nucleosides.

[0039] In one specific example, the antisense compound has a base sequence that is at least 70%, at least 80%, at least 90%, or completely complementary to any sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and the antisense compound has a base sequence that is from the start site 25 to the middle site 46, from the start site 284 to the middle site 305, from the start site 520 to the middle site 545, from the start site 2222 to the middle site 2344, from the start site 7334 to the middle site 9301, and from the start site 9506 to the middle site 9551 of the base sequence of SEQ ID NO: 1. The modified oligonucleotide may include a sequence having a base sequence containing at least eight consecutive adjacent nuclear bases that are completely complementary to any oligonucleotide base sequence selected from the group consisting of start site 9733 to middle site 10143, start site 10271 to middle site 10302, start site 10360 to middle site 10905, start site 10977 to middle site 11292, start site 11448 to middle site 11563, and start site 11633 to middle site 11773.

[0040] In one specific example, the antisense compound has a base sequence that is completely complementary to any sequence in the nucleic acid base sequence of the gene encoding WFDC2, and the antisense compound may include a modified oligonucleotide consisting of 10 to 30 consecutively linked nucleosides, comprising at least 8 or more adjacent nuclear bases that are completely complementary to any sequence in any one of the nucleic acid base sequences of SEQ ID NOs. 7 to 386.

[0041] According to one specific example, the antisense compound has a base sequence that is at least 70%, at least 80%, at least 90%, or completely complementary to any sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 20, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 65, SEQ ID NO: 83, SEQ ID NO: 86, SEQ ID NO: 89, SEQ ID NO: 109, SEQ ID NO: 113, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121 , SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 148, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 165, SEQ ID NO: 169, SEQ ID NO: 176, SEQ ID NO: 191, SEQ ID NO: 205, SEQ ID NO: 210, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 218, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240 , SEQ ID NOs: 243, 244, 245, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 264, 282, 284, 285, 286, 287, 289, 290, 291, 291, 292, 293, 295, 300 It may include a modified oligonucleotide having a base sequence containing at least eight consecutive adjacent nuclear bases that perfectly matches the base sequence of any one oligonucleotide selected from the group consisting of SEQ ID NOs: 310, 313, 314, 316, 317, 320, 330, 331, 336, 343, 376, 377, 378, 379, 380, 381, 382, ​​and 383.

[0042] In one specific example, the antisense compound may include a modified oligonucleotide consisting of one of the base sequences of SEQ ID NOs: 7 to 386.

[0043] According to one specific example, the antisense compound is SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 20, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 65, SEQ ID NO: 83, SEQ ID NO: 86, SEQ ID NO: 89, SEQ ID NO: 109, SEQ ID NO: 113, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 135, Sequence ID 136, Sequence ID 140, Sequence ID 141, Sequence ID 142, Sequence ID 148, Sequence ID 154, Sequence ID 155, Sequence ID 156, Sequence ID 157, Sequence ID 165, Sequence ID 169, Sequence ID 176, Sequence ID 191, Sequence ID 205, Sequence ID 210, Sequence ID 213, Sequence ID 214, Sequence ID 215, Sequence ID 218, Sequence ID 228, Sequence ID 229, Sequence ID 237, Sequence ID 238, Sequence ID 239, Sequence ID 240, Sequence ID 243, Sequence ID No. Sequence ID 244, Sequence ID 245, Sequence ID 247, Sequence ID 248, Sequence ID 249, Sequence ID 250, Sequence ID 251, Sequence ID 252, Sequence ID 253, Sequence ID 254, Sequence ID 255, Sequence ID 256, Sequence ID 257, Sequence ID 258, Sequence ID 259, Sequence ID 264, Sequence ID 282, Sequence ID 284, Sequence ID 285, Sequence ID 286, Sequence ID 287, Sequence ID 289, Sequence ID 290, Sequence ID 291, Sequence ID 291, Sequence ID 292, Sequence ID 29 3. A modified oligonucleotide having any one base sequence selected from the group consisting of SEQ ID NOs: 295, 300, 310, 313, 314, 316, 317, 320, 330, 331, 336, 343, 376, 377, 378, 379, 380, 381, 382, ​​and 383 may also be used.

[0044] In one specific example, the length of an antisense compound that can bind complementaryally to the nucleic acid base sequence in the transcription body of the gene encoding WFDC2 may include a modified oligonucleotide consisting of 10 to 30 consecutively linked nucleosides. Preferably, the antisense compound may consist of a modified oligonucleotide consisting of 12 to 28, 15 to 25, 18 to 24, 19 to 22, or 20 consecutively linked nucleosides, or preferably, the antisense compound may consist of a modified oligonucleotide consisting of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutively linked nucleosides.

[0045] In one specific example, the antisense compound may be single-stranded or double-stranded. If the antisense compound is double-stranded, the double-stranded compound may include a first modified oligonucleotide having a region complementary to the target nucleic acid and a second modified oligonucleotide having a region complementary to the first modified oligonucleotide.

[0046] hybridization The antisense compound can achieve the desired effect on regulating WFDC2 expression by selecting one or more target sites from the nucleic acid base sequence within the transcription of the gene encoding WFDC2, selecting oligonucleotides that are sufficiently complementary to the target, and hybridizing them sufficiently specifically with the target site.

[0047] In this specification, "hybridization" refers to hydrogen bonds that may be Watson-Crick, Hoogsteen, or reverse Hoogsteen hydrogen bonds between complementary nucleosides or nucleotide bases. For example, adenine and thymine are complementary nucleic acid bases that form a hydrogen bond and pair together.

[0048] In the present invention, the meaning of “hybridizable,” “complementary,” or “substantially complementary” means that the nucleotide sequences include such that, under appropriate in vitro and / or in vivo conditions of temperature and ionic strength of solution, nucleic acids (e.g., RNA, DNA) can be non-covalently bound to other nucleic acids in a sequence-specific, antiparallel manner (i.e., nucleic acids specifically bind to complementary nucleic acids), i.e., adenine (A) and thymidine (T) pairings, adenine (A) and uracil (U) pairings, and guanine (G) and cytosine (C) pairings, or “anneal,” or “hybridize.”

[0049] In one specific example, the hybridization occurs between the antisense compound disclosed herein and the nucleic acid base sequence within the transcript of the gene encoding WFDC2. The most common mechanism of hybridization involves hydrogen bonding between complementary nucleic acid bases of the nucleic acid molecule.

[0050] Hybridization can occur under a variety of conditions. Stringent conditions are sequence-dependent and determined by the properties and composition of the nucleic acid molecules being hybridized. Methods for verifying whether a sequence can specifically hybridize with a target nucleic acid are well-known in this industry.

[0051] Complementarity In this specification, the term “complementary” refers to the property of two nucleotides being able to form an elaborate pair. For example, if the base sequences of two different nucleic acids or oligonucleotides are described in the 5' to 3' direction, the two nucleic acids or oligonucleotides are said to be complementary if, when the base sequences of certain parts of one nucleic acid or oligonucleotide are aligned in the opposite direction, they non-covalently bind to certain parts of the other nucleic acid or oligonucleotide, i.e., form adenine (A) and thymidine (T), adenine (A) and uracil (U), and guanine (G) and cytosine (C).

[0052] Therefore, "specifically hybridizable" and "complementary" can be interpreted as terms used to indicate a sufficient degree of complementarity or sophisticated pairing that enables stable, specific binding between the oligonucleotide and the DNA or RNA target. It is known in the industry that the sequence of an antisense compound does not need to be 100% complementary to the sequence of the target nucleic acid to which it is specifically hybridized.

[0053] The antisense compound is interpreted as having sufficient complementarity to prevent non-specific binding of the antisense compound to non-target sequences under preferred conditions, i.e., physiological conditions in the case of in vivo analysis or therapy, and analytical conditions in the case of in vitro analysis.

[0054] In other words, if the antisense compound can be specifically hybridized with the target nucleic acid, then non-complementary nucleic acid bases between the antisense compound and the target nucleic acid are acceptable. Moreover, the antisense compound is hybridized with one or more nucleic acid moieties such that intervening or adjacent portions do not participate in the hybridization (e.g., loop structure, mismatch, or hairpin structure).

[0055] In one specific example, the antisense compound of the present invention or a modified oligonucleotide comprising the antisense compound may be at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to the nucleic acid sequence in the transcript of the gene encoding WFDC2 (e.g., SEQ ID NO: 1 or SEQ ID NO: 2). The percentage of complementarity of the antisense compound with the target nucleic acid can be determined by conventional methods known in the art. For example, an antisense compound in which 18 of the 20 nucleic acid bases of the antisense compound are complementary to the target region is specifically hybridizable and has 90% complementarity. In the above example, the remaining non-complementary nucleic acid bases do not need to be grouped with or located within the complementary nucleic acid bases, or adjacent to each other or to the complementary nucleic acid bases. Therefore, an antisense compound with a length of 18 nucleic acid bases, having four non-complementary nucleic acid bases positioned on both sides of two regions that are completely complementary to the target nucleic acid, is interpreted as falling within the scope of the present invention because it has 77.8% overall complementarity with the target nucleic acid. The percentage of complementarity of the antisense compound with the target nucleic acid region can usually be determined using the BLAST and PowerBLAST programs known in the art (Altschul et al., J.MoI. Biol., 1990, 215, 403 410; Zhang and Madden, Genome Res., 1997, 7, 649 656). On the other hand, homology percentages, sequence identity, or complementarity can be determined using Gap programs (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.) that utilize basic settings based on the Smith-Waterman algorithm (Adv.Appl.Math., 1981, 2, 482 489).

[0056] In one specific example, the antisense compound of the present invention or a modified oligonucleotide comprising the antisense compound may be 80% or more complementary to the nucleic acid base sequence in the transcript of the gene encoding WFDC2 (e.g., SEQ ID NO: 1 or SEQ ID NO: 2), preferably 90% or more, and most preferably fully complementary (100% complementary). In this specification, "fully complementary" means that each nucleic acid base of the antisense compound can form an elaborate base pair with the corresponding nucleic acid base of the target nucleic acid.

[0057] In one specific example, the non-complementary nucleic acid base can be located at the 5' or 3' end of the antisense compound. Alternatively, the non-complementary nucleic acid base or nucleic acid base can be located inside the antisense compound. If two or more non-complementary nucleic acid bases are present, they may be adjacent (i.e., bonded) or not adjacent. In one specific example, the non-complementary nucleic acid base can be located in the wing portion of a gapmer antisense oligonucleotide.

[0058] In one specific example, the antisense compound of the present invention may include those complementary to a portion of the nucleic acid base sequence within the transcript of the gene encoding WFDC2. In this specification, “portion” means a predetermined number of nucleic acid bases adjacent to (i.e., bound to) a region or portion of the target nucleic acid. The portion may also mean a certain number of adjacent nucleic acid bases of the antisense compound. In one specific example, the antisense compound may be complementary to at least eight nucleic acid base portions of the target portion, and may be complementary to at least twelve nucleic acid base portions, or at least fifteen nucleic acid base portions. Antisense compounds complementary to at least nine, ten, eleven, twelve, thirteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more nucleic acid base portions of the target portion, or any two of these values, are also interpreted as being included within the range.

[0059] Modified oligonucleotides According to one specific example of the present invention, the antisense compound may include a modified oligonucleotide, the modified oligonucleotide may include one or more modifications selected from one or more modified nucleosides comprising a linking group between one or more modified nucleosides, one or more modified nucleosides comprising a modified sugar moiety, and one or more modified nucleosides comprising a modified nuclear base.

[0060] A variation of sugar moiety In one specific example, the modified nucleoside may be a modified nucleoside comprising a non-bicyclic modified sugar moiety and / or a bicyclic or tricyclic sugar moiety and / or a sugar moiety modified with sugar substitutes or sugar pseudo-forms.

[0061] In one specific example, the modified nucleoside may include, but is not limited to, a sugar molecule into which one or more substituted derivatives selected from the group consisting of 2'-O-methyl (methyl) and 2'-O-alkyl (alkyl), 2'-O-alkoxyalkyl (alkoxyalkyl) such as 2'-O-methoxyethyl (methoxyethyl), 2'-amino (amino), 2'-allyl (allyl), 2'-fluoro (fluoro), 2'-arabino-fluoro (arabino)-fluoro (fluoro), 2'-ON-substituted acetamide such as 2'-OCH2C(=O)-NHCH3(NMA), 2'-O-benzyl (benzyl) and 2'-O-methyl (methyl)-4-pyridine (pyridine), 4'-O-methyl (methyl), 5'-methyl (methyl), 5'-vinyl (vinyl), and 5'-methoxy (methoxy).

[0062] An antisense compound according to one specific example of the present invention may include one or more modified nucleosides having selectively substituted or modified sugar moieties. Modification of the sugar moieties confers nuclease stability, binding affinity, or other advantageous biological properties to the antisense compound. The (pento)furanosyl sugar ring of the natural nucleosides can be modified in a variety of ways, including, but not limited to, the addition of substituents (especially at the 2' position); bridging of ring atoms at two other sites that form bicyclic nucleic acids (BNAs); and substitution of atoms or groups such as -S-, -N(R)- or -C(R1)(R2) on the 4'- ring oxygen. Modified sugar moieties include, but are not limited to, substituted sugars, particularly 2'-substituted sugars having 2'-F, 2'-OCH2(2'-OMe) or 2'-O(CH2)2-OCH3(2'-O-methoxyethyl or 2'-MOE) substituents; and biscyclic modified sugars (BNAs) having 4'-(CH2)nO-2'(n=1 or n=2) bridges. Methods for producing such modified sugars are known in the art. The base moiety in the nucleoside containing the modified sugar moiety can be maintained to hybridize with the target nucleic acid.

[0063] In one specific example, the modified nucleoside contains, at the 2' position, one of the following: F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; O-alkyl-O-alkyl; O-alkyl-O-alkyl-N(dialkyl); or O-alkyl-carboxylamide (where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C 10 Alkyl or C2-C 10 (These are alkenyls and alkynyls). O[(CH2)nO]mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, O(CH2)nO(CH2)nN[(CH2)mCH3]2, O(CH2)nC(=O)-NHCH3 and O(CH2)nON[(CH2)mCH3]2 are particularly preferred (where n and m are 0 to about 10). Other preferred modified oligonucleotides have C1-C at the 2' position.10 It may contain one of the following substituents: lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, or polyalkylamino. Preferably, the modification may include 2'-methoxyethoxy (2'-O-(2-methoxyethyl) or 2'-MOE, also known as 2'-O-CH2CH2OCH3 (Martin et al., He1v.Chim.Acta, 1995, 78, 486-504), i.e., an alkoxyalkoxy group, and the modification may include 2'-dimethylaminooxyethoxy (i.e., (CH2)2ON(CH3)2 group, also known as 2'-DMAOE), and 2'-dimethylaminoethoxyethoxy [i.e., 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE, also known as 2'-O(CH2)2O(CH2)2-N(CH3)2 group].

[0064] Further preferred variants include 2'-methoxy(2'-O-CH3), 2'-aminopropoxy(2'-OCH2CH2CH2NH2), 2'-allyl(2'-CH2-CH=CH2), 2'-O-allyl(2'-O-CH2-CH=CH2), and 2'-fluoro(2'-F). The 2'-variant may be at the arabino (upper) or ribo (lower) position. The preferred 2'-arabino variant is 2'-F, and other positions on the nucleoside (particularly the 3' position of the sugar in the 3'-terminal nucleoside or the 5' position in the 5'-terminal nucleoside) are similarly modifiable.

[0065] The bicyclic or tricyclic sugar moiety may be selected from, for example, the group consisting of locked nucleic acid (LNA), constrained ethyl bicyclic nucleic acid (cEt), 2'-O,4'-C-ethylene-bridged nucleic acid (ENA), and tricyclo-DNA, but is not limited thereto.

[0066] In one specific example, the modified nucleoside may include a sugar substitute having a six-membered ring or an acyclic moiety. The sugar substitute may be selected from, but is not limited to, the group consisting of a morpholino ring, such as phosphorodiamidate morpholino oligomer (PMO), a cyclohexenyl ring, a cyclohexyl ring, and a tetrahydropyranyl ring, such as hexitol, anitol, mannitol, or fluorhexitol. A variety of other bicyclic and tricyclic sugar-substituted ring systems used to modify the nucleoside introduced into the antisense compound according to the present invention are known in the art. Such ring systems can have their activity enhanced by various substitution processes.

[0067] Furthermore, the sugar substitute may, but is not limited to, an acyclic molecule such as unlocked nucleic acid (UNA) or peptide nucleic acid (PNA). Peptide nucleic acids (PNAs) are a type of nucleic acid analog in which the nuclear bases are linked by peptide bonds instead of phosphate bonds. Phosphodiester bonds are replaced by peptide bonds, and PNAs possess nuclear bases such as adenine, thymine, guanine, and cytosine, allowing them to specifically undergo hybridization reactions with nucleic acids. PNAs are not found in nature and are synthesized artificially using chemical methods. They can form double helixes through hybridization reactions with nucleic acids of complementary base sequences. Furthermore, because PNAs are electrically neutral, they are not only chemically stable but also biologically stable, as they are not degraded by nucleases or proteases. While the N-aminoethylglycine skeleton is the most widely used PNA, PNAs with modified skeletons are also usable, as is well known in the industry (PENielsen and M. Egholm, "An Introduction to PNA," in PENielsen (Ed.), "Peptide Nucleic Acids: Protocols and Applications," 2nd Ed., Page 9 (Horizon Bioscience, 2004)).

[0068] Unlocked nucleic acid (UNA) is a modified nucleoside that lacks a C2'-C3' bond in ribose. Its open-chain structure does not constrain its steric configuration, allowing for adjustment of the flexibility of oligonucleotides. It is known that the presence of UNA in antisense oligonucleotides can lower the Tm value by approximately 5°C to 10°C, thereby reducing off-target effects.

[0069] Variations of nucleobases To give one specific example, the modified nucleosides are pseudouridine, 2'-thiouridine, N6'-methyladenosine, 5'-methylcytidine, 5'-fluoro-2-deoxyuridine, N-ethylpiperidine 7'-EAA triazol modified adenine, and N-ethylpiperidine 6'-triazol modified adenine. The modified nucleoside may also contain one or more modified nucleobases selected from the group consisting of adenine, 6'-phenylpyrrolocytosine, 2',4'-difluorotoluylribonuleoside, and 5'-nitroindole.

[0070] Unmodified, or native, nucleic acid bases refer to the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U).

[0071] The modified nucleoside may also include nuclear base modification or substitution. Nuclear base modification or substitution is a structurally distinct form but functionally interchangeable with naturally occurring or synthetically unmodified nuclear bases. Both naturally occurring and modified nuclear bases can participate in hydrogen bonding. Such nuclear base modification confers nuclease stability, binding affinity, or other advantageous biological properties to the antisense compound. For example, certain nuclear base substitutions, such as 5-methylcytosine substitution, are known to enhance nucleic acid duplex stability up to 0.6–1.2°C and are particularly useful for increasing the binding affinity of antisense compounds to target nucleic acids.

[0072] For example, the modified nuclear bases include 5'-hydroxymethylcytosine, xanthine, hypoxanthine, 2'-aminoadenine, 6'-methyl and other alkyl derivatives of adenine and guanine, 2'-propyl and other alkyl derivatives of adenine and guanine, 2'-thiouracil, 2'-thiothymine and 2'-thiocytosine, 5'-halouracil and cytosine, 5'-propynyl(-C≡C-CH3)uracil and cytosine. and other alkynyl derivatives of pyrimidine bases, 6'-azouracil, cytosine and thymine, 5'-uracil (uracil-like), 4'-thiouracil, 8'-halo, 8'-amino, 8'-thiol, 8'-thioalkyl, 8'-hydroxy and other 8'-substituted adenine and guanine, 5'-halo (especially 5'-bromo), 5'-trifluoromethyl and other 5'-substituted uracil and cytosine, 7'-methylglycol Anine and 7'-methyladenine, 2'-F-adenine, 2'-aminoadenine, 8'-azaguanine and 8'-azaadenine, 7'-deazaguanine and 7'-deazaadenine and 3'-deazaguanine and 3'-deazaadenine, phenoxazinecytidine (IH-pyrimido[5,4-b][1,4]benzoxazine-2(3H)-one), phenothiazinecytidine (IH-pyrimido[5,4-b][1,4]benzothiadin- This includes, but is not limited to, G-clamps such as tricyclic pyrimidines (2(3H)-one), 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazine-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indole-2-one), and substituted phenoxazinecytidines such as pyridoindolecytidine (H-pyrimido[3',2':4,5]pyrrolo[2,3-d]pyrimidine-2-one).

[0073] Furthermore, heterocyclic base groups may also include heterocyclic base groups such as 7'-deaza-adenine, 7'-deazaguanosine, 2'-aminopyridine, and 2'-pyridone, in which purine or pyrimidine bases are substituted with other heterocyclic bases. Nucleic acid bases particularly useful for increasing the binding affinity of the antisense compound include, for example, 5'-substituted pyrimidines, 6'-azapyrimidines, and 2'-aminopropyladenine, 5'-propynyluracil, and 5'-propynylcytosine, and include, but are not limited to, N-2, N-6, and O-6 substituted purines. In addition, the modified nuclear bases include those disclosed in U.S. Patent No. 3,687,808, the literature [The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, JI, ed. John Wiley & Sons, 1990, Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613], and the literature [Sanghvi, YS, Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, STand Lebleu, B. ed., CRC Press, 1993].

[0074] Linking groups between deformed nucleosides In one specific example, in the antisense compound, the linking group between the modified nucleosides may be one or more modified nucleoside linking groups selected from the group consisting of phosphotriester, phosphoramidate, mesyl phosphoramidate, phosphorothioate, phosphorodithioate, methylphosphonate, and methoxypropyl-phosphonate.

[0075] As is well known in this industry, a nucleoside is a combination of a nuclear base and a sugar moiety. A nucleotide further contains a phosphate group that is covalently bonded to the sugar moiety of the nucleoside. In nucleotides containing pentofracinol sugars, the phosphate group can bond to the 2', 3', or 5' hydroxyl group of the bonded sugar. In the formation of oligonucleotides, the phosphate groups covalently bond to adjacent nucleosides to form a linear polymer compound. Sequentially, each end of the linear polymer structure also bonds to form a circular structure, although open linear structures are generally preferred. In oligonucleotide structures, the phosphate groups usually form the backbone between the nucleosides of the oligonucleotide, and the spontaneous bonding and backbone of RNA and DNA is a 3'-5' phosphodiester bond. An antisense compound, as a specific example, may contain one or more modified nucleoside bonds in addition to naturally occurring nucleoside bonds. These are preferred over antisense compounds containing naturally occurring nucleoside bonds due to properties such as enhanced cellular absorption, increased target nucleic acid affinity, and increased stability in the presence of nucleases.

[0076] A specific example of a preferred antisense compound used in the present invention is an oligonucleotide containing a modified skeleton or unnatural internucleoside bonds. As defined above, an oligonucleotide having a modified skeleton includes nucleotides containing a phosphorus atom in the skeleton and nucleotides not containing a phosphorus atom in the skeleton. In addition, as is incorporated by the art, a modified oligonucleotide that does not contain a phosphorus atom in the internucleoside skeleton is also interpreted herein as an oligonucleotide.

[0077] In an antisense compound according to one specific example, the deformed bond between nucleosides can include not only phosphate-containing nucleoside bonds but also phosphate-free nucleoside bonds. Typical phosphate-containing nucleoside bonds may include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotryesters, aminoalkyl phosphotryesters, 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, methyl and other alkyl phosphonates, phosphinates, 3'-aminophosphoramides and aminoalkyl phosphoramides having their normal 3'-5' bond and 2'-5' bond analogues, mesylphosphoramides, thionophosphoamides, thionoalkyl phosphonates, thionoalkyl phosphortryesters, selenophosphates and boranophosphates, and compounds having opposite polarity in which one or more nucleotide bonds are 3'-3', 5'-5', or 2'-2' bonds. Furthermore, oligonucleotides with opposite polarity contain a single 3'-3' bond in most nucleotide internucleotide bonds, i.e., a single inverted nucleoside residue (where a nucleic acid base is missing and a hydroxyl group is present instead), which can be a base-losing site. Various salts, salt mixtures, and free acid forms are also included.

[0078] Preferred modified oligonucleotide skeletons that do not contain phosphorus may be skeletons formed by single-chain alkyl or cycloalkyl nucleoside bonds, mixed heteroatoms and alkyl or cycloalkyl nucleoside bonds, or one or more single-chain heteroatoms or heterocyclic nucleoside bonds. These include, but are not limited to, skeletons having morpholine bonds (partially formed from the sugar portion of the nucleoside); siloxane skeletons; sulfide, sulfoxide and sulfone skeletons; formacetyl and thioformacetyl skeletons; methyleneformacetyl and thioformacetyl skeletons; riboacetyl skeletons; alkene-containing skeletons; sulfamate skeletons; methyleneimino and methylenehydrazino skeletons; sulfonate and sulfonamide skeletons; amide skeletons; and skeletons having mixed N, O, S and CH2 component elements. Methods for producing such phosphorus-containing nucleoside bonds and phosphorus-free nucleoside bonds are known in the industry.

[0079] In other specific examples, the antisense compound can have its 5'-terminal hydroxyl group replaced with one selected from the group consisting of 5'-(E)-vinylphosphonate, 5'-methylphosphonate, (S)-5'-C-methyl with phosphate, and 5'-phosphorothioate. Antisense compounds modified in this way are known to be readily loaded into RNA-induced silencing complexes (RISCs) and function as single-stranded short interfering RNAs (ss siRNAs) or double-stranded short interfering RNAs (ds siRNAs).

[0080] Antisense compound motif In one specific example, the antisense compound has a chimeric form in the form of Lx-Dy-Lz, where L may be a modified nucleoside. Here, D is DNA, x and z are any integers from 1 to 7, which may be the same or different, and y is any integer from 5 to 25. Preferably, x and z are any integers from 1 to 5 and y is any integer from 7 to 24, and more preferably, x and z are any integers from 3 to 5 and y is any integer from 8 to 23. At least the sugar moiety of the D region and the L region closest to it is modified, defining the boundary between the L region and the D region. Furthermore, in all the regions (L region and D region), the linking groups between each nucleoside include one or more phosphodiesters or the aforementioned linking groups between modified nucleosides (e.g., phosphorothioates), and the nuclear bases within the nucleoside may also include one or more native nuclear bases or the aforementioned modified nuclear bases.

[0081] In one specific example, the modified oligonucleotide comprises a gap segment consisting of linked deoxynucleosides, a 5' wing segment consisting of linked nucleosides, and a 3' wing segment consisting of linked nucleosides, wherein the gap segment is located between the 5' wing segment and the 3' wing segment, and the nucleosides of each wing segment may contain modified sugar moieties or sugar substitutes.

[0082] Chimeric antisense compounds typically include at least one region that has been modified to confer increased nuclease resistance, increased cellular uptake, increased binding affinity to a target nucleic acid, and / or increased inhibitory activity. Chimeric antisense compounds are formed in the form of a hybrid structure of two or more oligonucleotides or modified oligonucleotides. Such compounds are also referred to in the industry as hybrids or gapmers, and the manufacture of gapmer structures is disclosed in U.S. Patents 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922.

[0083] In a gapmer, the internal region having a number of nucleotides that assist in RNaseH cleavage is located between the nucleosides of the internal region and the external region having a number of nucleosides that are chemically distinct from each other. In the case of antisense oligonucleotides having a gapmer motif, the gap segment (the D region in the antisense compounds herein) assists in the splitting of the target nucleic acid, while the wing segment (the L region in the antisense compounds herein) may contain a modified oligonucleotide containing a modified nucleoside to improve stability, affinity, and exonuclease resistance. If necessary, the gap segment may also contain a modified oligonucleotide. The modified oligonucleotide may include one or more modifications selected from one or more linking groups between modified nucleosides, one or more modified nucleosides containing a modified sugar moiety, and one or more modified nucleosides containing a modified nuclear base, each of which is described above.

[0084] Preferably, each different region in the gapmer can contain a uniform sugar molecule. Furthermore, each different region is demarcated by a different sugar molecule, but the sugar molecules within each region may be in a mixmer form, freely selected from non-modified and modified nucleotides. According to one specific example of the present invention, such a wing-segment-gap-segment motif is represented in the form Lx-Dy-Lz (where x is the length of the 5' wing segment, y is the length of the gap segment, and z is the length of the 3' wing segment). An antisense compound according to one specific example may have a gapmer motif. In one specific example of an antisense compound, x, y, and z include, but are not limited to, 5-10-5, 3-10-3, 1-12-1, 2-10-3, 3-9-4, 3-8-3, 1-9-2, 2-13-5, 4-8-4, 4-12-3, 4-12-4, 3-14-3, 2-16-2, 1-18-1, 2-10-2, 1-10-1, or 2-8-2. In another specific example, the antisense compound may have a wing segment-gap segment or gap segment-wing segment structure, i.e., a "wingmer" motif when x or z is 0. The aforementioned wingmar structure includes, but is not limited to, 10-10, 8-10, 5-10, 8-4, 4-12, 12-4, 3-14, 16-2, 18-1, 10-3, 2-10, 1-10, or 8-2.

[0085] In one specific example, the characteristics of the 3'-wing segment and the 5'-wing segment of the antisense compound can be selected independently. Also, in the specific example, the monomer numbers of the 5'-wing segment (x in Lx) and the monomer numbers of the 3'-wing segment (z in Lz) may be the same or different. Furthermore, any deformation of the 5'-wing segment may be the same as or different from any deformation of the 3'-wing segment, and the monomeric linkages of the 5'-wing segment and the 3'-wing segment may be the same or different. In other words, the entire region is not to be uniformly deformed, and one or more of the deformations are introduced into one or more nucleotides inside the antisense oligonucleotide.

[0086] zygote One aspect of the present invention provides a conjugate in which an antisense compound is covalently bonded to one or more non-nucleotide molecules, in one specific example, the non-nucleotide molecule may include a protein, a fatty acid chain, a sugar residue, a glycoprotein, a polymer, or a combination thereof.

[0087] In this specification, “conjugate” means an antisense compound or antisense oligonucleotide covalently linked to a non-nucleotide moisture (conjugate moisture or region C or third region). Such conjugates can improve the pharmacology of antisense oligonucleotides by affecting, for example, the activity, cell distribution, cell absorption, or stability of the antisense oligonucleotide. In one specific example, the non-nucleotide moisture can alter or improve the pharmacokinetic properties of the antisense oligonucleotide by improving its cell distribution, bioavailability, metabolism, excretion, permeability, and / or cell absorption. The non-nucleotide moisture can also target the antisense oligonucleotide to specific organs, tissues, or cell types, thereby improving the efficacy of the antisense oligonucleotide in those organs, tissues, or cell types. In addition, the non-nucleotide moisture can reduce the activity of the antisense oligonucleotide in non-target cell types, tissues, or organs, such as off-target activity or activity in non-target cell types, tissues, or organs. International patent publications WO93 / 07883 and WO2013 / 033230 disclose preferred non-nucleotide moieties.

[0088] In one specific example, the non-nucleotide molecule includes, but is not limited to, intercalators, reporter molecules, polyamines, polyamides, peptides, carbohydrates, vitamin molecules, polyethylene glycol, thioethers, polyethers, cholesterol, cholic acid molecules, folates, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluorescein, rhodamine, coumarin, fluorophores, and dyes.

[0089] In one specific example, the non-nucleotide molecule may include active drug substances such as aspirin, warfarin, ketoprofen, carprofen, diazepine, antibacterial agents, and antibiotics.

[0090] In one specific example, the non-nucleotide molecule may further include an antibody.

[0091] In one specific example, the non-nucleotide moiety is ligated to the 5' or 3' end of the antisense compound or antisense oligonucleotide.

[0092] In one specific example, the non-nucleotide molecule may contain at least one to three N-acetylgalactosamine (GalNAc) molecules.

[0093] Dosage form To facilitate the use of oligonucleotides, a variety of dosage forms have been developed that allow oligonucleotides to be delivered to a target organism or cellular environment, for example, by minimizing degradation, facilitating delivery and / or absorption, or by providing other beneficial properties to the oligonucleotide in the dosage form.

[0094] In one specific example, an antisense oligonucleotide for reducing WFDC2 expression is suitably formulated so that a sufficient portion of the oligonucleotide enters the cells and reduces WFDC2 expression when administered to a subject in the environment facing the target cells or systemically. In one specific example, the antisense oligonucleotide is formulated in the form of a buffer solution, such as a phosphate-buffered saline solution, liposomes, micelle structures, or capsids. Alternatively, a naked oligonucleotide or its conjugate may be formulated in water or an aqueous solution (e.g., pH-adjusted water) or a basic buffered aqueous solution (e.g., PBS).

[0095] In one specific example, oligonucleotides can be easily introduced into cells using a dosage form containing cationic lipids. For example, cationic lipids such as lipofection, cationic glycerol derivatives, and polyvalent cationic molecules (e.g., polylysine) can be used. Suitable lipids include oligofectamine, lipofectamine (Life Technologies), NC388 (Ribozyme Pharmaceuticals, Inc.), or FuGene ((FuGene)6 (Roche)), all of which are available according to the manufacturer's protocol. Such dosage forms may include lipid nanoparticles.

[0096] Furthermore, the dosage form may include excipients. Excipients may include liposomes, lipids, lipid complexes, microspheres, microparticles, nanospheres, or nanoparticles, or may be differently shaped for administration to cells, tissues, organs, or bodies of subjects requiring them (see, for example, Remington: The Science and Practice of Pharmacy, 22nd edition, Pharmaceutical Press, 2013). Excipients impart to the composition improved stability, improved absorption, improved solubility, and / or therapeutic enhancement of the active ingredient. Excipients may also be buffers (e.g., sodium citrate, sodium phosphate, Tris base, or sodium hydroxide) or vehicles (e.g., buffer solutions, petrolatum, dimethyl sulfoxide, or mineral oil).

[0097] In some embodiments, oligonucleotides can be freeze-dried and then prepared in solution before use to extend their shelf life. Therefore, excipients in compositions containing oligonucleotides forming the antisense compound according to the present invention may be freeze-drying protectants (e.g., mannitol, lactose, polyethylene glycol, or polyvinylpyrrolidone) or disintegration temperature modifiers (e.g., dextran, picol, or gelatin).

[0098] Pharmaceutical compositions suitable for injection may include sterile aqueous solutions (if water-soluble) or dispersions and sterile powders for the in-situ preparation of sterile injection solutions or dispersions. For intravenous or subcutaneous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL.™ (BASF), or phosphate-buffered saline (PBS). Alternatively, the carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. In various cases, it is preferable to include isotonic agents in the composition, such as sugars, polyalcohols (e.g., mannitol, sorbitol), and sodium chloride. Sterile injection solutions are prepared by introducing the required amount of oligonucleotides into a selected solvent, along with one or a combination thereof as needed from the listed components, and then subjecting the mixture to sterile filtration. The pharmaceutical composition may contain at least about 0.1% of the therapeutic agent (e.g., an antisense oligonucleotide for reducing WFDC2 expression) or a higher dose, but the percentage of the active ingredient is preferably about 1% to about 80% of the total weight or volume of the composition. Factors such as solubility, bioavailability, biological half-life, route of administration, and product shelf life, as well as other pharmacological considerations, will be taken into account in the manufacture of the dosage form.

[0099] Treatment of diseases and methods The antisense compound or conjugate containing the same according to the present invention is a pharmaceutical composition for the prevention or treatment of cancer and can be used as a cancer treatment agent. "Cancer" refers to a group of diseases characterized by the over-proliferation and infiltration of surrounding tissues of cells when the balance of normal apoptosis is disrupted. "Treatment" refers to all actions by which the symptoms of cancer are improved, enhanced, or advantageously altered by the administration of the pharmaceutical composition according to the present invention.

[0100] In one specific example, the cancer may be one or more selected from the group consisting of carcinomas derived from epithelial cells such as stomach cancer, esophageal cancer, ovarian cancer, head and neck cancer, brain tumor, thyroid cancer, lung cancer, laryngeal cancer, colorectal cancer, liver cancer, gallbladder cancer, bile duct cancer, bladder cancer, pancreatic cancer, breast cancer, uterine cancer, cervical cancer, prostate cancer, kidney cancer, and skin cancer; sarcomas derived from connective tissue cells such as bone cancer, muscle cancer, lipoma, and fibrocarcinoma; hematological cancers derived from hematopoietic cells such as leukemia, lymphoma, and multiple myeloma; and tumors occurring in nerve tissue. Preferably, the cancer may be a solid tumor.

[0101] A therapeutic method using a pharmaceutical composition according to the present invention involves administering an effective amount of the composition to a subject, i.e., an amount that can produce a desirable therapeutic outcome. The therapeutically acceptable amount is preferably an appropriate dose that can treat the disease, and this can depend on certain factors, including the size of the subject, body surface area, age, the specific composition administered, the active ingredient in the composition, the time and route of administration, overall health, and other drugs administered simultaneously. The compositions according to the present invention can be administered, for example, orally (e.g., gastric tube, duodenal tube, gastrostomy, or rectally), parenterally (e.g., subcutaneous injection, intravenous injection or infusion, intra-arterial injection or infusion, intramuscular injection), topically (e.g., epidermally, by inhalation, eye drops, or mucous membrane), or by direct injection into a target organ (e.g., the liver of the subject). Preferably, the antisense compounds according to the present invention can be administered intravenously or subcutaneously and are available in doses ranging from 0.1 mg / kg to 50 mg / kg, 0.1 mg / kg to 30 mg / kg, 0.1 mg / kg to 20 mg / kg, 0.1 mg / kg to 5 mg / kg, or 0.5 mg / kg to 5 mg / kg. In some specific examples, the target of treatment is preferably a human or non-human primate or other mammalian subject, but may include dogs, cats, horses, cattle, pigs, sheep, goats, chickens, mice, rats, guinea pigs, or hamsters.

[0102] Combination therapy According to one specific example of the present invention, the pharmaceutical composition can be administered together with one or more other agents. According to one specific example, the one or more other agents can be designed to treat the same disease or condition as the target of treatment of the present invention. Alternatively, the one or more other agents can be designed to treat an undesirable effect of one or more pharmaceutical compositions of the present invention, or can be administered together with other agents that treat an undesirable effect of other therapeutic agents.

[0103] In one specific example, the pharmaceutical composition of the present invention may be administered simultaneously with one or more other drugs, or at different times. Furthermore, the pharmaceutical composition of the present invention and one or more other drugs may be manufactured together in a single dosage form, or separately.

[0104] In one specific example, a drug administered together with the pharmaceutical composition of the present invention can enhance the therapeutic effect, resulting in a superior therapeutic effect, i.e., a synergistic effect. In other words, the present invention can provide a pharmaceutical composition comprising an antisense compound and one or more drugs acting by a non-antisense mechanism. The drugs may be chemotherapeutic agents, and examples of such chemotherapeutic agents include daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, maphosphamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosourea, busulfan, mitomycin C, actinomycin D, mitramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosulfane This may also include, but is not limited to, urea, nitrogen mustard, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-azacitidine, hydroxyurea, deoxycoformycin, 4-hydroxyphenoxycyclophosphoramide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide (VP-16), trimethrexate, irinotecan, topotecan, gemcitabine, teniposide, cisplatin, or diethylstilbestrol (DES). When the aforementioned chemotherapeutic agents are used together with the antisense compounds of the present invention, they can be used individually (e.g., 5-FU and oligonucleotides), sequentially (e.g., after using 5-FU and oligonucleotides for a certain period, MTX and oligonucleotides), or in combination with one or more other chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotides, or 5-FU, radiotherapy and oligonucleotides).Anti-inflammatory agents (including, but not limited to, nonsteroidal anti-inflammatory drugs and corticosteroids) and antiviral agents (including, but not limited to, ribavirin, vidarabine, acyclovir, and ganciclovir) can also be mixed into the composition of the present invention. [Examples]

[0105] One or more specific examples will be described in more detail below through the examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

[0106] Experimental method 1.Cell culture Human-derived gastric cancer cell line SNU638 and glioblastoma cell line SF268 were cultured in RPM1-1640 (#Sh30027.01, Hyclone) containing 10% (v / v) FBS (#SH30084.03HI, Hyclone) and 1% (v / v) antibiotic (Penicillin-Streptomycin, #LS202-02, Welgene) in an incubator at 37°C with a carbon dioxide concentration of 5% (v / v).

[0107] PANC-1 cell lines derived from pancreatic cancer epithelial tissue were cultured in Dulbecco's modified Eagle's Medium (DMEM) medium containing 10% FBS and 1% antibiotic (Penicillin-Streptomycin, #LS202-02, Welgene) at 37°C and a carbon dioxide concentration of 5% (v / v). Subculturing was carried out every 4 days, and only cells that had undergone 5 to 10 subculturing cycles were used for transformation experiments.

[0108] 2. Preparation of antisense oligonucleotides (ASOs) The antisense oligonucleotides (ASOs) used in this example were designed to target diverse regions of WFDC2 pre-mRNA (SEQ ID NO: 1) or mRNA (SEQ ID NO: 2). They were either custom-made using Integrated DNA technology or synthesized by repeatedly applying a standardized phosphoramidite chemistry cycle, as shown in Table 1 below, to universal linkers bound to a controlled-pore glass (CPG) solid support using an automated DNA synthesizer (BioAutomation model MerMade12) or an automated peptide synthesizer (Biotage model Syro1).

[0109] [Table 1]

[0110] For the phosphorothioate linker, instead of the oxidation step shown in Table 1, a 0.1 M pyridine solution of 3-[(Dimethylamino-methylidene)amino]-3H-1,2,4-dithiazole-3-thione (DDTT) or a 0.05 M pyridine-acetonitrile 1:1 solution was used.

[0111] Once oligonucleotide synthesis was complete, concentrated ammonia solution was added and the mixture was reacted at 60°C for 12-18 hours to cleave with CPGF and simultaneously remove all protection groups. Subsequently, the CPG was filtered out, the ammonia was appropriately concentrated, and the residue was filtered through Sephdex Model G-25 resin to desalt it. The mixture was then freeze-dried and used immediately, or purified using preparative high-performance liquid chromatography (prep-HPLC), and then precipitated in 2-3 times the volume of cooled ethanol in a 0.3 M sodium chloride (NaCl) or sodium acetate (NaOAc) solution before use.

[0112] The synthesized and purified oligonucleotides were analyzed by analytical high-performance liquid chromatography (Analytical HPLC) to confirm a purity of 80% or higher. Their absorbance at a wavelength of 260 nm was measured using a UV-VIS spectrometer for quantification, and then their molecular weight was confirmed using a MALDI-TOF or Q-TOF mass spectrometer before use.

[0113] 3. Analysis of WFDC2 mRNA expression levels in cells treated with ASO using quantitative real-time PCR (qRT-PCR). Cells that have undergone 5-10 subcultures one day prior to transformation are 9 cm long. 2 0.25 × 10⁻¹⁵ 6 Only cells were cultured. The next day, 9cm 2 After confirming that cells had proliferated to approximately 80% of the surface area of ​​the (6-well) culture dish, the transformation experiment was carried out. Lipofectamine 3000 (#L3000008, Thermofisher) was used as the transformation reagent. The culture solution was replaced with Opti-MEM (#A3635101, Gipco), and after treating with lipofectamine 3000 with 0 nM (control) to 100 nM ASO for 24 hours each, according to the protocol provided by the manufacturer, the cells were analyzed.

[0114] After transformation, the culture medium was removed from the culture dish and washed twice with PBS (#ML008-01, Welgene). Then, total RNA was extracted from the cells using TRIzol (#15596018, Ambion) according to the manufacturer's protocol. The extracted RNA was used as a template for ImProm-II. TMUsing the Reverse Transcription System (#A3800, Promega), cDNA was synthesized according to the protocol of the manufacturing company. Subsequently, using the synthesized cDNA, the primers in Table 2, and TB Green® Fast qPCR Mix (#RR430, Takara), qRT-PCR was carried out according to the protocol of the manufacturing company. The analysis machine was StepOne TM Based on the results of qRT-PCR using the Real-Time PCR System (#4376357, Applied biosystems), the expression level of WFDC2 in the ASO-treated group was converted to a percentage compared to 0 nM (control).

[0115]

Table 2

[0116] 4. Analysis of the expression level of WFDC2 protein in cells administered with ASO by Enzyme-Linked Immunosorbent Assay (ELISA) Cells that had undergone subculture within 5 to 10 passages 1 day before transformation were seeded at 5.0×10 2 (24-well) culture dishes (#30024, SPL). 4 cells only. The next day, 1.9 cm 2After confirming that cells had proliferated to approximately 80% of the surface area of ​​a (24-well) culture dish, the transformation experiment was carried out. Lipofectamine 3000 (#L3000008, Invitrogen) was used as the transformation reagent. The culture solution was transferred to Opti-MEM (#A3635101, Gipco), and ASO levels from 0 nM (control) to 400 nM were treated with lipofectamine 3000 for 48 hours each, according to the protocol provided by the manufacturer. After 48 hours, the cell culture solution was collected in a 1.5 ml microcentrifuge tube (MCT-150-C, AXYGEN), and the residue contained in the cell culture solution was settled by centrifugation at 1,000 rpm for 20 minutes at 4°C. Only the upper layer was collected in a 1.5 ml microcentrifuge tube, and the sample was stored in an ultra-low temperature freezer at -80°C.

[0117] The concentration of WFDC2 was measured using a Duoset ELISA kit for WFDC2 (DY6274-05, R&D system, Minneapolis, MN, USA) according to the following protocol provided to the manufacturer: Human WFDC2 capture Antibody (#844347, R&D system) was diluted with PBS, dispensed into a 96-well micro-plate (#DY990, R&D system), reacted at room temperature for 30 minutes, and then stored in a refrigerator at 4°C for 1 day. Subsequently, 100 μl of the cell culture medium diluted 2-fold with Reagent diluent (#DY995, R&D system) or 100 μl of WFDC2 recombinant protein standard solution were dispensed into the 96-well micro-plate and reacted at room temperature for 2 hours. Then, biotinylated Human WFDC2 detection antibody (#844348, R&D system), diluted with Reagent diluent, was dispensed into each 96-well microplate and reacted at room temperature for 2 hours. After that, 100 μl of Streptavidin-peroxidase solution (Streptavidin-HRP, #893975, R&D system) was dispensed and reacted at room temperature for 20 minutes. Subsequently, 100 μl of Tetramethylbenzidine solution (Substrate solution, #DY999, R&D system) was dispensed into each 96-well microplate and reacted for 7 minutes. After that, 50 μl of 2N sulfuric acid solution was dispensed to inhibit the reaction, and the absorbance at a wavelength of 450 nm was measured using a microplate reader. The concentration of WFDC2 in each sample was calculated by substituting the measured absorbance of each sample into a standard curve created from the absorbance of a known concentration of WFDC2 recombinant protein standard solution. The WFDC2 production suppression rate was calculated using the following formula compared to the negative control group.

[0118]

number

[0119] 5. Confirmation of the cancer growth inhibitory effect of ASO using the SNU638 Xenograft mouse model. SNU638 cells grown under the cell culture conditions described in the above example were suspended in a Matrigel (#354230, Corning) / PBS (#ML008-01, Welgene) 1:1 solution, and then fed to 8-week-old Male NOD.SCID mice (NOD.CB17-Prkdcsscid / NCrKoat) that had been respiratoryally anesthetized, in a 3 × 10⁶ solution. 6 Only cells were injected. Subsequently, the cells were monitored for 3 weeks after injection to monitor cancer cell engraftment and proliferation. Three weeks after cancer cell transplantation, compound 3 at concentrations of 7.5 mpk and 30 mpk were injected twice a week for 4 weeks (8 injections total) via tail vein injection (group IV) and subcutaneous injection (group SC). The N(N) for each group (IV7.5mpk, IV30mpk, SC7.5mpk, SC30mpk) was 8, while the control group consisted of 5. Cancer cell size (mm²) was measured using a vernier caliper for 28 days. 3 The cancer growth inhibitory effect of ASO was confirmed by measuring ( ).

[0120] 6. Confirmation of the cancer growth inhibitory effect of ASO using the SF268 Xenograft mouse model. SF268 cells grown under the cell culture conditions described in the above example were suspended in a Matrigel (#354230, Corning) / PBS (#ML008-01, Welgene) 1:1 solution, and then fed to 8-week-old Male NOD.SCID mice (NOD.CB17-Prkdcsscid / NCrKoat) that had been respiratoryally anesthetized, in a 5 × 10⁶ solution. 6 Only cells were injected. Subsequently, the cells were monitored for 3 weeks after injection to monitor for cancer cell engraftment and proliferation. Three weeks after cancer cell transplantation, compound 3 at 20mpk was injected three times a week for 4 weeks (total 12 injections) via the tail vein injection route (group IV). The N3 in the IV20mpk group was 8, while the control group consisted of 4. Cancer cell size (mm²) was measured using a vernier caliper for 24 days. 3 The cancer growth inhibitory effect of ASO was confirmed by measuring ( ).

[0121] 7. Statistical analysis All statistical analyses were performed using GraphPad prism 9.0.0, and significance was verified using the Two-way ANOVA Multiple Comparisons test. Values ​​indicating statistical significance are indicated by *, **, and ***. * indicates P ≤ 0.05, ** indicates P ≤ 0.01, and *** indicates P ≤ 0.001.

[0122] Experimental results 1. Manufacturing results of ASO Using the antisense oligonucleotide preparation method described above, a total of 380 types were synthesized, including 5-8-5 or 5-10-5 MOE gapmer antisense oligonucleotides in which the 5' and 3' wings are modified with five consecutive 2'-MOE nucleosides, the gap consists of eight to ten consecutive native DNA nucleosides, and all linking groups between nucleosides are modified with phosphorothioates, or 3-10-3 LNA gapmer antisense oligonucleotides in which the 5' and 3' wings are modified with three consecutive 2'-LNA nucleosides, the gap consists of ten consecutive native DNA nucleosides, and all linking groups between nucleosides are modified with phosphorothioates. The base sequences and gapmer motifs including the start and middle regions of WFDC2 pre-mRNA (SEQ ID NO: 1) or mRNA (SEQ ID NO: 2) are shown in Table 3 below.

[0123] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] [Table 3-9] [Table 3-10]

[0124] 2. Confirmation of the effects of ASO at the mRNA level We investigated whether the prepared ASO reduced WFDC2 mRNA expression in the human glioblastoma cell line SF268. Table 4 shows the results of converting the WFDC2 expression levels to percentages after treatment with 100 nM ASO compared to 0 nM (control).

[0125] [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5]

[0126] Furthermore, the compounds that showed a concentration-dependent inhibitory effect on mRNA production in the SNU638 and SF268 cell lines are shown in Tables 5 and 6.

[0127] [Table 5]

[0128] [Table 6]

[0129] 3. Confirmation of the effects of ASO at the protein level. We used ELISA to determine whether the ASO produced above reduces WFDC2 protein expression in human gastric cancer cell line SNU638 and pancreatic cancer cell line PANC1.

[0130] The degree of reduction in WFDC2 protein expression by ASO in gastric cancer cell line SNU638 and pancreatic cancer cell line PANC1 was confirmed by ELISA, and as shown in Tables 7 and 8, the following compounds were affected: Compound 2, Compound 3, Compound 32, Compound 43, Compound 59, Compound 80, Compound 103, Compound 113, Compound 114, Compound 115, Compound 116, Compound 117, Compound 125, Compound 130, Compound 136, Compound 142, Compound 148, Compound 149, Compound 151, Compound 159, Compound 163, Compound 199, Compound 204, Compound 208, Compound 209, Compound 223, Compound 23 2. We confirmed that compounds 233, 234, 237, 239, 244, 245, 246, 247, 248, 249, 279, 285, 286, 294, 304, 307, 308, 310, 311, 314, 324, 325, 330, 337, 370, 373, 374, 375, 376, and 377 significantly reduced expression in the SNU638 cell line and the PANC1 cell line, respectively.

[0131] [Table 7-1] [Table 7-2] [Table 7-3] Table 7-4 Table 7-5 Table 7-6 Table 7-7 Table 7-8 Table 7-9 Table 7-10

[0132] Table 8-1 Table 8-2 Table 8-3 Table 8-4 Table 8-5 Table 8-6 Table 8-7 Table 8-8 Table 8-9

[0133] Furthermore, as shown in Tables 9 and 10, it was confirmed that compounds 1, 3, 57, 58, 76, 77, 79, 80, 88, 113, 114, 115, 116, 117, 125, and 136 decreased WFDC2 expression in a concentration-dependent manner in the SNU638 cell line, while compounds 2, 14, 24, 30, 32, 36, 37, 40, 41, 42, and 43 decreased WFDC2 expression in a concentration-dependent manner in the PANC1 cell line.

[0134] [Table 9-1] [Table 9-2]

[0135] [Table 10]

[0136] 4. Suppressive effect of inhibiting WFDC2 expression on cancer growth When compound 3 was administered to the gastric cancer cell line SNU638 Xenograft mouse model, a statistically significant tumor growth inhibitory effect was confirmed in a concentration-dependent manner, regardless of the route of administration. In particular, it was confirmed that this effect was further enhanced with long-term administration (Figures 1-4).

[0137] Furthermore, when compound 3 was administered by tail vein injection to the glioblastoma cell line SF638 Xenograft mouse model, a statistically significant inhibitory effect on cancer growth was confirmed at a concentration of 20 mpk. In particular, it was confirmed that this effect would be further enhanced with long-term administration (Figures 5 and 6).

[0138] The present invention has been described above, focusing on its preferred embodiments. Those with ordinary skill in the art to which the present invention pertains will understand that the present invention can be realized in modified forms without departing from its essential characteristics. Therefore, the disclosed embodiments should be considered in an explanatory rather than restrictive manner. The scope of the present invention is shown in the claims, not in the above description, and all differences within an equivalent scope should be interpreted as being included in the present invention.

Claims

1. It binds complementaryally to the nucleic acid base sequence of SEQ ID NO: 1 or SEQ ID NO: 2, which is the nucleic acid base sequence within the transcription of the gene that encodes WFDC2 (WAP Four-Disulfide Core Domain 2), and SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 49, SEQ ID NO: 83, SEQ ID NO: 86, SEQ ID NO: 89, SEQ ID NO: 109, SEQ ID NO: 113, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 1 42, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 165, SEQ ID NO: 169, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 225, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245 , SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252, SEQ ID NO: 253, SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, SEQ ID NO: 282, SEQ ID NO: 283, SEQ ID NO: 284, SEQ ID NO: 285, SEQ ID NO: 286, SEQ ID NO: 289, SEQ ID NO: 290, SEQ ID NO: 291, SEQ ID NO: 292, SEQ ID NO: 293, SEQ ID NO: 294, SEQ ID NO: 295, SEQ ID NO: 300, SEQ ID NO: 305, SEQ ID NO: 307, SEQ ID NO: 309, SEQ ID NO: 310, SEQ ID NO: 311, An antisense compound comprising a modified oligonucleotide containing any one nucleic acid base sequence selected from the group consisting of SEQ ID NOs: 312, 313, 314, 315, 316, 317, 320, 327, 328, 330, 333, 334, 335, 336, 343, 376, 377, 380, 381, 382, ​​and 383.

2. The antisense compound according to claim 1, wherein the modified oligonucleotide comprises one or more modifications selected from one or more linking groups between one or more modified nucleosides, one or more modified nucleosides containing a modified sugar moiety, and one or more modified nucleosides containing a modified nuclear base.

3. The antisense compound according to claim 2, wherein the modified nucleoside is a modified nucleoside comprising one or more modified sugar moieties selected from the group consisting of sugar moieties substituted with 2'-O-methyl, 2'-O-methoxyethyl, 2'-amino, 2'-fluoro, 2'-arabino-fluoro, 2'-O-benzyl, or 2'-O-methyl-4-pyridine.

4. The antisense compound according to claim 2, wherein the modified nucleoside is one or more modified nucleosides selected from the group consisting of locked nucleic acid (LNA), constrained ethyl bicyclic nucleic acid (cEt), 2'-O,4'-C-ethylene-bridged nucleic acid (ENA), and tricyclo-DNA.

5. The antisense compound according to claim 2, wherein the modified nucleoside is a modified nucleoside comprising a sugar substitute having a six-membered ring or an acyclic moiety.

6. The modified nucleosides are pseudouridine, 2'-thiouridine, N6'-methyladenosine, 5'-methylcytidine, 5'-fluoro-2-deoxyuridine, N-ethylpiperidine 7'-EAA triazole-modified adenine, and N-ethylpiperidine 6'-triazole-modified adenine. The antisense compound according to claim 2, which is a modified nucleoside comprising one or more modified nuclear bases selected from the group consisting of adenine, 6'-phenylpyrrolocytosine, 2',4'-difluorotoluylribonucleoside, and 5'-nitroindole.

7. The antisense compound according to claim 2, wherein the linking group between the modified nucleosides is one or more modified linking groups between nucleosides selected from the group consisting of phosphotryster, phosphoramidate, mesylphosphoramidate, phosphorothioate, phosphorodioate, methylphosphonate, and methoxypropyl-phosphonate.

8. The antisense compound according to claim 1, wherein the modified oligonucleotide comprises a gap segment consisting of linked deoxynucleosides, a 5'-wing segment consisting of linked nucleosides, and a 3'-wing segment consisting of linked nucleosides, the gap segment being located between the 5'-wing segment and the 3'-wing segment, and the nucleosides of each wing segment comprising a modified sugar moiety or sugar substitute.

9. The modified oligonucleotide consists of a gap segment comprising 8 to 10 linked deoxynucleosides; A 5' wing segment consisting of 3 to 5 linked nucleosides; and A 3' wing segment consisting of 3 to 5 linked nucleosides. Includes, The antisense compound according to claim 1, wherein the gap segment is located between the 5' wing segment and the 3' wing segment, and the nucleosides of each wing segment contain a modified sugar moiety.

10. The antisense compound according to claim 1, wherein the antisense compound reduces one or more of the mRNA level and protein level of WFDC.

11. A conjugate in which one or more non-nucleotide moieties are covalently bonded to the antisense compound described in claim 1.

12. The conjugate according to claim 11, wherein the non-nucleotide moiety comprises a protein, a fatty acid chain, a sugar residue, a glycoprotein, a polymer, or any combination thereof.

13. A pharmaceutical composition for the prevention or treatment of cancer, comprising the antisense compound described in claim 1 or the conjugate described in claim 11 as an active ingredient.

14. The composition according to claim 13, wherein the cancer is selected from the group consisting of gastric cancer, esophageal cancer, bile duct cancer, ovarian cancer, cervical cancer, head and neck cancer, brain tumor, lung cancer, liver cancer, thyroid cancer, prostate cancer, bladder cancer, kidney cancer, gallbladder cancer, colorectal cancer, and pancreatic cancer.

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