CN-122012505-A - Double-stranded oligonucleotide of targeted MMP-7 gene and application thereof

CN122012505ACN 122012505 ACN122012505 ACN 122012505ACN-122012505-A

Abstract

The present disclosure belongs to the field of biological medicine, and in particular relates to double-stranded oligonucleotides targeting MMP-7 genes and applications thereof. Specifically, provided are double-stranded oligonucleotide agents or salts, conjugates, compositions thereof for inhibiting the expression of matrix metalloproteinase 7 (MMP-7), wherein the double-stranded oligonucleotide agents comprise a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand sequence comprises at least 15 contiguous nucleotides and NO more than 3 nucleotides of any of the sequences shown in SEQ ID NOS: 1-85, and/or the sense strand sequence comprises at least 15 contiguous nucleotides and NO more than 3 nucleotides of any of the sequences shown in SEQ ID NOS: 86-170. The double-stranded oligonucleotide agent or the salt thereof for inhibiting MMP-7 expression disclosed by the application can obviously inhibit MMP-7 expression and can be used for preventing and/or treating MMP-7 gene-mediated diseases or conditions.

Inventors

NIE AIHUA
HE JUNLIN
LU XIN
ZHANG YANZHONG
LUO HUAN
WU ERZHONG
WANG ZHIPENG
CHI SHUANGSHUANG

Assignees

北京阿尔纳科技有限公司

Dates

Publication Date: 20260512
Application Date: 20260316

Claims (10)

1. A double-stranded oligonucleotide agent or a salt thereof for inhibiting expression of matrix metalloproteinase 7 (MMP-7), wherein the double-stranded oligonucleotide agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand sequence comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23 consecutive nucleotides selected from the sequences set forth in any of SEQ ID nos. 1-85, and the nucleotide differences do not exceed 3, 2, 1 or 0, and/or the sense strand sequence comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 consecutive nucleotides selected from the sequences set forth in any of SEQ ID nos. 86-170, and the nucleotide differences do not exceed 3, 2, 1 or 0.
2. The double-stranded oligonucleotide agent or salt thereof of claim 1, wherein the nucleotide sequence of the antisense strand comprises a nucleotide sequence selected from any one of SEQ ID NOs 1 to 85 and the antisense strand is NO more than 25 nucleotides in length; Preferably, the sense strand comprises a nucleotide sequence that is at least partially reverse-complementary or substantially complementary to the antisense strand to form a double-stranded region; Preferably, the nucleotide sequence of the sense strand comprises a sequence selected from the group consisting of SEQ ID NOS: 86-170, and the sense strand is NO more than 23 nucleotides in length; More preferably, the nucleotide sequence of the antisense strand comprises the nucleotide sequence shown in any one of the antisense strands in Table 4a, the nucleotide sequence of the sense strand comprises the nucleotide sequence shown in the sense strand corresponding to the antisense strand in Table 4a, and the antisense strand is not more than 25 nucleotides in length, and the sense strand is not more than 23 nucleotides in length.
3. The double-stranded oligonucleotide agent or salt thereof of claim 1 or 2, wherein each nucleotide of the double-stranded oligonucleotide agent is independently selected from modified or unmodified nucleotides; Preferably, at least 1 nucleotide of the nucleotides of the sense strand and the antisense strand of the double-stranded oligonucleotide agent is a modified nucleotide; Preferably, at least 1 nucleotide of the nucleotides of the antisense strand is a modified nucleotide; preferably, all nucleotides in the antisense strand are modified nucleotides; preferably, at least 1 nucleotide in the nucleotides of the sense strand is a modified nucleotide; preferably, all nucleotides in the sense strand are modified nucleotides; Preferably, the nucleotides of the sense and antisense strands of the double-stranded oligonucleotide comprise at least 1 abasic nucleotide; Preferably, the modified nucleotides are each independently selected from the group consisting of 2 '-halo-modified nucleotides, 2' -deoxy nucleotides, 2 '-C-alkyl substituted modified nucleotides, 2' -O-alkyl substituted modified nucleotides, 2 '-amino modified nucleotides, 2' -substituted amino modified nucleotides, nucleoside phosphorothioates, or phosphate analogues; Preferably, the modified nucleotides are each independently selected from the group consisting of 2 '-fluoro nucleotides, 2' -methoxy nucleotides, 2 '-deoxy nucleotides, 2' -O-methoxyethyl nucleotides, abasic nucleotides, inverted abasic nucleotides (InvAb), inverted 2'-OMe nucleotides, inverted 2' -deoxy nucleotides; preferably, the modified nucleotide is a nucleotide analog, more preferably from a Peptide Nucleic Acid (PNA), a Morpholine Nucleic Acid (MNA), a Bridge Nucleic Acid (BNA), a Locked Nucleic Acid (LNA), a glycol nucleic acid/Glycerol Nucleic Acid (GNA), a Threose Nucleic Acid (TNA) or an Unlocked Nucleic Acid (UNA).
4. A double stranded oligonucleotide agent or salt thereof according to any one of claims 1-3, wherein the 3 'end and/or 5' end of the sense strand and/or antisense strand independently comprises one or more modifications selected from (E) -vinyl phosphonate ((E) -VP), methylphosphonate (MP) or phosphorothioate; Preferably, the sense strand and/or antisense strand of the double-stranded oligonucleotide comprises a 3 'overhang and/or a 5' overhang of at least 1 nucleotide; preferably, the 5' end of the antisense strand of the double stranded oligonucleotide comprises one (E) -vinyl phosphonate ((E) -VP); preferably, the sense strand and/or antisense strand independently comprise one or more phosphorothioate linkages; Preferably, the sense strand and/or antisense strand independently comprises one or more phosphorothioate linkages at the 3 'end and/or the 5' end; Preferably, the sense strand and/or antisense strand comprises two consecutive phosphorothioate linkages between the 3 'terminal and/or 5' terminal nucleotides.
5. The double-stranded oligonucleotide agent or salt thereof of any one of claims 1-4, wherein said antisense strand comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23 consecutive nucleotides of any one of SEQ ID NOs 171-278 and 377-438, or consecutive nucleotides not differing from said consecutive nucleotides by more than 3, 2, 1 or 0 nucleotides; preferably, the nucleotide sequence of the antisense strand comprises a nucleotide sequence selected from any one of SEQ ID NOS: 171-278 and 377-438, and the antisense strand is NO more than 25 nucleotides in length; Preferably, the sense strand comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 consecutive nucleotides of the sequence set forth in any one of SEQ ID NOs 279 to 367 and 439 to 500, or consecutive nucleotides that differ from the consecutive nucleotides by NO more than 3, 2, 1 or 0 nucleotides; Preferably, the nucleotide sequence of the sense strand comprises a nucleotide sequence selected from any one of SEQ ID NOs 279-367 and 439-500, and the sense strand is NO more than 23 nucleotides in length; more preferably, the nucleotide sequence of the antisense strand comprises the nucleotide sequence shown in any one of the antisense strands in Table 4b or Table 4c, the nucleotide sequence of the sense strand comprises the nucleotide sequence shown in the sense strand corresponding to the antisense strand in Table 4b or Table 4c, and the antisense strand is not more than 25 nucleotides in length, and the sense strand is not more than 23 nucleotides in length.
6. A conjugate of a double stranded oligonucleotide, characterized in that the conjugate comprises a double stranded oligonucleotide agent according to any one of claims 1-5 or a salt thereof and one or more ligands capable of binding to a cell receptor; Preferably, the ligand is selected from the group consisting of a ligand capable of binding to a cellular receptor or a lipophilic ligand, preferably, the ligand is a protein ligand, an antibody, a polypeptide, an aptamer, and a small molecule of an epithelial cell membrane protein; More preferably, the ligand is an integrin-targeting ligand, including an αvβ6 integrin-targeting ligand; preferably, the ligand is selected from the group consisting of targeting groups, ligands that bind to asialoglycoprotein receptors; Preferably, the targeting group comprises a lectin, glycoprotein, lipid, antibody, surfactant protein a, mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, multivalent fucose, glycosylated polyamino acid, multivalent galactose, transferrin, RGD peptide mimetic or aptamer, preferably, the ligand is a galactose cluster; preferably, the galactose cluster comprises a targeting group having 1-4 galactose or galactose derivatives; Preferably, the galactose cluster comprises a molecule having 1 to 4N-acetylgalactosamine (N-acetylgalactosamine, galNAc); preferably, the ligand is an N-acetylgalactosamine (GalNAc) derivative; Preferably, the ligand is a lipophilic ligand, the lipophilic ligand comprises a lipid or a lipid-based molecule; Preferably, the ligand is selected from cholesterol, steroid, bile acid, folic acid, vitamin E, vitamin B12; Preferably, the lipid or lipid-based molecule is selected from cholesterol, fatty hydrocarbon groups, fatty acids, bile acids, folic acid, steroids, phospholipids, sphingolipids, phospholipid analogues; preferably, the fatty acid is selected from saturated and unsaturated fatty acids containing 6 to 22 carbon atoms, more preferably saturated and unsaturated fatty acids containing sixteen, eighteen, twenty-two carbon atoms; Preferably, the aliphatic hydrocarbon groups are selected from saturated and unsaturated aliphatic hydrocarbon groups containing 6 to 22 carbon atoms, more preferably saturated and unsaturated hydrocarbon groups containing sixteen, eighteen, twenty-two carbon atoms; Preferably, the aliphatic hydrocarbon group is attached to the 2' -position of the nucleotide; preferably, one or more cholesterol molecules are linked at the ends of the sense strand and/or the antisense strand; preferably, one cholesterol molecule is attached to the 3' end of the sense strand; preferably, the ligand is conjugated to any position or positions on the sense strand and/or the antisense strand of the double-stranded oligonucleotide; preferably, the ligand is conjugated to the sixth and/or sixteenth position of the sense strand of the double stranded oligonucleotide starting from the 5' -end; preferably, the ligand is conjugated to the 3 'end and/or the 5' end of the sense strand and/or the antisense strand of the double-stranded oligonucleotide; preferably, the ligand conjugate is attached to the 3' end of the sense strand of the double-stranded oligonucleotide.
7. The conjugate of claim 6, wherein the double-stranded oligonucleotide has a combination of sense and antisense strands selected from any one of table 4b or table 4 c: Wherein, the lower case letter a, u, g, C represents a 2' -methoxy-3 ' -nucleotide having a corresponding base, the upper case letter A, U, G, C represents a 2' -fluoro-3 ' -nucleotide having a corresponding base, the lower case letter s represents a phosphorothioate bond between two nucleotides adjacent to the left and right thereof, invab represents that one nucleotide adjacent to the right thereof is a flipped abasic nucleotide, the lower case letter d represents that ribose in one nucleotide adjacent to the right thereof is deoxyribose, VPu represents 2' -OMe-5' - (E) -vinyl phosphonate-3 ' -uridylic acid, C16 represents a C16 hydrocarbon group, and chol represents a cholesterol group.
8. A composition comprising one or more of the double stranded oligonucleotide agent of any one of claims 1-5 or a salt thereof or the conjugate of claim 6 or 7; preferably, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable adjuvant or carrier; Preferably, the pharmaceutical composition further comprises a delivery formulation, preferably, the delivery formulation comprises Lipid Nanoparticles (LNP), lipid multipolymer (LPP), polymer Nanoparticles (PNP), inorganic Nanoparticles (INP), cationic Nanoemulsion (CNE), exosomes, biologicals; Preferably, the pharmaceutical composition further comprises a second therapeutic agent, preferably the second therapeutic agent is selected from the group consisting of an anti-fibrotic drug, a glucocorticoid, a neuromuscular blocker, an anticholinergic drug, a leukotriene receptor antagonist, a beta 2 receptor agonist, a bio-targeting agent, an anti-infective drug, more preferably the second therapeutic agent is selected from the group consisting of nintedanib, pirfenidone, and pharmaceutically acceptable salts or esters thereof.
9. Use of the double stranded oligonucleotide agent or salt thereof of any one of claims 1-5 and/or the conjugate of claim 6 or 7 and/or the composition of claim 8 in the manufacture of a medicament for the prevention and/or treatment of a MMP-7 gene mediated disease or disorder; preferably, the disease or condition is selected from the group consisting of Idiopathic Pulmonary Fibrosis (IPF), asthma, other types of fibrosis, chronic inflammation, interstitial Lung Disease (ILD), SARS-COV-2 or other types of infectious disease, acute Respiratory Distress Syndrome (ARDS) or other types of acute lung injury, pulmonary arterial hypertension, cancer, renal fibrosis and liver fibrosis; more preferably, the disease or disorder is idiopathic pulmonary fibrosis.
10. A method of reducing or inhibiting MMP-7 gene expression and/or activity, the method comprising the step of contacting a cell with any of: (1) The double-stranded oligonucleotide agent or salt thereof of any one of claims 1-5; (2) The conjugate of claim 6 or 7, and/or (3) The composition of claim 8.

Description

Double-stranded oligonucleotide of targeted MMP-7 gene and application thereof Technical Field The present disclosure belongs to the field of biological medicine, and in particular relates to a double-stranded oligonucleotide targeting MMP-7 gene and an application thereof. Background Matrix metalloproteinase 7 (Matrix Metallopeptidase, MMP-7) is the member of the family of Matrix Metalloproteinases (MMPs) that has the smallest molecular weight (about 28, 28 kDa). The family of proteins is capable of degrading various components (e.g., collagen, proteoglycans, etc.) in the extracellular matrix and cleaving various non-matrix proteins, thereby playing a key role in tissue remodeling and cell signaling. Its dysfunction is closely related to the pathogenesis of various diseases such as tumor, chronic inflammation and fibrosis. MMP7 is mainly expressed and secreted by epithelial cells and participates in the epithelial repair process. Studies have demonstrated that their expression is significantly up-regulated in the pathological fibrotic processes of organs such as lung, liver, kidney, etc. For example, in lung tissue, bronchoalveolar lavage fluid, and peripheral blood of patients with idiopathic pulmonary fibrosis (Idiopathic Pulmonary Fibrosis, IPF), MMP7 levels are significantly elevated and positively correlated with disease severity and progression, and are therefore considered potential serum biomarkers of IPF. On molecular mechanisms, MMP7 promotes fibrosis development by mediating epithelial-mesenchymal transition, abnormal matrix repair, tissue remodeling and the like. It can promote fibrosis signal transmission by cutting E-cadherin and other proteins and activating heparin binding epidermal growth factor precursor. Animal experiments show that the MMP7 gene knockout mice have remarkable resistance to the bleomycin-induced pulmonary fibrosis, and lung inflammation, fibrosis degree and death rate are reduced, so that inhibition of MMP7 can be an effective strategy for treating fibrotic diseases. At present, although the existing medicines can delay the progress of IPF diseases, the medicines cannot be radically cured. Inhibition of MMP7 expression or functional block is expected to provide new therapeutic direction for idiopathic pulmonary fibrosis and other fibrotic diseases. In this context, novel therapeutic strategies, represented by RNA interference (RNAi), exhibit unique potential. In particular, by utilizing a small interfering RNA (siRNA) technology, target gene mRNA can be efficiently and specifically silenced, so that MMP-7 protein expression can be accurately and downwardly regulated at the posttranscriptional level. Compared with the traditional action mechanism, the siRNA strategy can block the generation of pathogenic proteins from the source, has definite action mechanism and possibly provides longer-lasting curative effect, and opens up a new way with a prospect for developing a novel therapy for MMP-7 related diseases. In summary, in light of the urgent need for targeted core pathological mechanism therapies in the current relevant disease treatment field and the limitations of the existing intervention means, the development of an siRNA drug targeting MMP-7 mRNA with high efficiency, specificity and good safety has important scientific significance and clinical value. Disclosure of Invention The invention provides an oligonucleotide molecule targeting MMP-7 gene, which is used for interfering or inhibiting MMP-7 gene expression. In a first aspect, the present disclosure provides a double-stranded oligonucleotide agent or salt thereof for inhibiting expression of matrix metalloproteinase 7 (MMP-7), capable of mediating cleavage of an RNA transcript of an MMP-7 gene by an RNA-induced silencing complex (RISC), wherein the double-stranded oligonucleotide agent comprises a sense strand and an antisense strand that form a double-stranded region, the sense strand and the antisense strand being complementary or substantially complementary. The antisense strand is at least partially complementary or substantially complementary to a nucleotide sequence in an mRNA of MMP-7. By substantially complementary is meant that the sense strand and the antisense strand or the antisense strand and the mRNA of MMP-7 differ by no more than 3 nucleotides in their double-stranded complementary regions, preferably each nucleotide of the double-stranded oligonucleotide is independently selected from modified or unmodified nucleotides. In a second aspect, the present disclosure provides a conjugate comprising the aforementioned double-stranded oligonucleotide agent or salt thereof and one or more ligands capable of binding to a cellular receptor. In a third aspect, the present disclosure provides a single stranded oligonucleotide targeting an MMP-7 gene, the single stranded oligonucleotide comprising a sequence of the aforementioned double stranded oligonucleotide agent that is at least partially complementary