Double-stranded oligonucleotide targeting MMP-7 gene and application thereof

Abstract

The invention belongs to the field of biological medicine, and particularly relates to double-stranded oligonucleotide of a targeted MMP-7 gene and application of the double-stranded oligonucleotide. Specifically, provided are a double-stranded oligonucleotide agent or a salt, conjugate, composition thereof for inhibiting the expression of matrix metalloproteinase 7 (MMP-7) wherein the double-stranded oligonucleotide agent comprises one sense strand and one antisense strand forming a double-stranded region; wherein the antisense strand sequence comprises at least 15 contiguous nucleotides of the sequence shown in any one of SEQ ID NO: 1-85 and the difference is not greater than 3 nucleotides, and / or the sense strand sequence comprises at least 15 contiguous nucleotides of the sequence shown in any one of SEQ ID NO: 86-170 and the difference is not greater than 3 nucleotides. The double-strand oligonucleotide agent for inhibiting the expression of the MMP-7 or the salt thereof disclosed by the invention can obviously inhibit the expression of the MMP-7, and can be used for preventing and / or treating MMP-7 gene mediated diseases or symptoms.

Description

Technical Field

[0001] This disclosure pertains to the field of biomedicine, specifically relating to a double-stranded oligonucleotide targeting the MMP-7 gene and its applications. Background Technology

[0002] Matrix metalloproteinase 7 (MMP-7) is the smallest member of the matrix metalloproteinase (MMP) family (approximately 28 kDa). Proteins in this family can degrade various components of the extracellular matrix (such as collagen and proteoglycans) and cleave a variety of non-matrix proteins, thus playing a crucial role in tissue remodeling and cell signaling regulation. Abnormalities in its function are closely related to the pathogenesis of various diseases, including tumors, chronic inflammation, and fibrosis.

[0003] MMP7 is mainly expressed and secreted by epithelial cells and participates in the epithelial repair process. Studies have confirmed that its expression is significantly upregulated in the pathological fibrosis process of organs such as the lungs, liver, and kidneys. For example, in the lung tissue, bronchoalveolar lavage fluid, and peripheral blood of patients with idiopathic pulmonary fibrosis (IPF), MMP7 levels are significantly elevated and positively correlated with disease severity and progression, thus it is considered a potential serum biomarker for IPF.

[0004] At the molecular level, MMP7 promotes fibrosis by mediating epithelial-mesenchymal transition, abnormal matrix repair, and tissue remodeling. It can promote fibrosis signaling by cleaving proteins such as E-cadherin and activating heparin-binding epidermal growth factor precursors. Animal experiments have shown that MMP7 knockout mice exhibit significant resistance to bleomycin-induced pulmonary fibrosis, with reduced lung inflammation, fibrosis severity, and mortality, suggesting that inhibiting MMP7 may be an effective strategy for treating fibrotic diseases.

[0005] Currently, although existing drugs can slow the progression of IPF, there is no cure. Inhibition or functional blockade of MMP7 expression holds promise for providing new therapeutic directions for idiopathic pulmonary fibrosis and other fibrotic diseases. Against this backdrop, novel therapeutic strategies, represented by RNA interference (RNAi), have shown unique potential. In particular, the use of small interfering RNA (siRNA) technology can efficiently and specifically silence target gene mRNA, thereby precisely downregulating MMP-7 protein expression at the post-transcriptional level. Compared with traditional mechanisms of action, the siRNA strategy can block the production of pathogenic proteins at the source, with a clear mechanism of action and potentially more durable efficacy, opening a promising new avenue for developing novel therapies for MMP-7-related diseases.

[0006] In conclusion, given the urgent need for targeted therapies that address the core pathological mechanisms in the treatment of related diseases, and the limitations of existing intervention methods, developing a highly efficient, specific, and safe siRNA drug that targets MMP-7 mRNA has significant scientific and clinical value. Summary of the Invention

[0007] This invention provides an oligonucleotide molecule that targets the MMP-7 gene for interfering with or inhibiting MMP-7 gene expression.

[0008] In a first aspect, this disclosure provides a double-stranded oligonucleotide agent or a salt thereof for inhibiting matrix metalloproteinase 7 (MMP-7) expression, which can mediate the cleavage of the MMP-7 gene RNA transcript via the RNA-induced silencing complex (RISC); wherein the double-stranded oligonucleotide agent comprises a sense strand and an antisense strand forming 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 the MMP-7 mRNA. Substantialloying means that the difference between the sense strand and the antisense strand, or between the antisense strand and the MMP-7 mRNA, in their double-stranded complementary region does not exceed 3 nucleotides; preferably, each nucleotide of the double-stranded oligonucleotide is independently selected from modified or unmodified nucleotides.

[0009] Secondly, this disclosure provides a conjugate comprising the aforementioned double-stranded oligonucleotide agent or a salt thereof and one or more ligands capable of binding to a cell receptor.

[0010] Thirdly, this disclosure provides a single-stranded oligonucleotide targeting the MMP-7 gene, wherein the single-stranded oligonucleotide comprises a sequence of the aforementioned double-stranded oligonucleotide that is at least partially complementary or substantially complementary to a nucleotide sequence in the mRNA of MMP-7.

[0011] Fourthly, this disclosure provides a composition comprising one or more of the aforementioned double-stranded oligonucleotide agents or their salts or conjugates; Preferably, the composition is a pharmaceutical composition, which further comprises pharmaceutically acceptable excipients or carriers; Preferably, the pharmaceutical composition further comprises a delivery formulation; preferably, the delivery formulation comprises lipid nanoparticles (LNP), lipid-polymer hybrid nanoparticles (LPP), polymer nanoparticles (PNP), inorganic nanoparticles (INP), cationic nanoemulsions (CNE), exosomes, and biological microvesicles; Preferably, the pharmaceutical composition further comprises a second therapeutic agent; preferably, the second therapeutic agent is selected from antifibrotic drugs, glucocorticoids, neuromuscular blocking agents, anticholinergic drugs, leukotriene receptor antagonists, β2 receptor agonists, biological targeting agents, and anti-infective drugs; more preferably, the second therapeutic agent is selected from nintedanib, pirfenidone, and pharmaceutically acceptable salts or esters thereof.

[0012] Fifthly, this disclosure provides the use of the aforementioned double-stranded oligonucleotide agent or its salt in the preparation of medicaments for the prevention and / or treatment of MMP-7 gene-mediated diseases or conditions; Preferably, the disease or condition is selected from idiopathic pulmonary fibrosis (IPF), asthma, other types of fibrosis, chronic inflammation, interstitial lung disease (ILD), SARS-CoV-2 or other types of infectious diseases, 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 condition is idiopathic pulmonary fibrosis.

[0013] Sixthly, this disclosure provides the use of the aforementioned composition in the preparation of a medicament for the prevention and / or treatment of MMP-7 gene-mediated diseases or conditions; Preferably, the disease or condition is selected from idiopathic pulmonary fibrosis (IPF), asthma, other types of fibrosis, chronic inflammation, interstitial lung disease (ILD), SARS-CoV-2 or other types of infectious diseases, acute respiratory distress syndrome (ARDS) or other types of acute lung injury, pulmonary hypertension, cancer, renal fibrosis, and liver fibrosis; More preferably, the disease or condition is idiopathic pulmonary fibrosis.

[0014] In a seventh aspect, this disclosure provides a method for reducing or inhibiting MMP-7 gene expression and / or activity, the method comprising the step of contacting a cell with any of the following: (1) The aforementioned double-stranded oligonucleotide agent or its salt; (2) The aforementioned conjugates; and / or (3) The aforementioned composition.

[0015] Eighthly, this disclosure provides a method for preventing and / or treating MMP-7 gene-mediated diseases or conditions, said method comprising administering to a subject an effective amount of any of the following: (1) The aforementioned double-stranded oligonucleotide agent or its salt; (2) The aforementioned conjugates; and / or (3) The aforementioned composition; Preferably, the disease or condition is selected from idiopathic pulmonary fibrosis (IPF), asthma, other types of fibrosis, chronic inflammation, interstitial lung disease (ILD), SARS-CoV-2 or other types of infectious diseases, acute respiratory distress syndrome (ARDS) or other types of acute lung injury, pulmonary hypertension, cancer, renal fibrosis, and liver fibrosis; More preferably, the disease or condition is idiopathic pulmonary fibrosis.

[0016] Compared with the prior art, this disclosure has at least the following beneficial effects: The double-stranded oligonucleotide or its conjugate or composition thereof disclosed in this application can significantly inhibit MMP-7 expression and can effectively treat diseases related to the MMP-7 gene.

[0017] The double-stranded oligonucleotide agent or its salt disclosed herein achieves advantages such as high gene silencing activity, low immunogenicity, high stability and low off-target risk through comprehensive structural optimization and chemical modification, making it suitable for highly effective gene silencing therapy.

[0018] The sequences disclosed herein can also be conjugated to targeted delivery systems or ligands to better enhance targeting capabilities to specific tissues or cells, and to better prolong half-life and survival time in target organs. Attached Figure Description

[0019] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this specification and, together with the description, serve to explain the principles of this specification.

[0020] Figure 1 IC ABN007-08 is shown. 50 picture.

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[0092] Figure 73 IC ABN007-146 is shown. 50 picture. Detailed Implementation

[0093] I. Definition In this disclosure, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. To better understand this disclosure, definitions and explanations of relevant terms are provided below.

[0094] When used herein, the term “and / or” should be considered as a specific disclosure of each of the two specified features or components in the presence or absence of the other. Thus, as used herein in phrases such as “A and / or B”, the term “and / or” is intended to include “A and B”; “A or B”; “A” (alone); and “B” (alone). Similarly, as used in phrases such as “A, B, and / or C”, the term “and / or” is intended to cover each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0095] As used herein, the term “about” or “approximately” when applied to one or more target values ​​means a value similar to the reference value. In some embodiments, unless otherwise stated or otherwise apparent from the context, the term “approximately” or “about” means falling within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the reference value in any direction (unless such a number would exceed 100% of the possible value). As used herein, “optional” or “optionally” means that the event or condition described thereafter may or may not occur, and the description includes both cases in which the event or condition occurs and cases in which it does not occur.

[0096] As used herein, the terms “oligonucleotide,” “polynucleotide,” or “oligomery” are used interchangeably and refer to polymers of nucleotides, including but not limited to single-stranded, double-stranded, or partially double-stranded nucleic acid molecules of DNA, RNA, or DNA / RNA hybrids, oligonucleotide chains containing regular and irregular alternating deoxyribosyl and ribosyl moieties, and modified and / or naturally occurring frameworks for such oligonucleotides.

[0097] The terms siRNA, "RNA interference agent," "RNAi agent," and "interfering RNA (iRNA)" are used interchangeably and all refer to preparations containing RNA components that can mediate targeted cleavage of RNA transcripts through the RNA-induced silencing complex (RISC) pathway and achieve sequence-specific degradation of messenger RNA (mRNA) through RNA interference mechanisms. These preparations can regulate (e.g., inhibit) the expression of genes such as MMP-7 within cells (e.g., in mammalian subjects). In some embodiments, oligonucleotide agents may be in single-stranded or double-stranded form and are introduced into cells or organisms to inhibit target mRNAs.

[0098] As used herein, the term "double-stranded oligonucleotide" refers to an oligonucleotide that is substantially in a double-stranded form. In some embodiments, complementary base pairings are formed between antiparallel oligonucleotide sequences in one or more double-stranded regions of the double-stranded oligonucleotide. In some embodiments, complementary base pairings are formed from a single nucleic acid strand folded (e.g., via a hairpin) to provide complementary antiparallel sequences of nucleotides that are paired together. In some embodiments, the double-stranded oligonucleotide comprises two separate nucleic acid strands that are completely complementary to each other. However, in some embodiments, the double-stranded oligonucleotide comprises two partially complementary nucleic acid strands, such as nucleic acid strands having protrusions at one or both ends. In some embodiments, the double-stranded oligonucleotide comprises antiparallel sequences of oligonucleotides that are partially complementary, and therefore may have one or more mismatches, which may include internal mismatches or terminal mismatches.

[0099] As used herein, the term "dsRNA" refers to a ribonucleic acid molecular complex with a double-stranded structure comprising two antiparallel and substantially complementary nucleic acid strands. These two strands have "sense" and "antisense" directions relative to the target RNA (such as the MMP-7 gene). In some embodiments of this disclosure, dsRNA mediates the degradation of the target RNA (such as mRNA) through a post-transcriptional gene silencing mechanism of RNA interference.

[0100] Generally, most nucleotides in each strand of a dsRNA molecule are ribonucleotides, but as described in detail herein, each or both strands may also contain one or more non-ribonucleotides, such as modified nucleotides. Additionally, as used herein, “iRNA” may contain chemically modified ribonucleotides; iRNA may contain substantial modifications at multiple nucleotide sites.

[0101] As used herein, the term "modified nucleotide" refers to a nucleotide that independently has a modified sugar moiety, a modified phosphate group, or a modified base, or any combination thereof. Therefore, the term modified nucleotide encompasses substitutions, additions, or removals of phosphate groups, sugar moieties, and / or bases, such as functional groups or atoms. Modifications applicable to pharmaceuticals disclosed herein include all types of modifications disclosed herein or known in the art. For the purposes of this specification and claims, any such modifications are covered by the term "double-stranded oligonucleotide agent," as used in siRNA-type molecules.

[0102] As used herein, the term "antisense oligonucleotide" or "ASO" refers to a single-stranded oligonucleotide, etc., having, comprising, or consisting of a specific base sequence that is capable of hybridizing with a target molecule (such as another nucleic acid, a modified nucleic acid, or a nucleic acid analog) through base pairing (e.g., Watson-Crick base pairing or non-Watson-Crick base pairing). In some embodiments, the antisense oligonucleotide is fully complementary or nearly fully complementary to the target molecule. In some embodiments, any oligonucleotide of any type described herein or known in the art may be used as an antisense oligonucleotide. In various embodiments, the antisense oligonucleotide may perform or participate in any of the various biological functions described herein or known in the art, including RNA interference, RNase H-mediated cleavage, exon jumping, exon jumping prevention, enhancing or inhibiting agents, or any other biological function performed by the antisense oligonucleotide.

[0103] As used herein, the term "siRNA" is a class of double-stranded RNA molecules that can mediate the silencing of a target RNA (e.g., mRNA, such as transcripts of genes encoding proteins) that is complementary to it. siRNA is typically double-stranded, consisting of an antisense strand complementary to the target RNA and a sense strand complementary to that antisense strand. For convenience, such mRNA is also referred to herein as the mRNA to be silenced. Such genes are also referred to as target genes. Typically, the RNA to be silenced is an endogenous gene or a pathogen gene. In addition, RNAs other than mRNA (e.g., tRNA) and viral RNA can also be targeted.

[0104] As used in this article, the term "antisense strand" refers to a strand of siRNA that contains regions that are fully or substantially complementary to the target sequence.

[0105] As used herein, the term "sense chain" refers to another chain of siRNA, which includes regions substantially complementary to the region referred to herein as the antisense chain.

[0106] As used herein, the term "complementary" refers to a structural relationship between nucleotides (e.g., two nucleotides on opposing nucleic acids or on opposing regions of a single nucleic acid strand) that allows them to form base pairs with each other. For example, a purine nucleotide of one nucleic acid can pair with a pyrimidine nucleotide of the opposing nucleic acid by forming hydrogen bonds. In some embodiments, complementary nucleotides may pair bases in a Watson-Crick manner or in any other manner that allows the formation of a stable duplex. In some embodiments, the two nucleic acids may have nucleotide sequences that are complementary to each other to form complementary regions.

[0107] As described herein, the term "complementary region" refers to a region on the antisense strand that is completely or substantially complementary to the target mRNA sequence. In cases where the complementary region is not completely complementary to the target sequence, mismatches can occur within the molecule or at the ends. Typically, the most tolerant mismatches are located in the end regions, for example, within 5, 4, 3, 2, or 1 nucleotides at the 5' and / or 3' ends. The antisense strand portion most sensitive to mismatches is called the "seed region." For example, in a siRNA containing a 19-nt strand, the 19th position (from 5' to 3') can tolerate some mismatches. The terms "complementary," "completely complementary," and "substantially complementary" can be used relative to the base pairing between the sense and antisense strands of the siRNA, or between the antisense strand of the siRNA reagent and the target sequence.

[0108] As used herein, the term "difference" refers to a "nucleotide difference" between one nucleotide sequence and another, meaning that the type of base pairs at the paired nucleotide positions has changed compared to the latter. In some embodiments, a nucleotide difference at a position may also be considered to have occurred when a baseless nucleotide or a nucleotide analog is used instead of the nucleotide at that position. Such nucleotide analogs include, but are not limited to, peptide nucleic acid (PNA), morpholino (MNA), bridged nucleic acid (BNA), locked nucleic acid (LNA), glycol nucleic acid (GNA), threose nucleic acid (TNA), or unlocked nucleic acid (UNA).

[0109] As used herein, the terms "comprising / including" and other forms of use are not restrictive. The terms "having" and other forms of use are not restrictive. As used herein, whether in transitional phrases or in the body of the claims, the term "comprising / including" shall be construed as having an open-ended meaning. That is, the above term shall be interpreted synonymously with the phrases "at least having" or "at least including." For example, when used in the context of a process, the term "comprising" means that the process includes at least the listed steps, but may also include additional steps. When used in the context of a compound, composition, or device, the term "comprising" means that the compound, composition, or device includes at least the listed features or components, but may also include additional features or components. When used above in the context of a combination of double-stranded oligonucleotides and / or dsRNA, the term "having" means that the sense / antisense strand includes at least the listed sequences, but may also include additional sequences.

[0110] As used herein, a "nucleoside" is a compound composed of two structural units: a purine or pyrimidine base and a ribose or deoxyribose. A "ribonucleotide," also called a "nucleotide," is a compound composed of three structural units: a purine or pyrimidine base, a ribose or deoxyribose, and a phosphate group. An "oligonucleotide" refers to a nucleic acid molecule (RNA or DNA) having, for example, fewer than 100, 200, 300, or 400 nucleotides. Oligonucleotides can contain ribonucleotides, deoxyribonucleotides, and / or modified nucleotides, including, for example, modified ribonucleotides. Oligonucleotides can be single-stranded or double-stranded and may or may not have double-stranded regions.

[0111] As used in this article, a "base" is one of the basic building blocks of nucleosides, nucleotides, and nucleic acids. Its constituent elements include nitrogen, hence it is also called a "nitrogenous base." In this article, unless otherwise specified, the uppercase letters A, U, T, G, and C, and their corresponding lowercase letters, represent the base composition of nucleotides: adenine, uracil, thymine, guanine, and cytosine, respectively.

[0112] As used herein, the terms “modified base” or “non-natural base” mean any base other than adenine, cytosine, guanine, thymine (also known as 5-methyluracil), or uracil.

[0113] In some implementations, those skilled in the art can interchange thymine and uracil depending on whether the nucleotide is RNA or DNA.

[0114] In some embodiments, the modified bases are selected from 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines.

[0115] In some embodiments, the modified base is selected from: 5-methylcytosine, 5-hydroxymethylcytosine, 2-thiouracil, 2-thiothymidine and 2-thiocytosine, 5-propynyl (-C≡C-CH3)uracil, 5-propynylcytosine, pyrimidines containing 5-halogenated (especially 5-bromine) and / or 5-trifluoromethyl substituted, 6-azauracil, 6-azacytosine, 6-azathymidine, pseudouracil, 4-thiouracil, xanthine, hypoxanthine, 2-aminoadenine, 6-N-methyladenine Purines, 2-propyladenine, 2-aminopropyladenine, purines including 8-halogenated, 8-amino, 8-mercapto, 8-thioalkyl, 8-hydroxy, 8-aza and other 8-substituted purines, 2-F-adenine, 2,6-diaminopurine, 7-deadenine, 7-substituted 7-deadenine, 7-deadenine, 7-substituted 7-deadenine, 3-deadenine, 3-deadenine, universal bases, hydrophobic bases, hybrid bases, size-amplified bases, and fluorinated bases.

[0116] In some embodiments, the modified bases also include heterocyclic structures, such as 1,3-diazaphenoxazin-2-one, 1,3-diazaphenthiazin-2-one and 9-(2-aminoethoxy)-1,3-diazaphenoxazin-2-one (G-clamp), 2-aminopyridine, and 2-pyridone.

[0117] As used herein, the term "deoxyribonucleotide" refers to a nucleotide in which the hydroxyl group at the 2'-position of the pentose sugar is replaced by hydrogen compared to a ribonucleotide. A modified deoxyribonucleotide is a deoxyribonucleotide having one or more modifications or substitutions (including modifications or substitutions in sugars, phosphate groups, or bases, or modifications or substitutions in sugars, phosphate groups, or bases) at positions other than the 2'-position.

[0118] As used herein, the term "modified nucleotide" means a nucleotide having independently modified sugar groups, modified phosphate groups, and / or modified bases, as well as combinations of various modifications.

[0119] As used herein, the term "modified oligonucleotide" means an oligonucleotide comprising at least one modified phosphate group, a modified sugar group and / or a modified base and a combination of various modifications.

[0120] As used herein, the term “inhibition” is used interchangeably with “reduction,” “silence,” “downregulation,” and other similar terms, and includes any level of inhibition.

[0121] As used herein, the term "treatment" includes suppressing, slowing, stopping, or reversing existing symptoms or the progression or severity of a patient's condition. Therefore, treatment includes prevention, treatment, and / or cure. Furthermore, "treatment method," "treatment," "relief," or "improvement" may be used interchangeably herein. Prevention refers to preventing potential disease and / or preventing the worsening of symptoms or the development of disease. "Improvement" refers to at least one indicator of the reduction, slowing, cessation, or reversal of symptoms or the severity of a disease. The severity of an indicator can be determined by subjective or objective measures known to those skilled in the art. As used herein, "efficacy" refers to the effect resulting from treatment of an individual that alters, improves, or enhances the symptoms of a disease or condition, or cures a disease or condition.

[0122] As used herein, a "therapeutic effective amount" or "therapeutic effective dose" refers to an amount of substance, compound, material, or composition containing a compound that, when applied to a subject, is at least sufficient to produce a therapeutic effect. Therefore, it is the amount necessary to prevent, cure, improve, block, or partially block the symptoms of a disease or condition. As used herein, a "preventive effective amount" or "preventive effective dose" refers to an amount of substance, compound, material, or composition containing a compound that, when applied to a subject, would have the intended preventive effect, for example, preventing or delaying the occurrence or recurrence of a disease or symptom, or reducing the likelihood of the occurrence or recurrence of a disease or symptom. A fully preventive effective dose does not necessarily occur through the application of a single dose and may occur only after the application of a series of doses. Therefore, a preventive effective amount may be applied in one or more administrations.

[0123] As used herein, the term "patient" refers to any animal, such as a mammal or marsupial. The subject matter of this disclosure includes, but is not limited to, humans, non-human primates (e.g., rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats, and any kind of poultry.

[0124] As used herein, the term "MMP-7 gene-mediated disease or condition" refers to a pathological state in which abnormal expression, function, or regulation of the MMP-7 gene is the core pathogenic link or key contributing factor. MMP-7 gene-mediated diseases or conditions include, but are not limited to: idiopathic pulmonary fibrosis, asthma, other types of fibrosis, chronic inflammation, interstitial lung disease, SARS-CoV-2 or other types of infectious diseases, acute respiratory distress syndrome or other types of acute lung injury, pulmonary hypertension, cancer, renal fibrosis, and liver fibrosis.

[0125] II. Detailed Description of Implementation Methods [Double-stranded oligonucleotides or their salts targeting the MMP-7 gene] In a first aspect, this disclosure provides a double-stranded oligonucleotide agent or a salt thereof that targets MMP-7 mRNA, wherein the double-stranded oligonucleotide agent or the salt thereof can effectively inhibit the expression of the MMP-7 gene, thereby treating related diseases (e.g., idiopathic pulmonary fibrosis). The double-stranded oligonucleotide agent or the salt thereof can effectively inhibit the expression of the MMP-7 gene in vivo and in vitro.

[0126] Specifically, this disclosure provides a double-stranded oligonucleotide agent or its salt targeting the MMP-7 gene, characterized in that the double-stranded oligonucleotide agent or its salt comprises a sense strand and an antisense strand, 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 the MMP-7 mRNA. Substantialloyment means that the mismatch between the sense strand and the antisense strand in their complementary regions does not exceed 3 nucleotides. Preferably, the sense strand and the antisense strand independently comprise 17-25 nucleotides, more preferably, the sense strand and the antisense strand each independently comprise 19, 20, 21, 22, or 23 nucleotides.

[0127] Further, the antisense strand sequence of the double-stranded oligonucleotide 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, or at least 23 consecutive nucleotides selected from any of the sequences shown in SEQ ID NO: 1-85, and the nucleotide differences do not exceed 3 (e.g., 3, 2, 1, or 0).

[0128] Further, the antisense strand sequence of the double-stranded oligonucleotide 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, or at least 23 consecutive nucleotides selected from any of the sequences shown in SEQ ID NO: 1-85, and the nucleotide differences do not exceed 3 (e.g., 3, 2, 1, or 0); the length of the antisense strand does not exceed 26 nucleotides (e.g., 21, 22, 23, 24, 25, or 26). More preferably, the nucleotide sequence of the antisense strand comprises any of the nucleotide sequences shown in SEQ ID NO: 1-85; and the length of the antisense strand does not exceed 21, 22, 23, 24, 25, or 26 nucleotides. More preferably, the nucleotide sequence of the antisense strand is selected from any of the nucleotide sequences shown in SEQ ID NO: 1-85.

[0129] Furthermore, the positive strand sequence of the double-stranded oligonucleotide comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 consecutive nucleotides selected from any of the sequences shown in SEQ ID NO: 86-170, and the nucleotide differences do not exceed 3 (e.g., 3, 2, 1, or 0).

[0130] Further, the positive strand sequence of the double-stranded oligonucleotide comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 consecutive nucleotides selected from any of the sequences shown in SEQ ID NO: 86-170, and the nucleotide differences do not exceed 3 (e.g., 3, 2, 1, or 0); the length of the positive strand does not exceed 24 nucleotides (e.g., 19, 20, 21, 22, 23, or 24). More preferably, the nucleotide sequence of the positive strand comprises any of the nucleotide sequences shown in SEQ ID NO: 86-170; and the length of the positive strand does not exceed 19, 20, 21, 22, 23, or 24 nucleotides. More preferably, the nucleotide sequence of the positive strand is selected from any of the nucleotide sequences shown in SEQ ID NO: 86-170.

[0131] Further, the double-stranded oligonucleotide agent or its salt comprises a sense strand and an antisense strand, wherein the sense strand comprises a nucleotide sequence that is at least partially anticomplementary or substantially complementary to the antisense strand to form a double-stranded region; the substantially complementary nature means that the difference between the sense strand and the antisense strand in the double-stranded region is no more than 3 nucleotides (e.g., 3, 2, 1, or 0). In some embodiments, the difference between the antisense strand and the target sequence is no more than 5, 4, 3, 2, or 1 nucleotide. In some embodiments, the antisense strand is completely anticomplementary to the target sequence.

[0132] In some implementations, the sense and antisense strands are at least partially anticomplementary to form a double-stranded region. In some implementations, the difference between the sense and antisense strands is no more than 5, 4, 3, 2, or 1 nucleotide. In some implementations, the sense and antisense strands are completely anticomplementary.

[0133] Furthermore, the nucleotide sequence of the antisense strand described in this disclosure is selected from any of the nucleotide sequences shown in SEQ ID NO: 1-85, and the nucleotide sequence of the sense strand is a sequence selected from SEQ ID NO: 86-170 that is complementary to any of the above-mentioned antisense strands (the correspondence is shown in Table 4a).

[0134] Furthermore, each nucleotide of the double-stranded oligonucleotide agent or its salt described in this disclosure is independently selected from modified or unmodified nucleotides.

[0135] Furthermore, in the double-stranded oligonucleotide agent or its salt described in this disclosure, at least one nucleotide in the sense strand and / or antisense strand is a modified nucleotide. Preferably, at least one nucleotide in the antisense strand is a modified nucleotide; more preferably, all nucleotides in the antisense strand are modified nucleotides. Preferably, at least one nucleotide in the sense strand is a modified nucleotide; more preferably, all nucleotides in the sense strand are modified nucleotides.

[0136] Furthermore, the nucleotides of the sense and antisense strands of the double-stranded oligonucleotides described in this disclosure contain at least one base-free nucleotide.

[0137] Furthermore, each of the modified nucleotides described in this disclosure is independently selected from 2'-halogenated nucleotides, 2'-deoxynucleotides, 2'-C-alkyl-modified nucleotides, 2'-C-substituted alkyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, 2'-O-substituted alkyl-modified nucleotides, 2'-amino-modified nucleotides, 2'-substituted amino-modified nucleotides, nucleoside thiophosphates, or phosphate ester analogs.

[0138] Preferably, the modified nucleotides described in this disclosure are nucleotide analogs, more preferably peptide nucleic acid (PNA), morpholino nucleic acid (MNA), bridged nucleic acid (BNA), locked nucleic acid (LNA), ethylene glycol nucleic acid / glycerol nucleic acid (GNA), threocyanate nucleic acid (TNA), or unlocked nucleic acid (UNA).

[0139] Preferably, the modified nucleotides described in this disclosure are each independently selected from 2'-fluoronucleotides, 2'-methoxynucleotides, 2'-deoxynucleotides, 2'-O-methoxyethylnucleotides, abase-free nucleotides, reverse nucleotides, reverse abase-free nucleotides (InvAb), reverse 2'-OMe nucleotides, reverse 2'-deoxynucleotides, or nucleotides containing non-natural bases or modified bases.

[0140] Furthermore, the 3' and / or 5' ends of the sense and / or antisense strands of the double-stranded oligonucleotide agent or its salt described in this disclosure independently contain one or more ingredients selected from (…). E )-Vinylphosphonate (( E Modification with 1-VP), methylphosphonate (MP), or thiophosphate.

[0141] Furthermore, the sense and / or antisense strands of the double-stranded oligonucleotide agent or its salts disclosed herein comprise a 3' overhang and / or a 5' overhang having at least one nucleotide. Preferably, the overhang has at least two, three, four, or five nucleotides. Preferably, the overhang is located at the 3' end of the antisense strand.

[0142] Furthermore, the sense and / or antisense strands of the double-stranded oligonucleotide agent or its salts disclosed herein independently contain one or more phosphate thioester bonds. Preferably, the sense and / or antisense strands independently contain one or more phosphate thioester bonds at the 3' and / or 5' ends. More preferably, the sense and / or antisense strands contain two consecutive phosphate thioester bonds between the 3' and / or 5' nucleotides.

[0143] In this disclosure, unless otherwise specified, uppercase letters C, G, U, A, T and corresponding lowercase letters denote the base composition of nucleotides; when modified nucleotides are involved, the nucleotides or modified nucleotides contained in the antisense or sense strands described in this disclosure are represented in their nucleotide sequences as shown in Table 1 below: Table 1 Further, the double-stranded oligonucleotide agent or its salt described in this disclosure comprises a sense strand and an antisense strand, wherein the nucleotide sequence of the 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, or at least 23 consecutive nucleotides selected from any one of SEQ ID NO: 171-278 and 377-438, and the nucleotide difference does not exceed 3 (e.g., 3, 2, 1, or 0). More preferably, the nucleotide sequence of the antisense strand is selected from any one of the nucleotide sequences shown in SEQ ID NO: 171-278 and 377-438.

[0144] Further, 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, or at least 23 consecutive nucleotides selected from any one of SEQ ID NO: 171-278 and 377-438, with no more than 3 nucleotide differences (e.g., 3, 2, 1, or 0); the length of the antisense strand does not exceed 26 nucleotides (e.g., 21, 22, 23, 24, 25, or 26). More preferably, the nucleotide sequence of the antisense strand comprises a nucleotide sequence selected from any one of SEQ ID NO: 171-278 and 377-438; and the length of the antisense strand does not exceed 21, 22, 23, 24, 25, or 26 nucleotides.

[0145] Furthermore, the antisense strand comprises at least 16 consecutive nucleotides of any of the sequences shown in SEQ ID NO: 171-278 and 377-438, or consecutive nucleotides differing from the consecutive nucleotides by no more than 3 nucleotides.

[0146] Further, the antisense strand comprises at least 16 consecutive nucleotides of any of the sequences shown in SEQ ID NO: 171-278 and 377-438, or consecutive nucleotides differing from the consecutive nucleotides by no more than 2 nucleotides.

[0147] Further, the antisense strand comprises at least 16 consecutive nucleotides of any of the sequences shown in SEQ ID NO: 171-278 and 377-438, or consecutive nucleotides differing from the consecutive nucleotides by no more than 1 nucleotide.

[0148] Further, the positive strand sequence of the double-stranded oligonucleotide agent or its salt is selected from at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 consecutive nucleotides of any sequence in SEQ ID NO: 279-367 and 439-500, and the nucleotide differences do not exceed 3 (e.g., 3, 2, 1, or 0). More preferably, the nucleotide sequence of the positive strand is selected from any of the nucleotide sequences shown in SEQ ID NO: 279-367 and 439-500.

[0149] Further, the positive strand sequence comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 consecutive nucleotides selected from any one of SEQ ID NO: 279-367 and 439-500, with no more than 3 nucleotide differences (e.g., 3, 2, 1, or 0); the length of the positive strand does not exceed 24 nucleotides (e.g., 19, 20, 21, 22, 23, or 24). More preferably, the nucleotide sequence of the positive strand comprises nucleotide sequences selected from any one of SEQ ID NO: 279-367 and 439-500; and the length of the positive strand does not exceed 19, 20, 21, 22, 23, or 24 nucleotides.

[0150] Furthermore, the positive chain comprises at least 16 consecutive nucleotides of any of the sequences shown in SEQ ID NO: 279-367 and 439-500, or consecutive nucleotides differing from the consecutive nucleotides by no more than 3 nucleotides.

[0151] Furthermore, the positive chain comprises at least 16 consecutive nucleotides of any of the sequences shown in SEQ ID NO: 279-367 and 439-500, or consecutive nucleotides differing from the consecutive nucleotides by no more than 2 nucleotides.

[0152] Furthermore, the positive chain comprises at least 16 consecutive nucleotides of any of the sequences shown in SEQ ID NO: 279-367 and 439-500, or consecutive nucleotides differing from the consecutive nucleotides by no more than 1 nucleotide.

[0153] Furthermore, the nucleotide sequence of the antisense strand described in this disclosure is selected from any of the nucleotide sequences shown in SEQ ID NO: 171-278 and 377-438, and the nucleotide sequence of the sense strand is a sequence selected from SEQ ID NO: 279-367 and 439-500 that is complementary to any of the above-mentioned antisense strands.

[0154] Furthermore, the double-stranded oligonucleotide agents described in this disclosure have nucleotide sequence combinations of the sense and antisense strands as shown in Table 4b or Table 4c.

[0155] Furthermore, the double-stranded oligonucleotide includes the sense strand-related sequence as described in this invention, while the sense strand length does not exceed 24 nucleotides, and / or includes the antisense strand-related sequence as described in this invention, while the antisense strand length does not exceed 26 nucleotides.

[0156] Furthermore, the double-stranded oligonucleotide includes the sense strand-related sequence as described in this invention, while the sense strand length does not exceed 22 nucleotides, and / or includes the antisense strand-related sequence as described in this invention, while the antisense strand length does not exceed 24 nucleotides.

[0157] Furthermore, the double-stranded oligonucleotide includes the sense strand-related sequence as described in this invention, while the sense strand length does not exceed 20 nucleotides, and / or includes the antisense strand-related sequence as described in this invention, while the antisense strand length does not exceed 22 nucleotides.

[0158] [Double-stranded oligonucleotide conjugates targeting the MMP-7 gene] In a second aspect of this disclosure, a double-stranded oligonucleotide conjugate is provided, characterized in that the conjugate comprises one or more ligands capable of binding to a cell receptor.

[0159] Further, the ligand conjugate is attached to the sense strand and / or antisense strand of the double-stranded oligonucleotide; more preferably, the ligand conjugate is attached to the 3' end and / or 5' end of the sense strand and / or antisense strand of the double-stranded oligonucleotide; even more preferably, the ligand conjugate is attached to the 3' end of the sense strand of the double-stranded oligonucleotide.

[0160] Furthermore, the ligand includes a targeting group, such as a cell or tissue target. Examples of ligands include, but are not limited to, lectins, glycoproteins, lipids, proteins (such as glycoproteins, peptides, or antibodies that bind to specific cell types), thyroid-stimulating hormone, melanocyte-stimulating hormone, lectins, glycoproteins, surfactant protein A, mucin carbohydrates, polylactose, polygalactose, N-acetylgalactosamine, N-acetylglucosamine, polymannose, polyfucose, glycosylated polyamino acids, polygalactose, transferrin, bisphosphonates, polyglutamate, polyaspartate, lipids, cholesterol, steroids, bile acids, folic acid, vitamin B12, biotin, RGD peptides, RGD peptide mimics, or aptamers.

[0161] Further, the ligand is a protein ligand, antibody, peptide, aptamer, or small molecule of an epithelial cell membrane protein; further, the ligand is a ligand targeting integrins, such as an integrin-targeting ligand with affinity for integrin αvβ6. Integrins are a family of transmembrane receptors that promote cell-extracellular matrix (ECM) adhesion. Among them, alpha-v-beta-6 (αvβ6) is an epithelial-specific integrin. αvβ6 is known to be a receptor for ECM proteins and TGF-β latency-related peptides and is expressed in various cells and tissues. Moreover, integrin αvβ6 is highly upregulated in damaged lung epithelial cells.

[0162] Further, the ligand is a lipid or lipid-based molecule, such as a long-chain hydrocarbon group, cholesterol, fatty acids, bile acids, folic acid, steroids, phospholipids, sphingolipids, or phospholipid analogs. Preferably, the long-chain hydrocarbon group is a C6-C30 straight-chain, branched, or cyclic structure containing a functional group; and the functional group is selected from hydroxyl, amino, aldehyde, ketone, carboxyl, sulfonic acid, phosphate, thiol, azide, and alkynyl groups. More preferably, the hydrocarbon group is linked to the double-stranded oligonucleotide by one or more linkers, the linkers being biodegradable or non-biodegradable, such as ethers, amides, hydrazones, acetals, hemiacetals, ureas, esters, sulfonates, maleamide-thioethers, thioureas, disulfide bonds, and click-reactive linkers of azide-alkynyl groups; more preferably, the lipid group is linked to the 2' position, phosphate group, thiophosphate group, or base of the nucleotide unit of the double-stranded oligonucleotide; more preferably, the lipid group is linked to the 2' position of the nucleotide unit by an ether bond; more preferably, the nucleotide unit containing the lipid group is located at any position on the sense and antisense strands of the double-stranded oligonucleotide; more preferably, the nucleotide unit containing the lipid group is located at the sixth and / or sixteenth position counting from the 5' end on the sense strand of the double-stranded oligonucleotide.

[0163] Furthermore, the ligand is a fatty acid, including fatty acids containing twelve carbon atoms, fourteen carbon atoms, sixteen carbon atoms, eighteen carbon atoms, twenty carbon atoms, and twenty-two carbon atoms.

[0164] Furthermore, the ligand is a fatty acid, attached to either the sense or antisense strand; Furthermore, the ligand is a fatty acid, attached to any intrachain position of the positive chain, such as the sixth position and / or the sixteenth position starting from the 5' end; Furthermore, the ligand is a fatty acid, attached to the 3' and / or 5' ends of the sense and antisense chains; Furthermore, the ligand is a long-chain hydrocarbon group, such as a hydrocarbon group containing twelve carbon atoms, fourteen carbon atoms, sixteen carbon atoms, eighteen carbon atoms, twenty carbon atoms, or twenty-two carbon atoms. More preferably, the ligand is a hydrocarbon group containing sixteen carbon atoms (C16) or a hydrocarbon group containing twenty-two carbon atoms (C22).

[0165] Further, the hydrocarbon group is attached to the 2'-position of the nucleotide unit; more preferably, the hydrocarbon group is attached at the 2'-position as a 2'-O-hydrocarbon group and / or a 2'-hydrocarbon group; even more preferably, the hydrocarbon group is attached at the 2'-position as a 2'-O-hydrocarbon group; even more preferably, the hydrocarbon group is C16 and / or C22 at the 2'-position.

[0166] Further, the ligand is cholesterol, preferably, one or more cholesterol molecules are attached to the ends of the sense and / or antisense strands; preferably, one cholesterol molecule is attached to the 3' end of the sense strand.

[0167] Further, the ligand is a ligand that binds to the desialyl glycoprotein receptor. Preferably, the ligand is selected from galactose clusters; the galactose clusters contain targeting groups having 1-4 (e.g., 1, 2, 3, or 4) galactose or galactose derivatives; the galactose derivatives are galactose derivatives with an affinity for the desialyl glycoprotein receptor equal to or greater than that of galactose. More preferably, the galactose clusters contain molecules having 1-4 (e.g., 1, 2, 3, or 4) N-acetylgalactosamine (GalNAc) molecules, i.e., the ligand is preferably an N-acetylgalactosamine (GalNAc) derivative. More preferably, the ligand can be a known GalNAC derivative ligand such as the L96 ligand.

[0168] Furthermore, the ligand is directly covalently linked to the oligonucleotide or linked via a linker. The linker can be a straight-chain linker or a branched-chain linker.

[0169] Furthermore, when the ligand is an liposome, it is conjugated to oligonucleotides through monovalent, divalent, or trivalent branched head.

[0170] Furthermore, when the ligand is a GalNAc derivative, it is conjugated to oligonucleotides via monovalent, divalent, or trivalent branched head links.

[0171] Furthermore, the sense and / or antisense strands of the double-stranded oligonucleotide agent or its salt are coupled to an optional ligand at their 3' ends via a phosphate ester bond or a thiophosphate bond.

[0172] Furthermore, the sense chain and / or antisense chain are optionally coupled to the ligand at their 5' or 3' ends.

[0173] Furthermore, the sense and / or antisense chains are coupled to an aliphatic group at their 5' or 3' ends.

[0174] Furthermore, the sense chain and / or antisense chain are coupled to an aliphatic group at any position within the chain.

[0175] Furthermore, an aliphatic group is introduced at the sixth and / or sixteenth position counting from the 5' end of the justice chain.

[0176] Furthermore, the justice chain is coupled to the L96 ligand at its 3' end.

[0177] Taking the L96 ligand as an example, the structural diagram of its connection to siRNA is shown below (this is only an illustrative representation of the conjugate structure of siRNA and ligand and should not be taken as a limitation on the conjugate structure; there will be some differences for different ligands and / or different siRNAs and connection methods): , in, This indicates siRNA.

[0178] Furthermore, the sense and / or antisense strands of the siRNA are optionally coupled to the ligand via a phosphate ester bond or a thiophosphate bond at their 5' or 3' ends.

[0179] Furthermore, the double-stranded oligonucleotide agents or their salts described in this disclosure have corresponding combinations of sense and antisense strands as shown in Table 4b or Table 4c.

[0180] Single-stranded oligonucleotides targeting the MMP-7 gene In a third aspect of this disclosure, a single-stranded oligonucleotide targeting the MMP-7 gene is provided, said single-stranded oligonucleotide comprising a sequence of the aforementioned double-stranded oligonucleotide that is at least partially complementary or substantially complementary to a nucleotide sequence in the mRNA of MMP-7.

[0181] Further, the single-stranded oligonucleotide of this disclosure 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, or at least 23 consecutive nucleotides of the sequence shown in SEQ ID NO: 1-85 or SEQ ID NO: 171-278 and 377-438, or consecutive nucleotides differing from said nucleotides by no more than 3 nucleotides (e.g., 3, 2, 1, or 0 nucleotides). More preferably, the nucleotide sequence of said single-stranded oligonucleotide is selected from the nucleotide sequences shown in SEQ ID NO: 1-85 or SEQ ID NO: 171-278 and 377-438.

[0182] Furthermore, the single-stranded oligonucleotide is an antisense oligonucleotide that can perform or participate in any of the various biological functions described herein or known in the art, including RNA interference, RNase H-mediated cleavage, exon jumping, exon jumping prevention, enhancing or inhibiting agents, or any other biological function performed by the antisense oligonucleotide.

[0183] [Composition / Pharmaceutical Composition] In a fourth aspect, this disclosure provides a composition comprising the double-stranded oligonucleotide agent or a salt thereof. Preferably, the composition of this disclosure is a pharmaceutical composition, further comprising a pharmaceutically acceptable excipient or carrier.

[0184] Preferably, the composition further comprises one or more additional therapeutic components.

[0185] Preferably, the composition further comprises a delivery formulation; preferably, the delivery formulation comprises lipid nanoparticles (LNP), lipid polypolymers (LPP), polymer nanoparticles (PNP), inorganic nanoparticles (INP), cationic nanoemulsions (CNE), exosomes, and biological microvesicles.

[0186] Preferably, the composition is packaged in a box, container, packaging material, dispenser, pre-filled syringe, or vial.

[0187] Preferably, the pharmaceutical composition further comprises a delivery formulation; preferably, the delivery formulation comprises lipid nanoparticles (LNP), lipid polypolymers (LPP), polymer nanoparticles (PNP), inorganic nanoparticles (INP), cationic nanoemulsions (CNE), exosomes, or biological microvesicles.

[0188] Preferably, the pharmaceutical composition further comprises a second therapeutic agent; preferably, the second therapeutic agent is selected from antifibrotic drugs, glucocorticoids, neuromuscular blocking agents, anticholinergic drugs, leukotriene receptor antagonists, β2 receptor agonists, biological targeting agents, and anti-infective drugs; more preferably, the second therapeutic agent is selected from nintedanib, pirfenidone, and pharmaceutically acceptable salts or esters thereof.

[0189] Preferably, the composition is formulated for administration via ocular, vaginal, rectal, nasal, transdermal, subcutaneous, intravenous, intra-arterial, intralymphatic, intrabronchial, intrapleural, intraperitoneal, cerebrospinal, or intramuscular injection, or for administration via the lungs, intrathecal, or intracardiac.

[0190] This disclosure also provides cell, carrier, and pharmaceutical compositions comprising any of the double-stranded oligonucleotide agents of this disclosure or salts thereof. The double-stranded oligonucleotide agent may be formulated in a non-buffered solution (such as saline or water) or a buffered solution (such as a solution comprising acetate, citrate, protamine, carbonate, or phosphate, or any combination thereof). In one embodiment, the buffered solution is phosphate-buffered saline (PBS).

[0191] This disclosure also provides salts comprising any of the double-stranded oligonucleotide agents disclosed herein, wherein the salts are preferably sodium or potassium salts.

[0192] [Uses for treating diseases] In a fifth aspect, this disclosure provides the use of the double-stranded oligonucleotide agent of this disclosure or a salt thereof in the preparation of a medicament for the prevention and / or treatment of MMP-7 gene-mediated diseases or conditions. Preferably, the disease or condition is selected from idiopathic pulmonary fibrosis (IPF), asthma, other types of fibrosis, chronic inflammation, interstitial lung disease (ILD), SARS-CoV-2 or other types of infectious diseases, acute respiratory distress syndrome (ARDS) or other types of acute lung injury, pulmonary hypertension, cancer, renal fibrosis, and liver fibrosis. More preferably, the disease or condition is idiopathic pulmonary fibrosis.

[0193] Furthermore, in a sixth aspect of this disclosure, the present disclosure provides the use of the composition of the third aspect in the preparation of a medicament for the prevention and / or treatment of MMP-7 gene-mediated diseases or conditions. Preferably, the disease or condition is selected from idiopathic pulmonary fibrosis (IPF), asthma, other types of fibrosis, chronic inflammation, interstitial lung disease (ILD), SARS-CoV-2 or other types of infectious diseases, acute respiratory distress syndrome (ARDS) or other types of acute lung injury, pulmonary hypertension, cancer, renal fibrosis, and liver fibrosis. More preferably, the disease or condition is idiopathic pulmonary fibrosis.

[0194] Furthermore, the MMP-7 gene-mediated diseases or conditions described in this disclosure are diseases or conditions caused by abnormal MMP-7 gene expression. More specifically, they are diseases or conditions caused by abnormally elevated MMP-7 gene expression.

[0195] Methods to reduce or inhibit MMP-7 gene expression or activity In a seventh aspect of this disclosure, the disclosure provides a method for reducing or inhibiting the expression and / or activity of the MMP-7 gene.

[0196] Preferably, the method is a method for reducing or inhibiting the expression and / or activity of the MMP-7 gene in vivo.

[0197] Preferably, the method includes the step of contacting cells with an effective amount of the aforementioned double-stranded oligonucleotide agent or its salt and / or the aforementioned composition.

[0198] The method includes the step of contacting the cell with any of the following: (1) The double-stranded oligonucleotide agent or its salt disclosed herein; (2) The conjugate of this disclosure; (3) The compositions disclosed herein; and / or (4) The pharmaceutical composition disclosed herein.

[0199] Treatment methods for the disease In a seventh aspect, this disclosure provides a method for preventing and / or treating MMP-7 gene-mediated diseases or conditions, said method comprising administering to a subject an effective amount of any one of the following: (1) The double-stranded oligonucleotide agent or its salt disclosed herein; (2) The conjugate of this disclosure; (3) The compositions disclosed herein; and / or (4) The pharmaceutical composition disclosed herein.

[0200] Furthermore, the method for preventing and / or treating diseases or conditions mediated by the MMP-7 gene is to prevent and / or treat diseases or conditions caused by abnormal MMP-7 gene expression, and more specifically, to prevent and / or treat diseases or conditions caused by abnormally elevated MMP-7 gene expression.

[0201] Preferably, the disease or condition is selected from idiopathic pulmonary fibrosis (IPF), asthma, other types of fibrosis, chronic inflammation, interstitial lung disease (ILD), SARS-CoV-2 or other types of infectious diseases, acute respiratory distress syndrome (ARDS) or other types of acute lung injury, pulmonary hypertension, cancer, renal fibrosis, and liver fibrosis. More preferably, the disease or condition is idiopathic pulmonary fibrosis.

[0202] Example For the purpose of clarity and concise description, the features are described herein as part of some identical or separate embodiments; however, it will be understood that the scope of this disclosure may include some embodiments having a combination of all or some of the features described.

[0203] Test reagents and equipment Unless otherwise stated, all reagents and instruments used in this disclosure are commercially available products from the manufacturers / suppliers listed in Table 2 below.

[0204] Table 2. Main Reagents and Instruments The primer sequences and batch information used in the examples are shown in Table 3, provided by Sangon Biotech (Shanghai) Co., Ltd.

[0205] Table 3. Primer and probe table Experimental result calculation method In the context of this disclosure, unless otherwise stated, the real-time quantitative PCR detection data of the activity evaluation experiments involved in this disclosure are all calculated using the ΔΔCt method to perform relative quantification of the target gene mRNA in each test group. The calculation method is summarized as follows: ΔΔCT = (CT target gene - CT internal reference gene) experimental group - (CT target gene - CT internal reference gene) negative control group, where the relative expression level of the target gene = 2 - ΔΔCT .

[0206] The inhibition rate of siRNA = (1 - relative expression level of the target gene) × 100%.

[0207] For the purpose of clarity and concise description, the features are described herein as part of some identical or separate embodiments; however, it will be understood that the scope of this disclosure may include some embodiments having a combination of all or some of the features described.

[0208] The present disclosure will now be described in more detail with reference to specific embodiments. However, the embodiments are for illustrative purposes only and are not intended to limit the scope of the present disclosure.

[0209] Example 1: siRNA sequence design Based on the mechanism of MMP-7 mRNA and siRNA, the naked sequence of the sense strand and the complementary naked sequence of the antisense strand, as shown in Table 4a, were designed. The lengths of the two strands are either 21 / 23 or 19 / 21.

[0210] Based on existing knowledge in this field, to avoid degradation of unmodified naked sequences during silencing activity evaluation, and to better reflect their silencing effect, their respective modification forms were designed, as shown in Table 4b. The 2'-O-methyl and 2'-fluoro modifications, and the terminal thiophosphate modification, are beneficial for siRNA to exert its silencing activity. Furthermore, to obtain even better silencing effects, a (…) was introduced at the 5' end of the antisense strand. E Modifications to the )-vinylphosphonate (VP) group. Further, to enhance the ability of siRNA to penetrate cell membranes, lipid groups, such as sixteen-carbon hydrocarbon groups and cholesterol, are introduced into the positive strand. For example, a C16 hydrocarbon group is introduced at the sixth position of the positive strand, and / or a cholesterol group (linked with a C6 hydrocarbon group) is introduced at the 3'-terminus, as shown in Table 4c.

[0211] On the one hand, the silencing ability evaluation results of modified siRNA can indicate whether unmodified siRNA can cleave the target fragment of MMP-7 mRNA through RNAi mechanism and achieve the result of silencing the mRNA; on the other hand, the modification types of the naked sequences shown in Table 4a are not limited to the modification methods presented in the embodiments of this patent.

[0212] Table 4a. siRNA naked sequence information Table 4b. Modified sequences based on bare sequences Table 4c. siRNAs with lipid group modifications Example 2: Synthesis of siRNA molecules 1. Solid-phase synthesis and ammonolysis The desired sequence was imported into an Oligo 48 synthesizer, and synthesized sequentially from the 3'-5' direction using Universal CPG / L96 GalNAc as the support. The phosphoramide monomer was dissolved in anhydrous acetonitrile (100 mM or 200 mM) and soaked in molecular sieves for at least 24 h. Each base linkage or modification involved four steps: deprotection, coupling, capping, and oxidation (or thiolation), ultimately yielding a nucleotide sequence with a solid support and protecting groups. Specific synthetic conditions are as follows: (1) The deprotection reaction was carried out using a 3% trichloroacetic acid solution in dichloromethane to remove the protecting group of the 5'-OH of the nucleotide attached to the solid support. The reaction time was 60 s.

[0213] (2) The coupling reaction uses 0.3M acetonitrile solution of 5-ethylthio-1H-tetrazole. The phosphoramide monomer or the phosphoramide monomer containing C16 reacts with the exposed 5'-OH to form a phosphate triester bond. The coupling time is 300 s.

[0214] (3) The capping reaction uses CAP A / CAP B (1 / 1, v / v) solution, where CAP A is a 20% N-methylimidazolium pyridine / acetonitrile mixed solution and CAP B is a 20% acetic anhydride acetonitrile solution to block unreacted 5'-OH groups, and the capping time is 20 s.

[0215] (4) The oxidation reaction used 0.05 M iodine solution for 25 s; the thiolation reaction used 0.2 M hydroflavin pyridine solution for 200 s.

[0216] (5) After the last nucleoside monomer is ligated, the dried solid support is added to a 25% ammonia solution and reacted at 55°C for 16 hours. The solid support is washed twice with ethanol / water, centrifuged and concentrated, dissolved in RNase-free water, filtered and purified.

[0217] 2. Purification Purification was performed using ion-pair reversed-phase chromatography (IP-RP-HPLC), where the target product and impurities were separated by gradient elution, and the desired components were collected and detected.

[0218] 3. QC testing and freeze-drying After purification, the collected samples were analyzed by HPLC and MS to verify their purity and molecular weight accuracy. The qualified fractions were quantified using a Nanodrop One instrument, and then freeze-dried to obtain the target product. The obtained double-stranded siRNA sample was freeze-dried to obtain a solid powder and stored at -65°C or below until analysis.

[0219] Structural information and characterization results are detailed in Table 5.

[0220] Table 5. Purity and molecular weight information of siRNA Example 3: In vitro activity verification of siRNA Using A549 cells (human non-small cell lung cancer cell line, Nanjing Kebai Biotechnology Co., Ltd., catalog number: CBP60084A549) with high MMP-7 RNA expression, transient transfection and qPCR were used to screen for siRNAs that could silence MMP-7 mRNA expression. The selected siRNAs were then subjected to IC50 assays. 50 The positive control sample was ABN007-55 (WO2025026220A1).

[0221] 1. Preparation of siRNA: The frozen siRNA was dissolved in enzyme-free sterile water, and the concentration was determined using an ultra-micro spectrophotometer and converted to molar concentration. After aliquoting, it was stored at -60°C or below for later use.

[0222] 2. Cell culture: After resuscitation, the cells were cultured and passaged in DMEM / F12 complete medium (DMEM / F12 + 10% FBS + 1% double antibiotics) at 37°C and 5% CO2.

[0223] 3. Cell plating and transfection: The A549 cell density was 1.00E+5 / mL. The cell suspension was transferred to a 96-well plate containing a mixture of siRNA-Opti-MEM and lipo-RNAiMAX, 80 μL / well. The cells were gently tapped to mix and then incubated in a 37°C, 5% CO2 incubator for 48–56 hours.

[0224] 4. RNA extraction and cDNA synthesis: Discard the culture medium from the transfected 96-well plate, add 200 μL of PBS buffer to each well for washing, discard the PBS buffer, add 200 μL of cell lysis buffer to each well, and incubate in a microplate shaker at 700 rpm for 5 min. Transfer all cell lysis buffer to a Binding Plate. Extract RNA using a nucleic acid extractor, and then reverse transcribe to synthesize cDNA.

[0225] 5. Real-time quantitative PCR (qPCR): Using diluted cDNA as a template, qPCR was performed using specific primers and SYBR Green premixed reagent. The reaction program was: 94℃ pre-denaturation for 30 seconds; followed by 40 cycles of amplification (94℃ for 5 seconds, 60℃ for 30 seconds). The CT values ​​of the target gene MMP-7 and the internal reference gene GAPDH were measured in each sample, and the inhibition rate and IC50 were calculated through data analysis. 50 The value shows an inhibition rate ranging from 25% to 96%, and the IC50 value is [value missing]. 50 The value ranges from 0.001 to 300 pM.

[0226] The inhibitory effects of siRNA transfection on MMP-7 mRNA in cells are shown in Tables 6 and 7, respectively. Figure 1 As shown.

[0227] Table 6. siRNA selection inhibition rate Table 7. siRNA IC 50 data Example 4: Evaluation of siRNA activity in mice AAV vectors or plasmids carrying human MMP 7-SEAP were intravenously injected into 6- to 8-week-old C57BL / 6 mice. Fourteen days post-injection, each mouse was subcutaneously injected with 1–3 mg / kg siRNA. Blood samples were collected before administration and on days 7 (D7), 14 (D14), and 28 post-administration to measure changes in SEAP levels and calculate the inhibition rate against MMP7 mRNA. The inhibition rate in the administered groups ranged from 20% to 80%. Safety was also assessed by measuring serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels.

[0228] Example 5: In vivo activity evaluation of siRNA in transgenic mice Using humanized MMP7 transgenic mice as the evaluation subjects, candidate siRNA molecules or physiological saline were administered to the lungs via nebulized injection at concentrations ranging from 0.5 mg / kg to 3 mg / kg. Treatment lasted for 3 or 7 days. After 14 days, mice were anesthetized and sacrificed. Lung tissue was harvested after perfusion to detect MMP7 mRNA and protein expression levels. In the treated groups, the inhibition rate against MMP7 mRNA ranged from 20% to 80%, and the inhibition rate against protein expression ranged from 5% to 60%.

[0229] The above is a further detailed description of this disclosure and should not be considered as a limitation on the specific implementation of this disclosure. For those skilled in the art, any simple deductions or substitutions that do not depart from the concept of this disclosure are within the scope of protection of this disclosure.

Claims

1. A double-stranded oligonucleotide agent or a salt thereof for inhibiting matrix metalloproteinase 7 (MMP-7) expression, characterized in that, 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, or at least 23 consecutive nucleotides selected from any of the sequences shown in SEQ ID NO: 1-85, and the nucleotide difference does 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, or at least 21 consecutive nucleotides selected from any of the sequences shown in SEQ ID NO: 86-170, and the nucleotide difference does not exceed 3, 2, 1, or 0.

2. The double-stranded oligonucleotide agent or its salt according to claim 1, characterized in that, The antisense strand has a nucleotide sequence comprising any of the nucleotide sequences shown in SEQ ID NO: 1-85, 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 anticomplementary or substantially complementary to the antisense strand to form a double-stranded region; Preferably, the nucleotide sequence of the positive strand comprises a sequence selected from SEQ ID NO: 86-170, and the length of the positive strand does not exceed 23 nucleotides; More preferably, the nucleotide sequence of the antisense strand comprises the nucleotide sequence shown in any of the antisense strands in Table 4a, and 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 length of the antisense strand does not exceed 25 nucleotides, and the length of the sense strand does not exceed 23 nucleotides.

3. The double-stranded oligonucleotide agent or its salt according to claim 1 or 2, characterized in that, Each nucleotide of the double-stranded oligonucleotide agent is independently selected from modified or unmodified nucleotides; Preferably, in the sense and antisense strands of the double-stranded oligonucleotide agent, at least one nucleotide is a modified nucleotide; Preferably, at least one nucleotide in the antisense strand is a modified nucleotide; Preferably, all nucleotides in the antisense strand are modified nucleotides; Preferably, at least one nucleotide in the positive strand is a modified nucleotide; Preferably, all nucleotides in the positive strand are modified nucleotides; Preferably, the nucleotides of the sense and antisense strands of the double-stranded oligonucleotide contain at least one base-free nucleotide. Preferably, the modified nucleotides are each independently selected from 2'-halogenated modified nucleotides, 2'-deoxynucleotides, 2'-C-alkyl-substituted modified nucleotides, 2'-O-alkyl-substituted modified nucleotides, 2'-amino-modified nucleotides, 2'-substituted amino-modified nucleotides, nucleoside thiophosphates or phosphate ester analogs. Preferably, each of the modified nucleotides is independently selected from 2'-fluoronucleotides, 2'-methoxynucleotides, 2'-deoxynucleotides, 2'-O-methoxyethylnucleotides, abase-free nucleotides, reverse nucleotides, reverse abase-free nucleotides (InvAb), reverse 2'-OMe nucleotides, and reverse 2'-deoxynucleotides. Preferably, the modified nucleotide is a nucleotide analog, more preferably peptide nucleic acid (PNA), morpholino nucleic acid (MNA), bridged nucleic acid (BNA), locked nucleic acid (LNA), ethylene glycol nucleic acid / glycerol nucleic acid (GNA), threocyanate nucleic acid (TNA), or unlocked nucleic acid (UNA).

4. The double-stranded oligonucleotide agent or its salt according to any one of claims 1-3, characterized in that, The 3' and / or 5' ends of the justice chain and / or antisense chain independently contain one or more components selected from ( E )-Vinylphosphonate (( E Modification with 1-VP), methylphosphonate (MP), or thiophosphate; Preferably, the sense strand and / or antisense strand of the double-stranded oligonucleotide includes a 3' overhang and / or a 5' overhang having at least one nucleotide. Preferably, the 5' end of the antisense strand of the double-stranded oligonucleotide contains a ( E )-Vinylphosphonate (( E )-VP); Preferably, the sense chain and / or antisense chain independently contain one or more thiophosphate bonds; Preferably, the sense and / or antisense strands independently contain one or more thiophosphate bonds at the 3' and / or 5' ends; Preferably, the sense strand and / or antisense strand contain two consecutive phosphate thioester bonds between the 3' and / or 5' end nucleotides.

5. The double-stranded oligonucleotide agent or its salt according to any one of claims 1-4, characterized in that, The 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, or at least 23 consecutive nucleotides of any of the sequences shown in SEQ ID NO: 171-278 and 377-438, or consecutive nucleotides differing from the consecutive nucleotides by no 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 NO: 171-278 and 377-438, and the length of the antisense strand does not exceed 25 nucleotides; Preferably, the positive chain comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 consecutive nucleotides of any of the sequences shown in SEQ ID NO: 279-367 and 439-500, or consecutive nucleotides differing from the consecutive nucleotides by no more than 3, 2, 1, or 0 nucleotides; Preferably, the nucleotide sequence of the positive strand comprises a nucleotide sequence selected from any one of SEQ ID NO: 279-367 and 439-500, and the length of the positive strand does not exceed 23 nucleotides; More preferably, the nucleotide sequence of the antisense strand comprises the nucleotide sequence shown in any of the antisense strands in Table 4b or Table 4c, and 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 length of the antisense strand does not exceed 25 nucleotides, and the length of the sense strand does not exceed 23 nucleotides.

6. A conjugate of a double-stranded oligonucleotide, characterized in that, The conjugate comprises a double-stranded oligonucleotide agent or a salt thereof according to any one of claims 1-5 and one or more ligands capable of binding to a cell receptor; Preferably, the ligand is selected from ligands capable of binding to cell receptors or lipophilic ligands; preferably, the ligand is a protein ligand of an epithelial cell membrane protein, an antibody, a polypeptide, an aptamer, or a small molecule; More preferably, the ligand is a ligand that targets integrin, including a ligand that targets αvβ6 integrin; Preferably, the ligand is selected from ligands that target groups and bind to desialyl glycoprotein receptors; Preferably, the targeting group includes lectins, glycoproteins, lipids, antibodies, surfactant protein A, mucin carbohydrates, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine polymannose, polyvalent fucose, glycosylated polyamino acids, polyvalent galactose, transferrin, RGD peptide, RGD peptide mimics, or aptamers; 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 molecules having 1-4 N-acetylgalactosamine (GalNAc); Preferably, the ligand is an N-acetylgalactosamine (GalNAc) derivative; Preferably, the ligand is a lipophilic ligand; the lipophilic ligand includes lipids or lipid-based molecules; Preferably, the ligand is selected from cholesterol, steroids, bile acids, folic acid, vitamin E, and vitamin B12; Preferably, the lipid or lipid-based molecule is selected from cholesterol, aliphatic hydrocarbons, fatty acids, bile acids, folic acid, steroids, phospholipids, sphingolipids, and phospholipid analogs. Preferably, the fatty acid is selected from saturated and unsaturated fatty acids containing 6-22 carbon atoms, more preferably from saturated and unsaturated fatty acids containing 16, 18, 20, or 22 carbon atoms. Preferably, the aliphatic hydrocarbon group is selected from saturated and unsaturated aliphatic hydrocarbon groups containing 6-22 carbon atoms, and more preferably from saturated and unsaturated hydrocarbon groups containing sixteen, eighteen, twenty, or 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 attached to the ends of the sense and / or antisense strands; Preferably, a cholesterol molecule is attached to the 3' end of the positive chain; Preferably, the ligand is conjugated to any one or more positions on the sense and / or antisense strands of the double-stranded oligonucleotide; Preferably, the ligand is conjugated to the sixth and / or sixteenth position, starting from the 5' end, of the positive strand of the double-stranded oligonucleotide; Preferably, the ligand is conjugated to the 3' and / or 5' ends of the sense and / or antisense strands of the double-stranded oligonucleotide; Preferably, the ligand is conjugated to the 3' end of the positive strand of the double-stranded oligonucleotide.

7. The conjugate according to claim 6, characterized in that, The double-stranded oligonucleotide has a combination of sense and antisense strands selected from either group in Table 4b or Table 4c: In this context, lowercase letters a, u, g, and c represent 2'-methoxy-3'-nucleotides with the corresponding bases; uppercase letters A, U, G, and C represent 2'-fluoro-3'-nucleotides with the corresponding bases; lowercase letter s indicates that the two nucleotides adjacent to it on the left and right are linked by a phosphate thioester bond; invab indicates that the nucleotide adjacent to it on the right is an inverted, baseless nucleotide; lowercase letter d indicates that the ribose in the nucleotide adjacent to it on the right is deoxyribose; and VPu represents 2'-OMe-5'-( E )-Vinylphosphonate-3'-uridine acid; C16 represents the C16 hydrocarbon group; chol represents the cholesterol group.

8. A composition comprising one or more of the double-stranded oligonucleotide agent according to any one of claims 1-5 or a salt thereof, or the conjugate according to claim 6 or 7; Preferably, the composition is a pharmaceutical composition, which further comprises pharmaceutically acceptable excipients or carriers; Preferably, the pharmaceutical composition further comprises a delivery formulation; preferably, the delivery formulation comprises lipid nanoparticles (LNP), lipid polypolymers (LPP), polymer nanoparticles (PNP), inorganic nanoparticles (INP), cationic nanoemulsions (CNE), exosomes, and biological microvesicles. Preferably, the pharmaceutical composition further comprises a second therapeutic agent; preferably, the second therapeutic agent is selected from antifibrotic drugs, glucocorticoids, neuromuscular blocking agents, anticholinergic drugs, leukotriene receptor antagonists, β2 receptor agonists, biological targeting agents, and anti-infective drugs; more preferably, the second therapeutic agent is selected from nintedanib, pirfenidone, and pharmaceutically acceptable salts or esters thereof.

9. Use of the double-stranded oligonucleotide agent of any one of claims 1-5 or a salt thereof and / or the conjugate of claim 6 or 7 and / or the composition of claim 8 in the preparation of a medicament for the prevention and / or treatment of diseases or conditions mediated by the MMP-7 gene; Preferably, the disease or condition is selected from idiopathic pulmonary fibrosis (IPF), asthma, other types of fibrosis, chronic inflammation, interstitial lung disease (ILD), SARS-CoV-2 or other types of infectious diseases, acute respiratory distress syndrome (ARDS) or other types of acute lung injury, pulmonary hypertension, cancer, renal fibrosis, and liver fibrosis; More preferably, the disease or condition is idiopathic pulmonary fibrosis.

10. A method for reducing or inhibiting MMP-7 gene expression and / or activity, the method comprising the step of contacting a cell with any of the following: (1) The double-stranded oligonucleotide agent or its salt according to any one of claims 1-5; (2) The conjugate according to claim 6 or 7; and / or (3) The composition according to claim 8.

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