MMP7-targeting siRNA, siRNA conjugates, and their pharmaceutical use

Abstract

This invention relates to siRNAs, siRNA conjugates, and their pharmaceutically acceptable uses that target MMP7. It also relates to pharmaceutical compositions, cells, or reagent kits containing such siRNAs, and to methods of using such siRNAs or siRNA conjugates for the treatment and / or prevention of related disorders in subjects.

Description

[Technical Field]

[0001] This disclosure claims priority to China Application No. 202310696482.8 filed on 13 June 2023 and China Application No. 202410074824.7 filed on 18 January 2024, the entire contents of the aforementioned patent applications being incorporated herein by reference.

[0002] This disclosure belongs to the biopharmaceutical field and specifically relates to siRNAs, conjugates, compositions, and their pharmaceutically acceptable uses that target matrix metalloproteinase 7 (MMP7). [Background technology]

[0003] Matrix metalloproteinase 7 (MMP7) is the smallest member (28 kDa) of the metalloproteinase family (MMPs). The MMP family comprises 23 members, each with different substrates and functions, capable of degrading all components of the extracellular matrix (e.g., elastin, proteoglycans, type IV collagen, fibronectin, etc.) as well as many non-matrix proteins such as cytokines. These functional roles in extracellular matrix remodeling and cytokine signaling regulation link MMP family members to common pathogenic mechanisms causing cancer, chronic inflammation, and fibrosis. MMP7 is primarily expressed and secreted in epithelial cells of systemic organs, playing a crucial role in epithelial cell repair. Increased MMP7 expression is associated with pathogenic fibrosis of the lung, liver, and kidney. In idiopathic pulmonary fibrosis (IPF) lung tissue, MMP7 gene and protein expression levels are significantly elevated and also significantly upregulated in bronchoalveolar lavage fluid (BAL). Serum expression of MMP7 is an effective serum biomarker for IPF and is associated with the severity and progression of IPF.

[0004] MMP7 is associated with pathogenic fibrosis through multiple potential mechanisms, including the promotion of epithelial-interstitial transition (EMT), extracellular matrix degradation, abnormal matrix repair, and tissue remodeling. MMP7 promotes fibrosis by cleaving E-cadherin to activate epithelial cells and hydrolyzing proteins to activate heparin-bound epidermal growth factor precursor (pro-HB-EGF), thereby releasing active HB-EGF. Studies have shown that MMP7 gene knockout mice exhibit protective effects against bleomycin-induced pulmonary fibrosis, demonstrating reduced lung inflammation, fibrosis, and mortality, suggesting that MMP7 knockdown may have therapeutic effects against pulmonary fibrosis. [Overview of the Initiative]

[0005] This disclosure provides siRNA. In some embodiments, this disclosure provides siRNA targeting MMP7, comprising a sense strand and an antisense strand forming a double-stranded region.

[0006] In some embodiments, the sense strand includes a sequence that differs from any one of the nucleotide sequences of SEQ ID NOs. 1 to 10 and SEQ ID NOs. 61 to 151 by three or fewer nucleotides (e.g., 0, 1, 2, 3), and includes at least 15 consecutive nucleotides (e.g., 16, 17, 18, 19, 20, 21). The above antisense strand contains a sequence that differs from any one of the nucleotide sequences of SEQ ID NOs. 31 to 40 and SEQ ID NOs. 152 to 242 by three or fewer nucleotides (e.g., 0, 1, 2, 3), and contains at least 15 consecutive nucleotides (e.g., 16, 17, 18, 19, 20, 21).

[0007] In some embodiments, the antisense strand is at least partially reverse-complementary to the target sequence in order to mediate RNA interference. In some embodiments, there are 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 or fewer mismatches between the antisense strand and the target sequence. In some embodiments, the antisense strand is completely reverse-complementary to the target sequence.

[0008] In some embodiments, the sense strand is at least partially inversely complementary to the antisense strand in order to form a double-stranded region. In some embodiments, there are 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, and 1 or fewer mismatches between the sense strand and the antisense strand. In some embodiments, the sense strand is completely inversely complementary to the antisense strand.

[0009] In some embodiments, the siRNA relating to this disclosure includes one or two blunt ends.

[0010] In some embodiments, each siRNA chain contains a protruding end formed independently of one or two unpaired nucleotides.

[0011] In some embodiments, the siRNA relating to this disclosure includes a 3' end overhang of the siRNA antisense strand.

[0012] In some embodiments, the sense strand and the antisense strand each independently have 16-35, 16-34, 17-34, 17-33, 18-33, 18-32, 18-31, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 19-25, 19-24, or 19-23 nucleotides (for example, 19, 20, 21, 22, or 23 nucleotides).

[0013] In some embodiments, the sense strand and the antisense strand are the same length or different, with the sense strand having 19 to 23 nucleotides and the antisense strand having 19 to 26 nucleotides. The sense strand to antisense strand length ratio of the siRNA provided in this disclosure may be 19 / 19, 19 / 20, 19 / 21, 19 / 22, 19 / 23, 19 / 24, 19 / 25, 19 / 26, 20 / 19, 20 / 20, 20 / 21, 20 / 22, 20 / 23, 20 / 24, 20 / 25, 20 / 26, 21 / 20, 21 / 21, 21 / 22, 21 / 23, 21 / 24, 21 / 25, 21 / 26, 22 / 20, 22 / 21, 22 / 22, 22 / 23, 22 / 24, 22 / 25, 22 / 26, 23 / 20, 23 / 21, 23 / 22, 23 / 23, 23 / 24, 23 / 25, or 23 / 26. In some embodiments, the sense strand to antisense strand length ratio of the siRNA is 19 / 21, 21 / 23, or 23 / 25.

[0014] In some specific embodiments, the length ratio of the sense strand to the antisense strand of the siRNA is 19 / 21.

[0015] In some embodiments, the sense strand contains at least 15 consecutive nucleotides and differs by two or fewer nucleotides from the nucleotide sequence shown in any one of SEQ ID NOs: 1 to 10 and SEQ ID NOs: 61 to 151. In some embodiments, it differs by one or fewer nucleotides from the nucleotide sequence, and in some embodiments, it differs by one nucleotide.

[0016] In some embodiments, the antisense strand comprises at least 15 consecutive nucleotides and differs by two or fewer nucleotides from the nucleotide sequence shown in any one of SEQ ID NOs. 31 to 40 and SEQ ID NOs. 152 to 242; in some embodiments, it differs by one or fewer nucleotides from the nucleotide sequence; and in some embodiments, it differs by one nucleotide.

[0017] In some embodiments, the sense strand comprises at least 15 consecutive nucleotides of any one nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 10 and SEQ ID NO: 61 to SEQ ID NO: 151. In some embodiments, the sense strand comprises at least 16 consecutive nucleotides of any one nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 10 and SEQ ID NO: 61 to SEQ ID NO: 151. In some embodiments, the sense strand comprises at least 17 consecutive nucleotides of any one nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 10 and SEQ ID NO: 61 to SEQ ID NO: 151. In some embodiments, the sense strand comprises at least 19 consecutive nucleotides of any one nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 10 and SEQ ID NO: 61 to SEQ ID NO: 151.

[0018] In some embodiments, the antisense strand comprises at least 15 consecutive nucleotides of any one nucleotide sequence of SEQ ID NO: 31 to SEQ ID NO: 40 and SEQ ID NO: 152 to SEQ ID NO: 242. In some embodiments, the antisense strand comprises at least 17 consecutive nucleotides of any one nucleotide sequence of SEQ ID NO: 31 to SEQ ID NO: 40 and SEQ ID NO: 152 to SEQ ID NO: 242. In some embodiments, the antisense strand comprises at least 19 consecutive nucleotides of any one nucleotide sequence of SEQ ID NO: 31 to SEQ ID NO: 40 and SEQ ID NO: 152 to SEQ ID NO: 242. In some embodiments, the antisense strand comprises at least 20 consecutive nucleotides of any one nucleotide sequence of SEQ ID NO: 31 to SEQ ID NO: 40 and SEQ ID NO: 152 to SEQ ID NO: 242. In some embodiments, the antisense strand comprises at least 21 consecutive nucleotides of any one nucleotide sequence of SEQ ID NO: 31 to SEQ ID NO: 40 and SEQ ID NO: 152 to SEQ ID NO: 242.

[0019] In some embodiments, the above sense strand comprises any one nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 10 and SEQ ID NO: 61 to SEQ ID NO: 151, or is selected therefrom.

[0020] In some embodiments, the antisense strand includes or is selected from any one of the nucleotide sequences of SEQ ID NOs: 31 to 40 and SEQ ID NOs: 152 to 242.

[0021] In some embodiments, the siRNA is Group 1), which consists of the sense strand indicated by Sequence ID 1 and the antisense strand indicated by Sequence ID 31, Group 2), which consists of the sense strand indicated by Sequence ID No. 2 and the antisense strand indicated by Sequence ID No. 32, Group 3), which consists of the sense strand indicated by Sequence ID No. 3 and the antisense strand indicated by Sequence ID No. 33, Group 4), which consists of the sense strand indicated by Sequence ID No. 4 and the antisense strand indicated by Sequence ID No. 34, Group 5), which consists of the sense strand indicated by Sequence ID No. 5 and the antisense strand indicated by Sequence ID No. 35, Group 6), which consists of the sense strand indicated by Sequence ID No. 6 and the antisense strand indicated by Sequence ID No. 36, Group 7), which consists of the sense strand indicated by Sequence ID 7 and the antisense strand indicated by Sequence ID 37, Group 8), which consists of the sense strand indicated by Sequence ID No. 8 and the antisense strand indicated by Sequence ID No. 38, The sense strand indicated by Sequence ID No. 9 and the antisense strand indicated by Sequence ID No. 39 (group 9), and It includes, or is selected from, a sense strand and an antisense strand from any one of the following groups: group 10), which consists of a sense strand indicated by sequence number 10 and an antisense strand indicated by sequence number 40.

[0022] In some embodiments, at least one nucleotide of the sense strand and / or antisense strand is a modified nucleotide.

[0023] In some embodiments, all nucleotides are modified nucleotides.

[0024] In some embodiments, the sense strand of the siRNA consists of three consecutive nucleotides that are 2'-fluoromodified.

[0025] In some embodiments, in the sense strand of the siRNA, the nucleotides at positions 7, 8, and 9 of the sense strand are nucleotides modified with 2'-fluoropolymers, in the direction from the 5' end to the 3' end.

[0026] In some embodiments, in the sense strand of the siRNA, the nucleotides at positions 7, 8, and 9 of the sense strand are nucleotides modified with 2'-fluoro groups, in the direction from the 5' end to the 3' end, and all nucleotides at the remaining positions of the sense strand are nucleotides modified with 2'-methoxy groups.

[0027] In some embodiments, the nucleotides at positions 2, 6, 12, 14, and 16 of the antisense chain, in the direction from the 5' end to the 3' end, are each independently nucleotides modified with 2'-fluoro. In some embodiments, the nucleotides at positions 2, 4, 6, 10, 12, 14, 16, or 18 of the antisense chain are each independently nucleotides modified with 2'-fluoro. In some embodiments, the nucleotides at the remaining positions of the antisense chain are all nucleotides modified with 2'-methoxy groups.

[0028] In some embodiments, at least one phosphodiester group of the sense chain and / or antisense chain is a phosphodiester group having a modifying group. The modifying group provides the siRNA with improved stability in biological samples or the environment. In some embodiments, the phosphodiester group having the modifying group is a thiophosphodiester group.

[0029] In some embodiments, the phosphodiester group having the above-mentioned modifying group is Between the first and second nucleotides at the 5' end of the sense strand mentioned above, Between the second and third nucleotides at the 5' end of the sense strand mentioned above, Between the first and second nucleotides at the 5' end of the antisense strand mentioned above, Between the second and third nucleotides at the 5' end of the antisense strand mentioned above, Between the first and second nucleotides at the 3' end of the antisense strand, and It is located at at least one position selected from between the second and third nucleotides at the 3' end of the antisense strand mentioned above.

[0030] In some embodiments, the sense chain and / or antisense chain comprises a plurality of phosphodiester groups having modifying groups.

[0031] In some embodiments, the sense chain and / or antisense chain comprises a plurality of phosphodiester groups having a modifying group, and the phosphodiester groups having the modifying group are Between the first and second nucleotides at the 5' end of the sense strand mentioned above, Between the second and third nucleotides at the 5' end of the sense strand mentioned above, Between the first and second nucleotides at the 5' end of the antisense strand mentioned above, Between the second and third nucleotides at the 5' end of the antisense strand mentioned above, Between the first and second nucleotides at the 3' end of the antisense strand mentioned above, It is located between the second and third nucleotides at the 3' end of the antisense strand mentioned above.

[0032] In some embodiments, the sense chain is Selected from nucleotide sequences containing the formula 5'-NmsNmsNmNmNmNmNfNfNfNmNmNmNmNmNmNmNm-2', In some embodiments, the antisense chain is 5'-Nm'sNf'sNm'Nm'Nm'Nf'Nm'Nm'Nm'Nm'Nm'Nf'Nm'Nf'Nm'Nf'Nm'Nm'Nm'sNm'sNm'-3', or The nucleotide sequence is selected from those containing the expression represented by the formula 5'-Nm'sNf'Nm'Nf'Nm'Nm'Nm'Nm'Nm'Nm'Nf'Nm'Nf'Nm'Nf'Nm'Nf'Nm'sNm'sNm'-3'.

[0033] In the sense and antisense chains described above, Nm and Nm' represent any nucleoside modified with a 2'-methoxy group, for example, C, G, U, A, or T modified with a 2'-methoxy group, and Nf and Nf' represent any nucleoside modified with a 2'-fluoro group, for example, C, G, U, A, or T modified with a 2'-fluoro group. The lowercase letter 's' indicates that the two nucleotides adjacent to it are linked by a thiophosphodiester group. Unless otherwise specified, the two nucleosides adjacent to it are linked by a phosphodiester group.

[0034] In some embodiments, the antisense chain comprises at least one nucleoside at positions 2-8 of its 5' region (e.g., positions 2, 3, 4, 5, 6, 7, and 8) with a chemical modification represented by formula (I) or a pharmaceutically acceptable salt thereof, wherein the chemical modification represented by formula (I) is [ka] Selected from these, of which B represents a base at a position corresponding to the 2-8 position at the 5' end of the antisense chain, and a nucleoside containing the chemical modification shown in formula (I) above or a pharmaceutically acceptable salt thereof is linked to an adjacent nucleoside via a phosphorothioate group or a phosphorothioate group having a modifying group.

[0035] In some embodiments, the sense strand is selected from or includes nucleotide sequences represented by any one of the sequence numbers 11 to 27 and 243 to 333.

[0036] In some embodiments, the antisense strand is selected from or includes nucleotide sequences represented by any one of the sequence numbers 41 to 57 and 334 to 424.

[0037] In a more specific embodiment, this disclosure provides siRNAs comprising or selected from any one of the following groups: The sense strand contains the polynucleotide shown in SEQ ID NO: 11, and the antisense strand contains the polynucleotide shown in SEQ ID NO: 41. The sense strand contains the polynucleotide shown in SEQ ID NO: 12, and the antisense strand contains the polynucleotide shown in SEQ ID NO: 42. The sense strand described above contains the polynucleotide shown in SEQ ID NO: 13, and the antisense strand described above contains the polynucleotide shown in SEQ ID NO: 43. The sense strand contains the polynucleotide shown in SEQ ID NO: 14, and the antisense strand contains the polynucleotide shown in SEQ ID NO: 44. The sense strand contains the polynucleotide shown in SEQ ID NO: 15, and the antisense strand contains the polynucleotide shown in SEQ ID NO: 45. The sense strand contains the polynucleotide shown in SEQ ID NO: 16, and the antisense strand contains the polynucleotide shown in SEQ ID NO: 46. The sense strand described above contains the polynucleotide shown in SEQ ID NO: 17, and the antisense strand described above contains the polynucleotide shown in SEQ ID NO: 47. The sense strand contains the polynucleotide shown in SEQ ID NO: 18, and the antisense strand contains the polynucleotide shown in SEQ ID NO: 48. The sense strand described above contains the polynucleotide shown in SEQ ID NO: 19, and the antisense strand described above contains the polynucleotide shown in SEQ ID NO: 49. The sense strand contains the polynucleotide shown in SEQ ID NO: 20, and the antisense strand contains the polynucleotide shown in SEQ ID NO: 50. The sense strand contains the polynucleotide shown in SEQ ID NO: 21, and the antisense strand contains the polynucleotide shown in SEQ ID NO: 51. The sense strand described above contains the polynucleotide shown in SEQ ID NO: 22, and the antisense strand described above contains the polynucleotide shown in SEQ ID NO: 52. The sense strand described above contains the polynucleotide shown in SEQ ID NO: 23, and the antisense strand described above contains the polynucleotide shown in SEQ ID NO: 53. The sense strand described above contains the polynucleotide shown in SEQ ID NO: 24, and the antisense strand described above contains the polynucleotide shown in SEQ ID NO: 54. The sense strand described above contains the polynucleotide shown in SEQ ID NO: 25, and the antisense strand described above contains the polynucleotide shown in SEQ ID NO: 55. The sense strand contains the polynucleotide shown in SEQ ID NO: 26, and the antisense strand contains the polynucleotide shown in SEQ ID NO: 56. The sense strand contains the polynucleotide shown in SEQ ID NO: 27, and the antisense strand contains the polynucleotide shown in SEQ ID NO: 57.

[0038] This disclosure further provides an siRNA complex comprising any one of the above-mentioned siRNAs and a target ligand linked to the above-mentioned siRNA.

[0039] An "siRNA complex" refers to a compound formed by the linkage of one or more chemical moieties to an siRNA molecule.

[0040] In some embodiments, the siRNA covalently or noncovalently binds to the target ligand.

[0041] In some embodiments, the target ligand is ligated to the antisense strand of the siRNA. In some embodiments, the target ligand is ligated to the 5' end of the antisense strand of the siRNA. In some embodiments, the target ligand is ligated to the 3' end of the antisense strand of the siRNA.

[0042] In some embodiments, the target ligand is ligated to the sense strand of the siRNA. In some embodiments, the target ligand is ligated to the 5' end of the sense strand of the siRNA. In some embodiments, the target ligand is ligated to the 3' end of the sense strand of the siRNA.

[0043] In some embodiments, the target ligand is ligated to the siRNA terminus via a phosphodiester group, a thiophosphodiester group, or a phosphonic acid group, and in some embodiments, it is ligated to the siRNA terminus via a phosphodiester group.

[0044] In some embodiments, the target ligand is indirectly linked to the siRNA terminus via a phosphodiester group, a thiophosphodiester group, or a phosphonic acid group. In some embodiments, the target ligand is indirectly linked to the siRNA terminus via a phosphodiester group. In some embodiments, the target ligand is directly linked to the siRNA terminus via a phosphodiester group, a thiophosphodiester group, or a phosphonic acid group. In some embodiments, the target ligand is directly linked to the siRNA terminus via a phosphodiester group.

[0045] In some embodiments, the target ligand is directly ligated to the 3' end of the siRNA sense strand via a phosphodiester group or a thiophosphodiester group.

[0046] In some embodiments, one or more of the target ligands are ligated to the sense strand of the siRNA.

[0047] In some embodiments, the target ligand has affinity for cell receptors expressed on epithelial cells.

[0048] In some embodiments, the target ligand includes an integrin target ligand.

[0049] In some embodiments, the target ligand includes an αvβ6 integrin target ligand. The αvβ6 integrin target ligand may be any target ligand capable of binding to αvβ6 integrin. In some embodiments, the target ligand has the following structure: [ka] In some embodiments, the target ligand targets the liver.

[0050] In some embodiments, the target ligand binds to the asialoglycoprotein receptor (ASGPR).

[0051] In some embodiments, the target ligand comprises a galactose cluster or a galactose derivative cluster, the galactose derivative being selected from N-acetyl-galactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, Nn-butyrylgalactosamine, or N-isobutyrylgalactosamine.

[0052] Target ligands that bind to the asialocrypoprotein receptor (ASGPR) are known to be particularly useful for inducing the delivery of oligomeric compounds to the liver. The asialocrypoprotein receptor is expressed in large quantities in liver cells (hepatocytes). The target moiety of ASGPR-targeting cell receptors includes galactose and galactose derivatives. Specifically, galactose derivative clusters contain clusters consisting of 2, 3, 4, or 4 or more N-acetyl-galactosamine (GalNAc or NAG) molecules, which can promote the uptake of certain compounds in hepatocytes. The GalNAc cluster coupled to the oligomeric compound is intended to guide the composition to the liver, where the N-acetyl-galactosamine sugar can bind to the asialocrypoprotein receptor on the surface of liver cells. Binding to the asialocrypoprotein receptor is thought to activate receptor-mediated endocytosis, thereby promoting the entry of compounds into the cell.

[0053] In some embodiments, the target ligands provided herein are the compounds shown below or pharmaceutically acceptable salts thereof. [ka]

[0054] In some embodiments, the N-acetyl-galactosamine moiety of the target ligand can be substituted with N-trifluoroacetylgalactosamine, N-propionylgalactosamine, Nn-butyrylgalactosamine, or N-isobutyrylgalactosamine.

[0055] In some embodiments, the sense strand in the siRNA complex described herein comprises a nucleotide sequence represented by any one of SEQ ID NOs: 28 to 30, and / or the antisense strand comprises a nucleotide sequence represented by any one of SEQ ID NOs: 58 to 60.

[0056] In some specific embodiments, the siRNA complex of this disclosure is one selected from the following: The sense strand contains the polynucleotide shown in SEQ ID NO: 28, and the antisense strand contains the polynucleotide shown in SEQ ID NO: 58. The sense strand described above contains the polynucleotide shown in SEQ ID NO: 29, and the antisense strand described above contains the polynucleotide shown in SEQ ID NO: 59. The sense strand contains the polynucleotide shown in SEQ ID NO: 30, and the antisense strand contains the polynucleotide shown in SEQ ID NO: 60.

[0057] In another embodiment, the Disclosure provides a pharmaceutical composition comprising an siRNA described herein, an siRNA conjugate, and one or more pharmaceutically acceptable excipients such as a vehicle, carrier, diluent, and / or delivery polymer. The term "pharmaceutical composition" refers to a mixture of one or more compounds described herein or their physiologically medicinal salts or prodrugs and other chemical components, and other components such as physiologically medicinal carriers and excipients. The pharmaceutical composition is intended to facilitate administration to a living organism and contribute to the absorption of the active ingredient, thereby exerting further biological activity.

[0058] Various drug delivery systems are known and applicable to the siRNA or siRNA complex of this disclosure, such as packaging into liposomes, microparticles, microcapsules, recombinant cells capable of expressing the siRNA or siRNA complex, receptor-mediated cell endocytosis, reverse transcription viruses, or the construction of nucleic acids that become part of other vectors.

[0059] "Pharmacologically acceptable excipients" include, but are not limited to, any excipients, carriers, flow enhancers, sweeteners, diluents, preservatives, dyes / colorants, flavorings, surfactants, humectants, dispersants, suspending agents, stabilizers, isotonic agents, solvents, or emulsifiers approved by the U.S. Food and Drug Administration (FDA) for use in humans or livestock.

[0060] In another embodiment, the disclosure provides the use of the above-mentioned siRNA, siRNA complex, or pharmaceutical composition in the preparation of a drug for treating a disease of interest.

[0061] In another embodiment, the present disclosure provides a method for treating a target disease, comprising administering the siRNA, siRNA complex, or pharmaceutical composition to a target.

[0062] In another embodiment, the present disclosure provides a method for inhibiting mRNA expression in a subject's body, comprising administering the subject the siRNA, siRNA complex, or pharmaceutical composition.

[0063] In another embodiment, the present disclosure provides a method for delivering an expression-inhibiting oligomeric compound into the body, comprising administering the siRNA, siRNA complex, or pharmaceutical composition to a subject.

[0064] In some embodiments, the oligomeric compound is delivered to the liver.

[0065] In some embodiments, the oligomer compound is delivered extrahepatically.

[0066] In some embodiments, the oligomer compound is delivered to the lungs.

[0067] The siRNAs, siRNA complexes, or pharmaceutical compositions and methods disclosed herein can reduce the level of target mRNA in cells, cell populations, tissues, or subjects, and inhibit the expression of target mRNA in the subject by administering the siRNAs, siRNA complexes, or pharmaceutical compositions described herein to the subject. In some embodiments, the subject is administered an effective amount or effective dose of the siRNAs, siRNA complexes, or pharmaceutical compositions described herein.

[0068] In some embodiments, the subject has already been identified as having a pathogenic upregulation of the target gene in the target cells or tissues.

[0069] The subjects described in this disclosure are those diagnosed with (or suspected of having, or prone to having) a disease or condition that would benefit from the reduction or inhibition of target mRNA expression.

[0070] Delivery may be by local administration (e.g., direct injection, implantation, or local administration), systemic administration, or by subcutaneous, intravenous, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), intramuscular, percutaneous, airway (aerosol), nasal, oral, rectal, or local (including oral, cheek, and sublingual) administration.

[0071] In a selective embodiment, the pharmaceutical compositions provided herein may be administered by injection, for example, intravenous, intramuscular, intradermal, subcutaneous, duodenal, or intraperitoneal injection.

[0072] In a selective embodiment, after the target ligand is linked to the siRNA to form a complex, the complex may be packaged in a reagent kit.

[0073] In another embodiment, the Disclosure further provides pharmaceutical compositions comprising the siRNA or siRNA complex of the Disclosure.

[0074] In some embodiments, the pharmaceutical composition may further contain pharmaceutically acceptable additives and / or adjuvants, which may be one or more of the various formulations or compounds commonly used in the art. For example, the pharmaceutically acceptable additives may include at least one of pH buffers, protective agents, and osmotic regulators.

[0075] In some embodiments, the unit dose of the above pharmaceutical composition is 0.001 mg to 1000 mg.

[0076] In some embodiments, based on the total weight of the composition, the pharmaceutical composition contains 0.01 to 99.99% of the above compound or its pharmaceutically acceptable salt or its isotopic substitution. In some embodiments, the pharmaceutical composition contains 0.1 to 99.9% of the above compound or its pharmaceutically acceptable salt or its isotopic substitution. In some embodiments, the pharmaceutical composition contains 0.5 to 99.5% of the above compound or its pharmaceutically acceptable salt or its isotopic substitution. In some embodiments, the pharmaceutical composition contains 1 to 99% of the above compound or its pharmaceutically acceptable salt or its isotopic substitution. In some embodiments, the pharmaceutical composition contains 2 to 98% of the above compound or its pharmaceutically acceptable salt or its isotopic substitution.

[0077] Pharmaceutical compositions can be conveniently expressed in unit dosage forms. Typically, pharmaceutical compositions can be prepared in any suitable dosage form, such as injections, tablets, capsules, gels, etc., but are not limited to these. Pharmaceutical compositions include, but are not limited to, solutions, emulsions, and liposome-containing formulations.

[0078] In some embodiments, based on the total weight of the composition, the pharmaceutical composition contains 0.01% to 99.99% of pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical composition contains 0.1% to 99.9% of pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical composition contains 0.5% to 99.5% of pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical composition contains 1% to 99% of pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical composition contains 2% to 98% of pharmaceutically acceptable excipients.

[0079] In some embodiments, when the siRNA, siRNA complex, or pharmaceutical composition comes into contact with cells expressing a target gene, for example, by psiCHECK activity screening and luciferase reporter gene detection methods, as well as by methods based on PCR or branched DNA (bDNA), or protein-based methods such as immunofluorescence analysis methods including Western Blot or flow cytometry, the siRNA complex or pharmaceutical composition inhibits the expression of the target gene by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0080] In some embodiments, when the above-mentioned siRNA, siRNA complex, or pharmaceutical composition comes into contact with cells expressing a target gene, for example, when measured by immunofluorescence analysis methods such as psiCHECK activity screening and luciferase reporter gene detection, as well as by methods such as PCR, branched DNA (bDNA), or protein-based methods such as Western Blot or flow cytometry, the percentage of excess expression of the target gene mRNA induced by the above-mentioned siRNA complex or pharmaceutical composition is 99% or less, 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less.

[0081] In some embodiments, when the above-mentioned siRNA, siRNA complex, or pharmaceutical composition comes into contact with cells expressing a target gene, for example, when measured by immunofluorescence analysis methods such as Western Blot or flow cytometry, in addition to psiCHECK activity screening and luciferase reporter gene detection, the siRNA complex retains on-target activity while simultaneously reducing off-target activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%.

[0082] In some embodiments, when the above-mentioned siRNA, siRNA complex, or pharmaceutical composition comes into contact with cells expressing a target gene, for example, when measured by immunofluorescence analysis methods such as Western Blot or flow cytometry, in addition to psiCHECK activity screening and luciferase reporter gene detection, the siRNA complex reduces on-target activity by at least 20%, at least 19%, at least 15%, at least 10%, at least 5%, or more than 1%, while simultaneously reducing off-target activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%.

[0083] In some embodiments, when the above-mentioned siRNA, siRNA complex, or pharmaceutical composition comes into contact with cells expressing a target gene, for example, psiCHECK activity screening and luciferase reporter gene detection methods, as well as methods based on PCR or branched DNA (bDNA), or protein-based methods such as immunofluorescence analysis methods including Western Blot or flow cytometry, the siRNA complex increases on-target activity by at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%, while simultaneously decreasing off-target activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%.

[0084] This disclosure further provides cells comprising the siRNA and / or siRNA complex of this disclosure.

[0085] This disclosure further provides reagent kits comprising the siRNA and / or siRNA complexes and / or pharmaceutical compositions of this disclosure. In some embodiments, the reagent kit comprises one or more containers comprising the siRNA and / or siRNA complexes and / or pharmaceutical compositions of this disclosure.

[0086] This disclosure provides a method for silencing a target gene or mRNA of a target gene in a cell, further comprising the step of introducing an siRNA, siRNA complex, and / or pharmaceutical composition according to this disclosure into the cell.

[0087] This disclosure provides a method for silencing a target gene or mRNA of a target gene in cells in vivo or in vitro, further comprising the step of introducing an siRNA, siRNA complex and / or pharmaceutical composition according to this disclosure into the cells.

[0088] This disclosure further provides a method for reducing the mRNA expression of a target gene or a target gene, comprising administering the siRNA and / or siRNA complex and / or pharmaceutical composition of this disclosure to a subject requiring such reduction. In some embodiments, the method comprises administering an effective amount or effective dose of the siRNA and / or siRNA complex of this disclosure to a subject requiring such reduction.

[0089] In some embodiments, administration is carried out by methods of administration including intramuscular, intrabronchial, intrathoracic, intraperitoneal, intraarterial, lymphatic, intravenous, subcutaneous, cerebrospinal fluid, or a combination thereof.

[0090] In some embodiments, the effective amount or effective dose of siRNA, siRNA complex and / or pharmaceutical composition is about 0.001 mg / kg body weight to about 200 mg / kg body weight, about 0.01 mg / kg body weight to about 100 mg / kg body weight, or about 0.5 mg / kg body weight to about 50 mg / kg body weight.

[0091] In some embodiments, the target gene is matrix metalloproteinase 7 (MMP7).

[0092] This disclosure provides siRNA and / or pharmaceutical compositions and / or siRNA conjugates for treating and / or preventing diseases associated with the expression of the matrix metalloproteinase 7 (MMP7) gene in a subject. In some embodiments, the disease 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 infection, acute respiratory distress syndrome (ARDS) or other types of acute lung injury, pulmonary hypertension, cancer, renal fibrosis, and hepatic fibrosis.

[0093] This disclosure provides a method for treating and / or preventing a disease associated with the expression of the MMP7 gene in a subject, comprising administering to the subject an siRNA and / or siRNA complex and / or the pharmaceutical composition described herein. In some embodiments, the method comprises administering to the subject an effective amount or effective dose of the siRNA and / or siRNA complex and / or the pharmaceutical composition described herein. In some embodiments, the disease 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 infection, acute respiratory distress syndrome (ARDS) or other types of acute lung injury, pulmonary hypertension, cancer, renal fibrosis, and hepatic fibrosis.

[0094] This disclosure provides the use of the above-mentioned siRNA and / or pharmaceutical compositions and / or siRNA complexes in the preparation of agents for treating and / or preventing diseases associated with the expression of the MMP7 gene. In some embodiments, the above-mentioned diseases are selected from idiopathic pulmonary fibrosis (IPF), asthma, other types of fibrosis, chronic inflammation, interstitial lung disease (ILD), SARS-CoV-2 or other types of infection, acute respiratory distress syndrome (ARDS) or other types of acute lung injury, pulmonary hypertension, cancer, renal fibrosis, and hepatic fibrosis.

[0095] This disclosure provides the use of the above-mentioned siRNA and / or pharmaceutical composition and / or siRNA complex in the preparation of a drug for inhibiting MMP7 expression.

[0096] This disclosure provides a method for inhibiting MMP7 expression, comprising administering the above-mentioned siRNA and / or pharmaceutical composition and / or siRNA complex to a subject. In some embodiments, the method comprises administering an effective amount or effective dose of the siRNA and / or pharmaceutical composition and / or siRNA complex to a subject.

[0097] This disclosure provides the above-mentioned siRNA and / or pharmaceutical compositions and / or siRNA complexes for treating and / or preventing diseases. In some embodiments, the above-mentioned diseases are selected from idiopathic pulmonary fibrosis (IPF), asthma, other types of fibrosis, chronic inflammation, interstitial lung disease (ILD), SARS-CoV-2 or other types of infection, acute respiratory distress syndrome (ARDS) or other types of acute lung injury, pulmonary hypertension, cancer, renal fibrosis, and hepatic fibrosis.

[0098] This disclosure provides a method for treating and / or preventing a disease, comprising administering the above-mentioned siRNA and / or pharmaceutical composition and / or siRNA complex to a subject. In some embodiments, the method comprises administering to a subject an effective amount or effective dose of the siRNA and / or pharmaceutical composition and / or complex of this disclosure. In some embodiments, the disease 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 infection, acute respiratory distress syndrome (ARDS) or other types of acute lung injury, pulmonary hypertension, cancer, renal fibrosis, and hepatic fibrosis.

[0099] This disclosure provides the use of the above-mentioned siRNA and / or pharmaceutical compositions and / or siRNA complexes in the preparation of agents for treating and / or preventing diseases. In some embodiments, the above-mentioned diseases are selected from idiopathic pulmonary fibrosis (IPF), asthma, other types of fibrosis, chronic inflammation, interstitial lung disease (ILD), SARS-CoV-2 or other types of infection, acute respiratory distress syndrome (ARDS) or other types of acute lung injury, pulmonary hypertension, cancer, renal fibrosis, and hepatic fibrosis.

[0100] This disclosure provides a method for delivering siRNA that inhibits the expression and / or replication of MMP7 into the body, comprising administering the siRNA and / or pharmaceutical composition and / or siRNA complex to a subject.

[0101] In some embodiments, the siRNA is delivered to the liver.

[0102] In some embodiments, the siRNA is delivered extrahepatically.

[0103] In some embodiments, the siRNA is delivered to the lungs.

[0104] This disclosure further provides a method for preparing siRNA or siRNA complexes, which includes synthesizing the siRNA or siRNA complexes described herein.

[0105] This disclosure further provides siRNA or siRNA complexes characterized by substituting base T for one or more bases U of any one siRNA or siRNA complex relating to this disclosure, for example, 1, 2, 3, 3, 5, 6, 7, 8, 9, or 10 bases U.

[0106] The medicinal salts of the compounds described herein are selected from inorganic salts or organic salts, and the compounds described herein can react with acidic or basic substances to produce the corresponding salts.

[0107] In another embodiment, where the configuration is not explicitly stated, the compounds of the Disclosure may have a specific geometric or stereoisomeric form. The Disclosure includes all cis-trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures thereof and other mixtures, such as mixtures rich in enantiomers or diastereomers, and all such compounds are intended to be within the scope of the Disclosure. Substituents such as alkyl groups may have other chiral carbon atoms. All such isomers and mixtures thereof are within the scope of the Disclosure.

[0108] Furthermore, unless otherwise specified, the compounds and intermediates of this disclosure may exist in different tautomer forms, and all such forms are included within the scope of this disclosure. The terms “tautomer” or “tautomer form” refer to structural isomers of different energies that can be interconverted over a low energy barrier.

[0109] All compounds in this disclosure can be described as either type A or type B. All tautomer forms are within the scope of this disclosure. The nomenclature of the compounds does not exclude any tautomers.

[0110] The compounds of this disclosure may be asymmetric, for example, having one or more stereoisomers. Unless otherwise specified, all stereoisomers include, for example, enantiomers and diastereomers. Compounds containing an asymmetric carbon atom of this disclosure can be isolated in the form of optically active pures or racemic forms. The optically active pures may be separated from racemic mixtures or synthesized using chiral starting materials or chiral reagents.

[0111] Optically active (R)- and (S)- isomers and D- and L isomers can be prepared by chiral synthesis, chiral reagents, or other conventional techniques. To obtain one enantiomer of a compound in this disclosure, it can be prepared by asymmetric synthesis or by induction with a chiral aid, where the resulting diastereomer mixture is isolated and the group splitting is assisted to provide the required pure enantiomer. Alternatively, if the molecule contains a basic functional group (e.g., an amino group) or an acidic functional group (e.g., a carboxyl group), a salt of the diastereomer is formed with a suitable optically active acid or base, and the diastereomer is split by conventional methods known in the art, and then recovered to obtain the pure enantiomer. The isolation of enantiomers and diastereomers is generally completed by chromatography, which uses a chiral stationary phase and is optionally combined with a chemical induction method (e.g., generating a carbamate from an amine).

[0112] This disclosure is the same as that described herein, but further includes some compounds of the present disclosure with isotope labels in which one or more atoms are replaced by atoms having an atomic weight or mass number different from the atomic weight or mass number usually found in nature. Examples of isotopes that can be bonded to the compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine. For example, 2 H, 3 H, 11 C, 13 C, 14 C, 13 N, 15 N, 15 O, 17 O, 18 O, 31 P, 32 P, 35 S, 18 F, 123 I, 125 I and 36 Cl, and so on.

[0113] Unless otherwise specified, where one position is specifically designated as deuterium (D), that position should be understood to be deuterium having an abundance at least 1000 times higher than the natural abundance of deuterium (0.015%) (i.e., at least 10% deuterium incorporated). Having an abundance higher than the natural abundance of deuterium in the example compounds may mean deuterium with an abundance of at least 1000 times, at least 2000 times, at least 3000 times, at least 4000 times, at least 5000 times, at least 6000 times, or even greater. This disclosure further includes compounds in various deuterated forms. Each available hydrogen atom bonded to a carbon atom may be independently substituted with a deuterium atom. Those skilled in the art can synthesize compounds in deuterated forms by referring to relevant literature. When preparing compounds in their deuterated form, commercially available deuterated starting materials may be used, or they may be synthesized using deuterated reagents by conventional techniques. Deuterated reagents include, but are not limited to, borane deuterated, borane-tetrahydrofuran trihydrogenated solution, lithium aluminum hydride deuterated, iodoethane deuterated, and iodomethane deuterated.

[0114] JPEG2026519860000004.jpg63170

[0115] The full texts of WO2022028462A1, WO2023274395A1, and WO2023208023A1 are incorporated into this disclosure.

[0116] Explanation of terms To make this disclosure more easily understood, several technical and scientific terms are defined below. Unless otherwise explicitly defined herein, all other technical and scientific terms used herein have the meanings that are ordinarily understood by those skilled in the art.

[0117] Unless otherwise specified, the terms “matrix metalloproteinase 7” and “MMP7” are interchangeable in the context of this disclosure. MMP7 includes, but is not limited to, human MMP7, cynomolgus monkey MMP7, mouse MMP7, and rat MMP7, and their amino acids, complete coding sequences, and mRNA sequences can be readily obtained using previously disclosed databases such as GenBank, UniProt, OMIM, and the Macaca Genome Project site.

[0118] The term "MMP7" also refers to naturally occurring DNA sequence variations in the MMP7 gene, such as single nucleotide polymorphisms (SNPs). Exemplary SNPs can be found in the dbSNP database.

[0119] The term "target sequence" refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during the MMP7 transcription period, including mRNA of the RNA processing product as the main transcription product. The targeted portion of the target sequence must be long enough to function as a substrate for iRNA-directed cleavage. In one embodiment, the target sequence is located within the protein-coding region of MMP7. In the context of this disclosure, "target sequence" and "target mRNA" are synonymous and are interchangeable.

[0120] The target groups described herein may include cell receptor ligands, such as integrin target ligands. Integrins are a family of transmembrane receptors that promote cell-extracellular matrix (ECM) adhesion. Among them, integrin alpha-v-beta-6 (αvβ6) is a type of epithelial-specific integrin, and αvβ6 is known to be a receptor for ECM proteins and TGF-β latency-related peptide (LAP), and is expressed in various cells and tissues. Furthermore, integrin αvβ6 is highly upregulated in damaged lung epithelial cells.

[0121] The MMP7 siRNAs described herein can be ligated to integrin target ligands having affinity for integrin αvβ6. As noted herein, the “αvβ6 integrin target ligand” is a compound having affinity for integrin αvβ6, which can be used as a ligand to facilitate the targeting and delivery of the ligated siRNA to desired cells and / or tissues (i.e., cells expressing integrin αvβ6).

[0122] As used herein, in the case of RNA-mediated gene silencing, the sense strand (also known as SS, SS strand, or significance strand) refers to the strand containing the same or essentially the same sequence as the target mRNA sequence, and the antisense strand (also known as AS or AS strand) refers to the strand containing a sequence complementary to the target mRNA sequence.

[0123] In the context of describing the siRNA sense strands described herein, the phrase "a sequence that differs by three or fewer nucleotides from any one of the nucleotide sequences of SEQ ID NOs. 1 to SEQ ID NOs. 10, and contains at least 15 consecutive nucleotides" is intended to indicate that the siRNA sense strands described herein contain at least 15 consecutive nucleotides from any one of the sense strands of SEQ ID NOs. 1 to SEQ ID NOs. 10, or a sequence that differs by three or fewer nucleotides from at least 15 consecutive nucleotides from any one of the sense strands of SEQ ID NOs. 1 to SEQ ID NOs. 10 (optionally, a sequence that differs by two or fewer nucleotides, or optionally, a sequence that differs by one nucleotide). Optionally, the siRNA sense strands described herein contain at least 16 consecutive nucleotides from any one of the sense strands of SEQ ID NOs. 1 to SEQ ID NOs. 10, or a sequence that differs by three or fewer nucleotides from at least 16 consecutive nucleotides from any one of the sense strands of SEQ ID NOs. 1 to SEQ ID NOs. 10 (optionally, a sequence that differs by two or fewer nucleotides, or optionally, a sequence that differs by one nucleotide).

[0124] In the context of describing the siRNA antisense strands described herein, the phrase "a sequence that differs by three or fewer nucleotides from any one of the antisense strands of SEQ ID NOs. 31 to SEQ ID NOs. 40, and contains at least 15 consecutive nucleotides" is intended to indicate that the siRNA antisense strands described herein contain at least 15 consecutive nucleotides from any one of the antisense strands of SEQ ID NOs. 31 to SEQ ID NOs. 40, or a sequence that differs by three or fewer nucleotides from at least 15 consecutive nucleotides from any one of the antisense strands of SEQ ID NOs. 31 to SEQ ID NOs. 40 (optionally, a sequence that differs by two or fewer nucleotides, or optionally, a sequence that differs by one nucleotide).

[0125] In this disclosure, the "5' region," i.e., the "5' end," and "5' terminus" of the sense strand or antisense strand are interchangeable. For example, the nucleotides at positions 2-8 of the antisense strand's 5' region may be substituted with the nucleotides at positions 2-8 of the antisense strand's 5' terminus. Similarly, the "3' region," "3' terminus," and "3' terminus" of the sense strand or antisense strand are also interchangeable.

[0126] As used in this disclosure, the term “2'-fluoro-modified nucleotide / nucleoside” refers to a nucleotide / nucleoside formed by substituting a fluoro group for the hydroxyl group at the 2' position of the ribosyl group of the nucleotide, and “non-2'-fluoro-modified nucleotide / nucleoside” refers to a nucleotide / nucleoside or nucleotide analogue formed by substituting a non-fluoro group for the hydroxyl group at the 2' position of the ribosyl group of the nucleotide.

[0127] As used in this disclosure, the term “2'-methoxy-modified nucleotide / nucleoside” refers to a nucleotide / nucleoside formed by substituting a methoxy group for the 2'-hydroxy group of a ribosyl group.

[0128] Unless otherwise specified, in the context of this disclosure, "G", "C", "A", "T", and "U" represent nucleotides containing the bases guanine, cytosine, adenine, thymidine, and uracil, respectively. It is well known to those skilled in the art that substitutions of the bases T and U do not significantly affect the properties of the siRNA sequence, and that U can be optionally substituted with T in the sequences of this disclosure, and that the resulting sequences are also protected within the scope of this disclosure. In the sequences of this disclosure, for the same nucleic acid chain, the direction from the 5' end to the 3' end is from left to right, the lowercase letter m indicates that the nucleoside adjacent to the left of the letter m is a nucleoside modified with a 2'-methoxy group, the lowercase letter f indicates that the nucleoside adjacent to the left of the letter f is a nucleoside modified with a 2'-fluoro group, and the lowercase letter s indicates that the two nucleosides adjacent to the letter s are linked by a thiophosphodiester group. Unless otherwise specified, “RNAi agents,” “nucleotides,” “compounds,” “chemical modifications,” “oligonucleotides,” “double-stranded RNAi inhibitor molecules,” “siRNA,” “siRNA complexes,” “dsRNA,” “nucleic acids,” and “RNAi” in this disclosure may each exist independently in the form of a salt, a mixed salt, or an unsalted form (e.g., a free acid or a free base). If they exist in the form of a salt or a mixed salt, they may be pharmaceutically acceptable salts. The term “pharmaceutically acceptable salt” includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts. If they exist in the form of a salt, some groups may be ionized to form anions / cations, for example, phosphodiester groups and thiophosphodiester groups may exist in anionic form, and the structures in salt form corresponding to each of the following structures are also within the scope of this disclosure. Unless otherwise specified, the 3' position of the first nucleotide at the 3' end of each strand is a hydroxyl group, and the 5' position of the first nucleotide at the 5' end of each strand is a hydroxyl group.

[0129] The above modifications and linking groups each have the structures shown in the table below, where Base indicates a base: [Table 1-1]

[0130] [Table 1-2]

[0131] As used herein, the terms “complementary” and “reverse complementary” are interchangeable and have meanings well known to those skilled in the art, where, in a double-stranded nucleic acid molecule, the bases of one strand pair complementaryly with the bases of the other strand. In DNA, the purine base adenine always pairs with the pyrimidine base thymine (or uracil in RNA), and the purine base guanine always pairs with the pyrimidine base cytosine. Each base pair contains one purine and one pyrimidine. When adenine in one strand always pairs with thymine (or uracil) in the other strand, and guanine always pairs with cytosine, the two strands are considered complementary, and the sequence of the strands can be inferred from the sequence of the complementary strands. Accordingly, “mismatch” in this art means that, in a double-stranded nucleic acid, the bases at corresponding positions do not exist paired in a complementary form.

[0132] As used herein, the term “inhibition” is interchangeable with “reduction,” “silencing,” “downregulate,” “suppression,” and other similar terms, and includes any level of inhibition. Inhibition can be assessed by a reduction in the absolute or relative level of one or more of these variables compared to a control level. The control level may be any type of control level used in this art, for example, a pre-administration baseline level, or a level determined from an untreated or control-treated subject, cell, or sample (e.g., buffer control or inactivator control only). For example, the degree of inhibition of target gene expression by siRNA can be expressed by the excess mRNA expression level. For instance, excess mRNA expression levels are 99% or less, 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less. The inhibition rate of target gene expression can be detected using the Dual-Glo® Luciferase Assay System, and the firefly chemiluminescence value (Fir) and sea cucumber chemiluminescence value (Ren) can be read, respectively, and the relative value Ratio = Ren / Fir can be calculated. In this disclosure, the ratio of excess mRNA expression (or excess activity %) = Ratio(siRNA-treated group) / Ratio(siRNA-free control group), and the inhibition rate (%) = 100% - excess mRNA expression (%).

[0133] Unless otherwise specified, the “compounds,” “ligands,” “nucleic acid ligand complexes,” “siRNA complexes,” “nucleic acids,” “complexes,” “chemical modifications,” “target ligands,” “dsRNA,” and “RNAi” of this disclosure may exist independently in the form of salts, mixed salts, or unsalted substances (e.g., free acids or free bases). If existing in the form of salts or mixed salts, they may be pharmaceutically acceptable salts.

[0134] The term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0135] A "pharmaceutically acceptable acid addition salt" refers to a salt formed with an inorganic or organic acid that can preserve the bioavailability of the free base without other side effects. Inorganic salts include, but are not limited to, hydrochloride, hydrobromide, sulfate, nitrate, and phosphate salts. Organic salts include, but are not limited to, formate, acetate, 2,2-dichloroacetate, trifluoroacetate, propionate, caproate, octanoate, decanoate, undecylenate, glycolate, gluconate, lactate, sebacinate, adipine, glutarate, malonate, oxalate, maleate, succinate, fumarate, tartrate, citrate, palmitate, stearate, oleate, cinnamate, laurate, malate, glutamate, pyroglutamate, aspartate, benzoate, mesylate, benzenesulfonate, p-toluenesulfonate, alginate, ascorbate, salicylate, 4-aminosalicylate, and naphthalenedisulfonate salts. These salts can be prepared by methods known in the art.

[0136] A "pharmaceutically acceptable base addition salt" refers to a salt formed with an inorganic or organic base that can maintain the bioavailability of the free acid without other side effects. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, and aluminum salts. Preferred inorganic salts are ammonium salts, sodium salts, potassium salts, calcium salts, and magnesium salts, with sodium salts being preferred. Salts derived from organic bases include, but are not limited to, the following salts: primary amines, secondary and tertiary amines, substituted amines including naturally substituted amines, cyclic amines, and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, glycine betaine, ethylenediamine, glucosamine, methylglucosamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, and polyamide resins. Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. These salts can be prepared by methods known in the art.

[0137] "Effective dose" or "effective amount" means the amount of drug, compound, or pharmaceutical composition required to obtain any one or more beneficial or necessary therapeutic outcomes. With respect to prophylactic use, beneficial or necessary outcomes include the elimination or reduction of risk, reduction of severity, or delay of attacks of the condition, and include biochemical, histological, and / or behavioral symptoms of the condition, its complications, and intermediate pathological phenotypes that appear in the progression of the condition. With respect to therapeutic use, beneficial or necessary outcomes include clinical outcomes, such as a reduction in the incidence of various target gene, target mRNA, or target protein-related conditions of this disclosure, or improvement of one or more symptoms of such conditions, a reduction in the dose of other drugs required to treat the condition, an improvement in the therapeutic effect of another drug, and / or delay in the progression of a patient's target gene, target mRNA, or target protein-related condition of this disclosure.

[0138] As used herein, “patient,” “subject,” or “individual” are interchangeable and include humans or non-human animals such as mammals, e.g., humans or monkeys.

[0139] The siRNAs provided in this disclosure can be obtained by conventional preparation methods in the art (e.g., solid-phase synthesis and liquid-phase synthesis). Of these, solid-phase synthesis is already available as a commercially available customized service. Modified nucleotide groups can be introduced into the siRNAs described in this disclosure using nucleoside monomers having the corresponding modifications, and methods for preparing nucleoside monomers having the corresponding modifications, and methods for introducing modified nucleotide groups into siRNAs, are well known to those skilled in the art.

[0140] The terms "chemical modification" or "modification" encompass all chemical changes to nucleotides, such as the addition or removal of a chemical moiety, or the substitution of one chemical moiety with another.

[0141] The term "base" includes any known DNA and RNA bases and base analogs, such as purines and pyrimidines, and also includes the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and their natural analogs.

[0142] The terms "flat end" and "blunt end" are interchangeable and refer to the absence of unpaired nucleotides or nucleotide analogs at a given end of an siRNA, i.e., no nucleotide protrusions. In most cases, siRNAs with both ends being blunt ends are double-stranded along their entire length.

[0143] The terms “approximately” or “nearly” mean that the numerical value is within the acceptable margin of error of a specific value measured by a person skilled in the art, as determined by how the numerical portion is measured or measured (i.e., the limits of the measuring system). For example, “approximately” may mean a standard deviation of 1 or greater than 1. Alternatively, “approximately” or “basically including” may mean that it varies within a range of up to 20%, for example, between 1% and 15%, between 1% and 10%, between 1% and 5%, between 0.5% and 5%, or between 0.5% and 1%. In this disclosure, the term “approximately” preceding a number or numerical range also includes a predetermined number of embodiments in each case. Unless otherwise specified, where specific values ​​appear in this application and claims, the meaning of “approximately” or “basically including” should be assumed to be within the acceptable margin of error of the specific value.

[0144] Unless otherwise specified, "optionally," "optionally," "selectively," or "selective" means that the event or situation described thereafter may or may not occur, and the description includes both cases in which the event or situation occurs and cases in which it does not. For example, "optionally, R1 and R2 are directly connected to form a ring" means that R1 and R2 may be directly connected to form a ring, but this does not necessarily have to be the case, and the description includes both cases in which R1 and R2 are directly connected to form a ring and cases in which R1 and R2 do not form a ring.

[0145] JPEG2026519860000007.jpg17170

[0146] In the context of this disclosure, [ka] Based on the premise [ka] This can be replaced with any group that can be linked to an adjacent nucleotide.

[0147] The term "covalent linkage" refers to a connection between two molecules, whether by covalent bonds or non-covalent bonds (e.g., hydrogen bonds or ionic bonds), and includes both direct and indirect linkages.

[0148] The term "direct linkage" refers to a linkage between a first compound or group and a second compound or group without the involvement of any atoms or groups of atoms.

[0149] The term "indirect linking" refers to the linking of a first compound or group and a second compound or group via an intermediate group, compound, or molecule (e.g., a linking group).

[0150] The term "substituted" indicates that one or more hydrogen atoms on a specified atom (typically carbon, oxygen, and nitrogen atoms) are substituted with any group not limited herein, provided that the substitution does not exceed the normal valence of the specified atom and that the substitution forms a stable compound. Non-exclusive examples of substituents include C1-C6 alkyl groups, C2-C6 alkenyl groups, C2-C6 alkynyl groups, cyano groups, hydroxyl groups, oxo groups, carboxyl groups, cycloalkyl groups, cycloalkenyl groups, heterocyclyl groups, heteroaryl groups, aryl groups, ketones, alkoxycarbonyl groups, aryloxycarbonyl groups, heteroaryloxycarbonyl groups, or halogens (e.g., F, Cl, Br, I). When the substituent is a ketone or oxo (i.e., =O), two (2) hydrogen atoms on the atom are substituted.

[0151] "Substituted with one or more substituents" means that the substituent may be replaced with one or more substituents. If it is replaced with multiple substituents, they may be multiple identical substituents, or a combination of one or more different substituents. [Modes for carrying out the invention]

[0152] The present disclosure will be further explained below in conjunction with examples, but these examples are not intended to limit the scope of the present disclosure. Experimental methods in the examples of the present disclosure for which specific conditions are not specified usually follow the usual conditions, such as those found in Cold Spring Harbor's Antibody Technology Experiment Manual, Molecular Cloning Manual, etc., or the conditions proposed by the raw material or product manufacturer. Reagents for which the specific source is not specified are common commercially available reagents.

[0153] Example 1. Design of human MMP7 siRNA Table 1B shows unmodified or modified 19 / 21nt siRNAs and siRNA complexes of the present disclosure, designed to satisfy general rules for active siRNA, with the human MMP7 gene (NM_002423.5) as the target gene.

[0154] [Table 2-1]

[0155] [Table 2-2]

[0156] [Table 2-3]

[0157] [Table 2-4]

[0158] Table 2-5

[0159] Table 2-6

[0160] Table 2-7

[0161] Table 2-8

[0162] Table 2-9

[0163] Table 2-10

[0164] Table 2-11

[0165] Table 2-12

[0166] Table 2-13

[0167] Table 2-14

[0168] Table 2-15

[0169] [Table 2-16]

[0170] [Table 2-17]

[0171] In Table 1B above, the sequences are arranged from left to right, from the 5' end to the 3' end. The lowercase letter m indicates that the nucleoside adjacent to the left of the letter m is a nucleoside modified with a 2'-methoxy group. The lowercase letter f indicates that the nucleoside adjacent to the left of the letter f is a nucleoside modified with a 2'-fluoro group. The lowercase letter s indicates that the linkage between the two nucleosides or linked nucleosides adjacent to the left and right of the letter s and the delivery group I-14 is a thiophosphodiester group linkage. Unless otherwise specified, two adjacent nucleosides are linked via a phosphodiester group. Unless otherwise specified, the 3' position of the first nucleotide at the 3' end of each chain is a hydroxyl group, and the 5' position of the first nucleotide at the 5' end of each chain is a hydroxyl group.

[0172] The structures of the 2'-methoxy-modified nucleoside, 2'-fluoro-modified nucleoside, thiophosphodiester group, phosphodiester group, and I-14 are shown in the following table. If the siRNA or siRNA complex of this disclosure exists in salt form, for example, in sodium salt form, the salt form structures corresponding to the structures in Table 2 below are also within the scope of this disclosure:

[0173] [Table 3]

[0174] Among them, "Base" indicates a base.

[0175] Example 2. Synthesis of the siRNA of the Disclosure

[0176] The synthesis of siRNA is no different from the conventional solid-phase synthesis method of phosphoramidites. The synthesis process is briefly described below. Using a Dr. Oligo48 synthesizer (Biolytic), nucleoside phosphoramidite monomers were linked one by one using a synthesis program, starting with a general-purpose CPG carrier. Nucleoside phosphoramidite monomers such as 2'-F RNA and 2'-O-methyl RNA, which are the raw materials for the nucleoside monomers, were purchased from Shanghai Zhaowei or Suzhou Jima. 5-ethylthio-1H-tetrazole (ETT) was used as an activator (0.6 M acetonitrile solution), a solution of 0.22 M PADS dissolved in acetonitrile and trimethylpyridine (Suzhou Kerema) in a 1:1 volume ratio was used as a sulfidating agent, and iodopyridine / aqueous solution (Kerema) was used as an oxidizing agent.

[0177] After solid-phase synthesis was complete, the oligo-ribonucleotides were dissolved from the solid support and immersed in a 3:1 28% aqueous ammonia and ethanol solution at 50°C for 16 hours. The mixture was then centrifuged, the supernatant was transferred to another centrifuge tube, concentrated, and evaporated. Purification was performed by C18 reverse-phase chromatography, with DMTr removed using a mobile phase of 0.1 M TEAA and acetonitrile, and then a 3% trifluoroacetic acid solution. The target oligonucleotides were collected, lyophilized, identified as the target product by LC-MS, and further quantified by UV (260 nm) using nanodrop.

[0178] The obtained single-stranded oligonucleotides were paired complementaryally in equimolar ratios, annealed, and finally the resulting double-stranded siRNA was dissolved in 1×PBS and adjusted to the concentration required for the experiment for use. The synthesis method for oligonucleotides containing I-14 is described in Examples 3 and 4 below.

[0179] Example 3. Synthesis of Ligand Compound I-12 The synthesis route for compound I-12 is as follows:

[0180] [ka]

[0181] 3-1 Synthesis of Compound I-2 To a solution of N-tert-butoxycarbonylglycine (28.00 g, 159.8 mmol, commercially available) in NN-dimethylformamide (200 mL), N,N-diisopropylethylamine (132 mL, 799.2 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (66.85 g, 175.8 mmol), and compound I-1 (78.93 g, 191.8 mmol, prepared according to the method described in patent application WO2019089765A1) were added, and the mixture was ventilated three times with nitrogen gas. The reaction was stirred at room temperature under a nitrogen gas atmosphere for 3 hours, and completion of the reaction was indicated by LCMS monitoring. Water was added to the reaction mixture to quench it, and it was extracted with ethyl acetate. The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. The concentrated crude product was purified by normal-phase silica gel column chromatography to obtain compound I-2 (60.0 g, yield 66%). LC-MS: MS(ESI) m / z = 513.6 [M+H-56] + .

[0182] 3-2 Synthesis of Compound I-3 To a solution of compound I-2 (2.30 g, 4.05 mmol) in methanol (30.0 mL), 10% palladium-carbon (0.23 g) was added, and the atmosphere was changed to hydrogen. The reaction was stirred at room temperature for 8 hours, and completion of the reaction was confirmed by monitoring with LC-MS. The reaction mixture was filtered and concentrated to obtain compound I-3 (1.80 g, 93% yield). 1H NMR (400MHz, DMSO) δ10.27(s, 1H), 8.39(d, J=8.4Hz, 1H), 8.30-8.09(m, 1H), 7.75(dd, J=6.4, 3.2Hz, 1H), 7.54-7.40(m, 4H), 7.37(d, J=8.4Hz, 2H), 7.23(d, J=7.6Hz, 1H), 6.95(dd, J=14.0, 6.8Hz, 2H), 5.32(q, J=7.6Hz, 1H ), 3.60(s, 3H), 3.57(d, J=6.0Hz, 2H), 2.88(t, J=6.0Hz, 2H), 1.39(s, 9H).

[0183] 3-3 Synthesis of Compound I-4 To a solution of compound I-3 (1.00 g, 2.09 mmol) in N,N-dimethylformamide (10.0 mL), azide-pentapolyethylene glycol-p-toluenesulfonyl ester (1.74 g, 4.18 mmol) and potassium carbonate (0.58 g, 4.18 mmol) were added. The reaction was heated to 80°C and stirred overnight, and completion of the reaction was confirmed by LC-MS monitoring. The reaction mixture was quenched with saturated sodium bicarbonate solution and extracted with ethyl acetate (3 times, 40 mL each time). The combined organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The concentrated crude product was purified by normal-phase silica gel column chromatography to obtain compound I-4 (1.30, yield 85%). LC-MS: MS(ESI) m / z = 724.5 [M + H] + .

[0184] 3-4 Synthesis of Compound I-5 To a solution of compound I-4 (1.30 g, 1.79 mmol) in dichloromethane (20.0 mL), a solution of 1,4-dioxane hydrochloride (20.0 mL) was added. The reaction was stirred overnight at room temperature, and completion of the reaction was confirmed by monitoring with LC-MS. The reaction mixture was concentrated to obtain compound I-5 (1.10 g, 98% yield). LC-MS: MS(ESI) m / z = 624.4 [M + H] + .

[0185] 3-5 Synthesis of Compound I-7 To a solution of compound I-6 (2.90 g, 20.11 mmol, commercially available) in tert-butyl alcohol (50.0 mL), di-tert-butyl dicarbonate (6.58 g, 30.17 mmol) was added. The reaction was stirred overnight at room temperature, and completion of the reaction was confirmed by detection using LC-MS. The reaction mixture was concentrated, and the resulting crude product was purified by normal-phase silica gel column chromatography to obtain compound I-7 (3.90 g, 79% yield). LC-MS: MS(ESI) m / z = 245.4 [M + H] + .

[0186] 3-6 Synthesis of Compound I-8 To a solution of compound I-7 (0.98 g, 4.00 mmol) in N,N-dimethylformamide (10.0 mL), sodium hydride (0.19 g, 4.80 mmol) was added at 0°C, and the mixture was stirred for 30 minutes while maintaining the temperature. Then, ethyl 4-bromobutyrate (0.93 mL, 4.81 mmol) was added. The reaction was stirred overnight at room temperature, and completion of the reaction was confirmed by LC-MS monitoring. The reaction mixture was quenched with water and extracted with ethyl acetate (3 times, 50 mL each time). The combined organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The concentrated crude product was purified by normal-phase silica gel column chromatography to obtain compound I-8 (1.30 g, 91% yield). LC-MS: MS(ESI) m / z = 359.5 [M + H] + .

[0187] 3-7 Synthesis of Compound I-9 Compound I-8 (1.30 g, 3.63 mmol) was dissolved in tetrahydrofuran (20.0 mL) and then in 1N lithium hydroxide (20.0 mL). The reaction was stirred at room temperature for 2 hours, and completion of the reaction was confirmed by LC-MS monitoring. The reaction mixture was adjusted to pH 2 with 1N HCl, extracted with ethyl acetate (3 times, 20 mL each time), washed the combined organic phase with saturated brine, and dried over anhydrous sodium sulfate. After concentrating the filtrate, compound I-9 (1.18 g, 98% yield) was obtained. LC-MS: MS(ESI) m / z = 331.1 [M + H] + .

[0188] 3-8 Synthesis of Compound I-10 Compound I-9 (0.20 g, 0.61 mmol) was added to NN-dimethylformamide (5.0 mL) with N,N-diisopropylethylamine (0.39 g, 3.03 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (0.28 g, 0.73 mmol), and compound I-5 (0.28 g, 0.72 mmol). The reaction was stirred at room temperature for 2 hours, and completion of the reaction was confirmed by LC-MS monitoring. The reaction mixture was quenched with water and extracted with ethyl acetate (3 times, 40 mL each time). The combined organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The concentrated crude product was purified by normal-phase silica gel column chromatography to obtain compound I-10 (0.30 g, yield 53%). LC-MS: MS(ESI) m / z = 936.7 [M + H] + .

[0189] 3-9 Synthesis of Compound I-11 A solution of compound I-10 (0.07 g, 0.08 mmol) in tetrahydrofuran (2.0 mL) was added to a solution of 1 N lithium hydroxide (2.0 mL). The reaction was stirred at room temperature for 2 hours, and completion of the reaction was confirmed by LC-MS monitoring. The reaction mixture was adjusted to pH 2 with 1 N HCl, extracted with ethyl acetate (3 times, 20 mL each time), washed the combined organic phase with saturated brine, and dried over anhydrous sodium sulfate. After concentrating the filtrate, compound I-11 (0.27 g, 91% yield) was obtained. LC-MS: MS(ESI) m / z = 922.1 [M + H] + .

[0190] 3-10 Synthesis of Compound I-12 To a solution of compound I-11 (0.27 g, 0.29 mmol) in dichloromethane (4.0 mL), trifluoroacetic acid (4.0 mL) was added. The reaction was stirred at room temperature for 3 hours, and completion of the reaction was confirmed by detection using LC-MS. The reaction mixture was concentrated, and the resulting crude product was purified by preparative high-performance liquid chromatography to obtain compound I-12 (0.09 g, yield 37%). LC-MS: MS(ESI) m / z = 822.1 [M + H] + . 1H NMR (400MHz, DMSO) δ8.97(s, 1H), 8.51(d, J=8.4Hz, 1H), 8.33-8.23(m, 1H), 8.15(t, J=5.6Hz, 1H), 7.88(d, J=8.4Hz, 1H), 7. 79-7.72(m, 1H), 7.63(d, J=8.4Hz, 1H), 7.51(m, 8H), 7.31(d, J=8.0Hz, 1H), 7.26-7.20(m, 1H), 7.04(d, J=8.0Hz, 1H), 6.91(s , 1H), 5.32(q, J=7.2Hz, 1H), 4.34-4.31(m, 2H), 3.95-3.91(m, 2H), 3.78(d, J=5.6Hz, 2H), 3.69(dd, J=6.0, 3.6Hz, 2H), 3.61- 3.50(m, 13H), 3.38-3.34(m, 2H), 3.29(t, J=7.2Hz, 2H), 2.80(d, J=7.2Hz, 2H), 2.29(t, J=7.2Hz, 2H), 1.86(p, J=7.2Hz, 2H).

[0191] When the binding ability of compound I-12 to αvβ6 protein was measured using the conventional fluorescence polarization (FP) method in this field, the IC of the binding of compound I-12 to human recombinant αvβ6 protein was found to be high. 50 The value was measured to be 79.86 nM, indicating that compound I-12 is a ligand capable of binding to the αvβ6 protein. The specific detection method is as follows: 3.5 μL of test compound I-12 was added to a 384-black well plate. The test compound was set to 12 concentration points, with the highest concentration being 10 μM, and then 3-fold gradient dilution was performed. A DMSO control well was used to determine the maximum signal, and a 500 nM compound CWHM-12 (MCE, HY-18644) control well was used as the minimum signal. Next, 3.5 μL of 4X human recombinant αvβ6 protein activator was added. After incubation at room temperature for 15 minutes, 7 μL of 2X fluorescent RGD peptide solution was added to each well. After incubation at room temperature for 1 hour, the FP signal was read by Envision. Inhibition curve fitting was performed using GraphPad Prism to obtain IC50. 50 Calculate the value, IC is calculated using the following formula 50The value (Z prime > 0.5) was calculated: Curve fitting formula: Y = Bottom + (Top - Bottom) / (1 + 10^((LogIC50 - X) * Hill Slope)) X: Log inhibitor concentration, Y: inhibition rate %.

[0192] Example 4. Synthesis of the siRNA complex of the present disclosure 4-1 Synthesis of sense strands linked by I-13' The sense chain containing I-13' in Table 3 was synthesized according to the same method as in Example 2, except that at the position corresponding to I-13', the phosphoramidite monomer I-13 containing an alkynyl group was prepared by the method described in patent application WO2019161213A1 and participated in the solid-phase synthesis to obtain the sense chain containing I-13'. The structures of I-13 and I-13' are as follows: [ka]

[0193] [Table 4]

[0194] 4-2 Synthesis of sense strands containing I-14 Stock solutions of 0.5 M tris(3-hydroxypropyltriazolylmethyl)amine (THPTA), 0.5 M Cu(II) sulfate pentahydrate (Cu(II)SO4·5H2O), and 2 M sodium ascorbate were prepared in deionized water. In addition, a 2 M TEAA solution (10,000 ng / μL) of I-13-28 (2.5 μmol) obtained in step 4-1, and a 25 mmol / L DMSO solution of I-12 (35 μmol) were prepared. A DMSO solution containing I-12 was added to a 2M TEAA solution containing I-13-28, and after shaking to mix homogeneously, 75 μL of 0.5M Cu(II)(Cu(II)SO4·5H2O), 75 μL of 0.5M THPTA, and 18 μL of 2M ascorbate were sequentially added to a centrifuge tube. After shaking in a constant-temperature reactor for 1 hour, the reaction was detected to be complete by LC-MS, and after centrifugation, the supernatant was taken and purified by hydrophobic chromatography (A: ammonium sulfate buffer, B: pure water) to obtain a sense chain containing I-14, as shown in SEQ ID NO: 28.

[0195] The sense strands indicated by sequence numbers 29 and 30 were obtained by preparing them in the same manner as described above, except that I-13-29 and I-13-30 were used to replace I-13-28 in the above step.

[0196] 4-3 Synthesis of siRNA complexes containing I-14 Following the same method as in Example 2, the sense strand containing I-14 obtained in step 4-2 and the corresponding antisense strand were paired equimolarly and complementaryly, and then annealed to obtain an siRNA complex containing I-14.

[0197] Example 5. On-target activity of siRNA psiCHECK at three concentration points. In HEK293A cells, in vitro molecular-level simulations and on-target activity screening were performed for the siRNAs disclosed herein using three concentration gradients (10 nM, 1 nM, and 0.1 nM).

[0198] HEK293A cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum at 37°C and 5% CO2. 24 hours prior to transfection, HEK293A cells were inoculated into 96-well plates at an inoculation density of 8 × 10³ cells / well, with 100 μL of medium per well.

[0199] Cells were co-transfected with siRNA and the corresponding full-length MMP7 plasmid using Lipofectamine 2000 (ThermoFisher, 11668019) according to the instructions for use, with 0.3 μL of Lipofectamine 2000 used in each well. The plasmid transfection rate was 40 ng / well. For the on-target sequence plasmid, three concentration points were set for the siRNA: 10 nM, 1 nM, and 0.1 nM, with two parallel wells for each concentration. On-target levels were detected 24 hours after transfection using a Dual-Luciferase Reporter Assay System (Promega, E2940). The activity results are shown in Table 4, and as shown in Table 4, the siRNA of this disclosure exhibits good MMP7 mRNA inhibitory activity in the psi-CHECK system.

[0200] [Table 5-1]

[0201] [Table 5-2] JPEG2026519860000033.jpg95164

[0202] [Table 5-3] JPEG2026519860000035.jpg109164

[0203] [Table 5-4] JPEG2026519860000037.jpg95164

[0204] [Table 5-5] JPEG2026519860000039.jpg110164

[0205] [Table 5-6]

[0206] Example 6. On-target activity of siRNA psiCHECK at 9 concentration points In HEK293A cells, in vitro molecular-level simulations and on-target activity screening were performed for the siRNAs disclosed herein using nine concentration gradients.

[0207] HEK293A cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum at 37°C and 5% CO2. 24 hours prior to transfection, HEK293A cells were inoculated into 96-well plates at an inoculation density of 1 × 10⁴ cells / well, with 100 μL of medium per well.

[0208] According to the instruction manual, cells were co-transfected with siRNA and the corresponding plasmid using Lipofectamine 2000 (ThermoFisher, 11668019), and 0.3 μL of Lipofectamine 2000 was used per well. The transfection amount of the plasmid was 20 ng / well. For the on-target sequence plasmid, a total of 9 concentration points were set for siRNA, the final concentration of the highest concentration point was 20 nM, and it was diluted in a 3-fold gradient to 20.0000 nM, 6.6667 nM, 2.2222 nM, 0.7407 nM, 0.2469 nM, 0.0823 nM, 0.0274 nM, 0.0091 nM, 0.0030 nM. 24 hours after transfection, the on-target level was detected using the Dual-Luciferase Reporter Assay System (Promega, E2940). The results are as shown in Table 5. As shown in Table 5, the siRNA of the present disclosure has good MMP7 mRNA on-target activity under the concentration conditions of 9 concentration points in the psi-CHECK system.

[0209]

Table 6-1

[0210]

Table 6-2

[0211]

Table 6-3

[0212]

Table 6-4

[0213] Example 7. On-target activity of siRNA at 7 endogenous concentration points In HCC38 cells, we performed in vitro molecular-level activity screening for siRNA using seven concentration gradients.

[0214] HCC38 cells were cultured in 1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2. 24 hours prior to transfection, HCC38 cells were inoculated into 96-well plates at an inoculation density of 1.5 × 10⁴ cells / well, with 100 μL of medium per well.

[0215] Cells were transfected with siRNA using Lipofectamine RNAi Max (Invitrogen, 13778-150) according to the instructions for use, with 0.3 μL of Lipofectamine RNAi Max used in each well. A total of seven concentration points were established for the siRNA, at 20.0000 nM, 10.0000 nM, 2.0000 nM, 0.4000 nM, 0.0800 nM, 0.0160 nM, and 0.0032 nM, respectively. 24 hours after transfection, RNA was extracted and siRNA activity was detected by Q-PCR. The results are shown in Table 6, and the siRNA disclosed herein exhibits good MMP7 mRNA-on-target activity in HCC38 cells.

[0216] [Table 7-1]

[0217] [Table 7-2]

[0218] [Table 7-3]

[0219] Example 8. On-target activity of siRNA at seven endogenous concentration points. In HCC38 cells, an in vitro molecular level activity screening was performed on siRNA using seven concentration gradients.

[0220] HCC38 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37 °C under 5% CO2. 24 hours before transfection, HCC38 cells were seeded in 96-well plates at an inoculation density of 1.5×104 cells / well, and each well contained 100 μL of medium.

[0221] According to the instructions, cells were transfected with siRNA using Lipofectamine RNAi Max (Invitrogen, 13778-150), and 0.3 μL of Lipofectamine RNAi Max was used per well. A total of seven concentration points were set for siRNA, which were 20.0000 nM, 4.0000 nM, 0.8000 nM, 0.1600 nM, 0.0320 nM, 0.0064 nM, and 0.0013 nM, respectively. 24 hours after transfection, RNA was extracted and siRNA activity was detected by Q-PCR. The results are as shown in Table 7, and the siRNA of the present disclosure shows good MMP7 mRNA on-target activity in HCC38 cells.

[0222]

Table 8-1

[0223]

Table 8-2

[0224]

Table 8-3

[0225] Example 9. On-target activity at nine concentration points of siRNA psiCHECK In HEK293A cells, we performed in vitro molecular-level simulations and on-target activity screening for siRNA using nine concentration gradients.

[0226] HEK293A cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum at 37°C and 5% CO2. 24 hours prior to transfection, HEK293A cells were inoculated into 96-well plates at an inoculation density of 1 × 10⁴ cells / well, with 100 μL of medium per well.

[0227] Cells were co-transfected with siRNA and the corresponding plasmid using Lipofectamine 2000 (ThermoFisher, 11668019) according to the instructions for use, with 0.3 μL of Lipofectamine 2000 used in each well. The plasmid transfection rate was 20 ng / well. For the on-target sequence plasmid, a total of nine concentration points were established for the siRNA, with the highest concentration point being 20 nM. After 3-fold gradient dilution, the concentrations were 20.0000 nM, 6.6667 nM, 2.2222 nM, 0.7407 nM, 0.2469 nM, 0.0823 nM, 0.0274 nM, 0.0091 nM, and 0.0030 nM. On-target levels were detected 24 hours after transfection using a Dual-Luciferase Reporter Assay System (Promega, E2940). The results are shown in Table 8, with NA indicating undetectable levels. As shown in Table 8, the siRNAs of this disclosure exhibit good MMP7 mRNA-on-target activity in the psiCHECK system.

[0228] [Table 9-1]

[0229] [Table 9-2]

[0230] Example 10. On-target activity of the siRNA complex at two endogenous concentration points.

[0231] In HCC38 cells, we performed in vitro molecular-level activity screening for siRNA complexes using two concentration gradients.

[0232] HCC38 cells were cultured in 1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2. 24 hours prior to transfection, HCC38 cells were inoculated into 96-well plates at an inoculation density of 1.5 × 10⁴ cells / well, with 100 μL of medium per well.

[0233] Following the instructions for use, cells were transfected with the siRNA complex using Lipofectamine RNAi Max (Invitrogen, 13778-150), with 0.3 μL of Lipofectamine RNAi Max used in each well. Two concentration points were established for the siRNA complex, at 10 nM and 2 nM, respectively. 24 hours after transfection, RNA was extracted and siRNA complex activity was detected by Q-PCR. The results are shown in Table 9, demonstrating that the siRNA complex of this disclosure exhibits good MMP7 mRNA-on-target activity in HCC38 cells.

[0234] [Table 10]

Claims

1. siRNA comprising a sense strand and an antisense strand that form a double-stranded region, The sense strand contains at least 15 consecutive nucleotides and differs from any one nucleotide sequence of SEQ ID NOs. 1 to SEQ ID NOs. 10 by 3 or fewer nucleotides, and The antisense strand contains at least 15 consecutive nucleotides and differs from any one nucleotide sequence of SEQ ID NOs. 31 to 40 by three or fewer nucleotides. siRNA.

2. The sense strand comprises at least 17 consecutive nucleotides that differ from any one nucleotide sequence of SEQ ID NOs. 1 to SEQ ID NOs. 10 by three or fewer nucleotides. The antisense strand comprises at least 17 consecutive nucleotides that differ by three or fewer nucleotides from any one nucleotide sequence of SEQ ID NOs. 31 to 40. Preferably, the sense strand contains at least 19 consecutive nucleotides that differ by three or fewer nucleotides from any one nucleotide sequence of SEQ ID NOs. 1 to SEQ ID NOs. 10, and preferably, the nucleotide sequences differ by one or fewer nucleotides, and / or The antisense strand comprises at least 21 consecutive nucleotides that differ by three or fewer nucleotides from any one nucleotide sequence of SEQ ID NOs. 31 to 40, preferably the nucleotide sequences differ by one or fewer nucleotides. The siRNA according to claim 1.

3. Group 1), which consists of the sense strand indicated by Sequence ID No. 1 and the antisense strand indicated by Sequence ID No.

31. Group 2), which consists of the sense strand shown in Sequence ID No. 2 and the antisense strand shown in Sequence ID No.

32. Group 3), which consists of the sense strand indicated by Sequence ID 3 and the antisense strand indicated by Sequence ID 33. Group 4), which consists of the sense strand indicated by Sequence ID No. 4 and the antisense strand indicated by Sequence ID No.

34. Group 5), which consists of the sense strand indicated by Sequence ID No. 5 and the antisense strand indicated by Sequence ID No.

35. Group 6), which consists of the sense strand indicated by Sequence ID No. 6 and the antisense strand indicated by Sequence ID No.

36. Group 7), which consists of the sense strand indicated by Sequence ID 7 and the antisense strand indicated by Sequence ID 37. Group 8), which consists of the sense strand indicated by Sequence ID No. 8 and the antisense strand indicated by Sequence ID No.

38. Group 9), which consists of the sense strand indicated by Sequence ID No. 9 and the antisense strand indicated by Sequence ID No. 39, and It includes a sense strand and an antisense strand from any one of the groups 10), which are the sense strand indicated by SEQ ID NO: 10 and the antisense strand indicated by SEQ ID NO: 40, or is selected from there. The siRNA according to claim 1 or 2.

4. At least one nucleotide of the sense strand and / or antisense strand is a modified nucleotide. The siRNA according to any one of claims 1 to 3.

5. The three consecutive nucleotides in the sense strand are nucleotides modified with 2'-fluoro, and / or From the 5' end to the 3' end, the nucleotides at positions 2, 6, 12, 14, and 16 of the antisense strand are each independently nucleotides modified with 2'-fluoropolymer. Preferably, the nucleotides at positions 2, 6, 12, 14, and 16 of the antisense chain are each independently modified with 2'-fluoronucleotides, or the nucleotides at positions 2, 4, 6, 10, 12, 14, 16, or 18 of the antisense chain are each independently modified with 2'-fluoronucleotides. Preferably, the nucleotides at positions 7, 8, and 9 of the sense strand are nucleotides modified with 2'-fluoropolymers in the direction from the 5' end to the 3' end. The nucleotides at the remaining positions in the sense strand and the antisense strand are nucleotides modified with a 2-methoxy group. The siRNA according to claim 4.

6. At least one phosphodiester group in the sense chain and / or antisense chain is a phosphodiester group having a modifying group, preferably a thiophosphodiester group. The siRNA according to any one of claims 1 to 5.

7. The phosphodiester group having the aforementioned modifying group is Between the first and second nucleotides at the 5' end of the sense strand, Between the second and third nucleotides at the 5' end of the sense strand, Between the first and second nucleotides at the 5' end of the antisense strand, Between the second and third nucleotides at the 5' end of the antisense strand, Between the first and second nucleotides at the 3' end of the antisense strand, They are located at one or more positions selected from between the second and third nucleotides at the 3' end of the antisense strand, Preferably, the sense chain and / or antisense chain comprises a plurality of phosphodiester groups having a modifying group. The siRNA according to claim 6.

8. The sense strand comprises a nucleotide sequence represented by any one of SEQ ID NOs: 11 to 27, and / or The antisense strand includes a nucleotide sequence represented by any one of SEQ ID NOs: 41 to 57. The siRNA according to any one of claims 1 to 7.

9. It is a siRNA complex, A siRNA according to any one of claims 1 to 8, The siRNA comprises a target ligand that is linked to the siRNA, The aforementioned target ligand has affinity for cell receptors expressed on epithelial cells. Preferably, the target ligand includes an integrin target ligand. More preferably, the target ligand includes an αvβ6 integrin target ligand. More preferably, the target ligand has the structure shown in I-14. 【Chemistry 1】 siRNA complex.

10. The target ligand is ligated to the sense strand of the siRNA, Preferably, the target ligand is ligated to the 5' end of the sense strand of the siRNA. The siRNA complex according to claim 9.

11. The sense strand comprises a nucleotide sequence represented by any one of SEQ ID NOs: 28 to 30, and / or The antisense strand includes a nucleotide sequence represented by any one of SEQ ID NOs. 58 to 60. The siRNA complex according to claim 9 or 10.

12. A siRNA according to any one of claims 1 to 8 or an siRNA complex according to any one of claims 9 to 11, and a pharmaceutically acceptable carrier, Pharmaceutical composition.

13. siRNA according to any one of claims 1 to 8, or A siRNA complex according to any one of claims 9 to 11, cell.

14. siRNA according to any one of claims 1 to 8, or The siRNA complex according to any one of claims 9 to 11, or A pharmaceutical composition according to claim 12, Reagent kit.

15. A method for reducing the expression of matrix metalloproteinase 7 (MMP7), The method includes administering to a subject the siRNA described in any one of claims 1 to 8, the siRNA complex described in any one of claims 9 to 11, or the pharmaceutical composition described in claim 12. method.

16. A method for treating and / or preventing a disease, The method involves administering to a subject the siRNA described in any one of claims 1 to 8, the siRNA complex described in any one of claims 9 to 11, or the pharmaceutical composition described in claim 12. Preferably, the disease is selected from asthma, fibrosis, chronic inflammation, interstitial lung disease, infection, acute lung injury, pulmonary hypertension, and cancer. Preferably, the infection is SARS-CoV-2. Preferably, the acute lung injury is acute respiratory distress syndrome. Preferably, the fibrosis is idiopathic pulmonary fibrosis, renal fibrosis, or hepatic fibrosis. method.

17. A method for delivering siRNA that inhibits the expression and / or replication of MMP7 into the body, The method involves administering to a subject the siRNA described in any one of claims 1 to 8, the siRNA complex described in any one of claims 9 to 11, or the pharmaceutical composition described in claim 12. Preferably, the siRNA is delivered outside the liver. More preferably, the siRNA is delivered to the lungs. method.

18. A method for preparing siRNA or a siRNA complex, The method includes synthesizing the siRNA described in any one of claims 1 to 8 or the siRNA complex described in any one of claims 9 to 11. method.

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