Sirna targeting MMP7, sirna conjugate and medical use thereof

Abstract

The present invention relates to an siRNA targeting MMP7, an siRNA conjugate and the medical use thereof. The present invention also relates to a pharmaceutical composition comprising the siRNA, a cell or a kit, and a method for treating and / or preventing a subject suffering from related disorders by using the siRNA and the siRNA conjugate.

Description

siRNA targeting MMP7, siRNA conjugates and medical uses thereof

[0001] This disclosure claims priority to Chinese application No. 202310696482.8 filed on June 13, 2023 and Chinese application No. 202410074824.7 filed on January 18, 2024, and the entire contents of the above patent applications are incorporated herein by reference. Technical Field

[0002] The present disclosure belongs to the field of biomedicine, and specifically relates to siRNA, conjugates, compositions and medical uses thereof targeting matrix metalloproteinase 7 (MMP7). Background Art

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

[0004] MMP7 is associated with pathogenic fibrosis through multiple potential mechanisms, including the promotion of epithelial-mesenchymal transition (EMT), extracellular matrix degradation, abnormal matrix repair, and tissue remodeling. MMP7 promotes fibrosis by cleaving E-cadherin to activate epithelial cells and proteolytically activating heparin-binding epidermal growth factor precursor (pro-HB-EGF) to release active HB-EGF. Studies have shown that MMP7 knockout mice are protected against bleomycin-induced pulmonary fibrosis, showing reduced lung inflammation, fibrosis, and mortality, suggesting that knockdown of MMP7 has a therapeutic effect on pulmonary fibrosis.

[0005] Summary of the Invention

[0006] The present disclosure provides an siRNA. In some embodiments, the present disclosure provides an siRNA targeting MMP7, comprising a sense strand and an antisense strand forming a double-stranded region.

[0007] In some embodiments, the sense strand comprises a sequence that differs from any one of SEQ ID NO: 1 to SEQ ID NO: 10 and SEQ ID NO: 61 to SEQ ID NO: 151 by no more than 3 (e.g., 0, 1, 2, 3) nucleotides and comprises at least 15 (e.g., 16, 17, 18, 19, 20, 21) consecutive nucleotides;

[0008] The antisense strand comprises a nucleotide sequence that differs from any one of SEQ ID NO: 31 to SEQ ID NO: 40 and SEQ ID NO: 152 to SEQ ID NO: 242 by no more than 3 (e.g., 0, 1, 2, 3) nucleotides and comprises at least 15 (e.g., 16, 17, 18, 19, 20, 21) consecutive nucleotides.

[0009] In some embodiments, the antisense strand is at least partially reverse complementary to the target sequence to mediate RNA interference. In some embodiments, there are no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 mismatch between the antisense strand and the target sequence. In some embodiments, the antisense strand is completely reverse complementary to the target sequence.

[0010] In some embodiments, the sense strand and the antisense strand are at least partially reverse complementary to form a double-stranded region. In some embodiments, there are no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 mismatch between the sense strand and the antisense strand. In some embodiments, the sense strand and the antisense strand are completely reverse complementary.

[0011] In some embodiments, the disclosed siRNAs comprise one or two blunt ends.

[0012] In some embodiments, each strand of the siRNA independently comprises an overhang of 1 to 2 unpaired nucleotides.

[0013] In some embodiments, the disclosed siRNA comprises an overhang located at the 3' end of the antisense strand of the siRNA.

[0014] In some embodiments, the sense and antisense strands each independently have 16 to 35, 16 to 34, 17 to 34, 17 to 33, 18 to 33, 18 to 32, 18 to 31, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 19 to 25, 19 to 24, or 19 to 23 nucleotides (e.g., 19, 20, 21, 22, 23 nucleotides).

[0015] In some embodiments, the sense strand and the antisense strand are the same or different in length, with the sense strand being 19-23 nucleotides in length and the antisense strand being 19-26 nucleotides in length. The ratio of the length of the sense strand to the antisense strand of the siRNA provided by the present disclosure can 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 ratio of the length of the sense strand to the antisense strand of the siRNA is 19 / 21, 21 / 23, or 23 / 25.

[0016] In some specific embodiments, the length ratio of the sense strand to the antisense strand of the siRNA is 19 / 21.

[0017] In some embodiments, the sense strand comprises at least 15 consecutive nucleotides and differs by no more than 2 nucleotides from the nucleotide sequence set forth in any one of SEQ ID NO: 1 to SEQ ID NO: 10 and SEQ ID NO: 61 to SEQ ID NO: 151. In some embodiments, the nucleotide sequence differs by no more than 1 nucleotide; in some embodiments, by 1 nucleotide.

[0018] In some embodiments, the antisense strand comprises at least 15 consecutive nucleotides and differs from the nucleotide sequence shown in any one of SEQ ID NO:31 to SEQ ID NO:40 and SEQ ID NO:152 to SEQ ID NO:242 by no more than 2 nucleotides; in some embodiments, the nucleotide sequence differs by no more than 1 nucleotide; in some embodiments, by 1 nucleotide.

[0019] In some embodiments, the sense strand comprises at least 15 consecutive nucleotides of any one of SEQ ID NO: 1 to SEQ ID NO: 10 and SEQ ID NO: 61 to SEQ ID NO: 151. In some embodiments, at least 16 consecutive nucleotides. In some embodiments, at least 17 consecutive nucleotides. In some embodiments, at least 19 consecutive nucleotides.

[0020] In some embodiments, the antisense strand comprises at least 15 consecutive nucleotides of any one of SEQ ID NO: 31 to SEQ ID NO: 40 and SEQ ID NO: 152 to SEQ ID NO: 242. In some embodiments, at least 17 consecutive nucleotides. In some embodiments, at least 19 consecutive nucleotides. In some embodiments, at least 20 consecutive nucleotides. In some embodiments, at least 21 consecutive nucleotides.

[0021] In some embodiments, the sense strand comprises or is selected from any one of the following nucleotide sequences: SEQ ID NO: 1 to SEQ ID NO: 10 and SEQ ID NO: 61 to SEQ ID NO: 151.

[0022] In some embodiments, the antisense strand comprises or is selected from any one of the following nucleotide sequences: SEQ ID NO:31 to SEQ ID NO:40 and SEQ ID NO:152 to SEQ ID NO:242.

[0023] In some embodiments, the siRNA comprises a sense strand and an antisense strand selected from any one of the following groups:

[0024] Group 1), the sense strand set forth in SEQ ID NO: 1 and the antisense strand set forth in SEQ ID NO: 31;

[0025] Group 2), the sense strand set forth in SEQ ID NO: 2 and the antisense strand set forth in SEQ ID NO: 32;

[0026] Group 3), the sense strand as shown in SEQ ID NO: 3 and the antisense strand as shown in SEQ ID NO: 33;

[0027] Group 4), the sense strand set forth in SEQ ID NO: 4 and the antisense strand set forth in SEQ ID NO: 34;

[0028] Group 5), the sense strand set forth in SEQ ID NO: 5 and the antisense strand set forth in SEQ ID NO: 35;

[0029] Group 6), the sense strand set forth in SEQ ID NO: 6 and the antisense strand set forth in SEQ ID NO: 36;

[0030] Group 7), the sense strand set forth in SEQ ID NO: 7 and the antisense strand set forth in SEQ ID NO: 37;

[0031] Group 8), the sense strand set forth in SEQ ID NO: 8 and the antisense strand set forth in SEQ ID NO: 38;

[0032] Group 9), the sense strand as shown in SEQ ID NO: 9 and the antisense strand as shown in SEQ ID NO: 39; and

[0033] Group 10), the sense strand shown in SEQ ID NO: 10 and the antisense strand shown in SEQ ID NO: 40.

[0034] In some embodiments, at least one nucleotide in the sense strand and / or the antisense strand is a modified nucleotide.

[0035] In some embodiments, all nucleotides are modified nucleotides.

[0036] In some embodiments, three consecutive nucleotides in the sense strand of the siRNA are 2'-fluoro-modified nucleotides.

[0037] In some embodiments, in the sense strand of the siRNA, in the direction from the 5' end to the 3' end, the 7th, 8th and 9th nucleotides of the sense strand are 2'-fluoro-modified nucleotides.

[0038] In some embodiments, in the sense strand of the siRNA, from the 5' end to the 3' end, the 7th, 8th and 9th nucleotides of the sense strand are 2'-fluoro-modified nucleotides, and the remaining positions of the sense strand are 2'-methoxy-modified nucleotides.

[0039] In some embodiments, 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 a 2'-fluoro-modified nucleotide. In some embodiments, the nucleotides at positions 2, 4, 6, 10, 12, 14, 16, or 18 of the antisense strand are each independently a 2'-fluoro-modified nucleotide. In some embodiments, the remaining nucleotides in the antisense strand are all 2'-methoxy-modified nucleotides.

[0040] In some embodiments, at least one phosphodiester group in the sense strand and / or antisense strand is a phosphodiester group with a modifying group. The modifying group allows the siRNA to have increased stability in a biological sample or environment. In some embodiments, the phosphodiester group with a modifying group is a thiophosphate diester group.

[0041] In some embodiments, the phosphodiester group having a modifying group is present in at least one position selected from the group consisting of:

[0042] between the first and second nucleotides at the 5' end of the sense strand;

[0043] between the second and third nucleotides at the 5' end of the sense strand;

[0044] between the first and second nucleotides at the 5' end of the antisense strand;

[0045] between the second and third nucleotides at the 5' end of the antisense strand;

[0046] between the first nucleotide and the second nucleotide at the 3' end of the antisense strand; and

[0047] between the second and third nucleotides at the 3' end of the antisense strand.

[0048] In some embodiments, the sense strand and / or the antisense strand comprises a plurality of phosphodiester groups having modification groups.

[0049] In some embodiments, the sense strand and / or antisense strand includes multiple phosphodiester groups with modifying groups, wherein the phosphorothioate diester groups are present in:

[0050] between the first nucleotide and the second nucleotide at the 5' end of the sense strand; and

[0051] between the second and third nucleotides at the 5' end of the sense strand; and

[0052] between the first nucleotide and the second nucleotide at the 5' end of the antisense strand; and

[0053] between the second and third nucleotides from the 5' end of the antisense strand; and

[0054] between the first nucleotide and the second nucleotide at the 3' end of the antisense strand; and

[0055] between the second and third nucleotides at the 3' end of the antisense strand.

[0056] In some embodiments, the sense strand is selected from a nucleotide sequence comprising the formula:

[0057] 5'-NmsNmsNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmNmNm-3',

[0058] In some embodiments, the antisense strand is selected from a nucleotide sequence comprising the following formula:

[0059] 5'-Nm'sNf'sNm'Nm'Nm'Nf'Nm'Nm'Nm'Nm'Nm'Nf'Nm'Nf'Nm'Nf'Nm'Nm'Nm'sNm'sNm'-3'; or

[0060] 5'-Nm'sNf'sNm'Nf'Nm'Nf'Nm'Nm'Nm'Nf'Nm'Nf'Nm'Nf'Nm'Nf'Nm'Nf'Nm'sNm'sNm'-3'.

[0061] In the above-mentioned sense strand and antisense strand, Nm and Nm' represent any nucleoside modified with 2'-methoxy, such as C, G, U, A, and T modified with 2'-methoxy; Nf and Nf' represent any nucleoside modified with 2'-fluoro, such as C, G, U, A, and T modified with 2'-fluoro;

[0062] The lowercase letter s indicates that the two nucleotides adjacent to the letter s are linked by a thiophosphate diester group. Unless otherwise specified, the two nucleosides adjacent to the letter s are linked by a phosphodiester group.

[0063] In some embodiments, at least one nucleoside in positions 2 to 8 (e.g., positions 2, 3, 4, 5, 6, 7, 8) of the antisense strand in its 5' region comprises a chemical modification represented by formula (I) or a pharmaceutically acceptable salt thereof, wherein the chemical modification represented by formula (I) is selected from:

[0064] Wherein: B represents the base corresponding to positions 2-8 at the 5' end of the antisense strand, and the nucleoside comprising the chemical modification represented by formula (I) or a pharmaceutically acceptable salt thereof is linked to the adjacent nucleoside via a phosphorothioate group or a phosphorothioate group having a modified group.

[0065] In some embodiments, the sense strand is selected from or comprises the nucleotide sequence shown in any one of SEQ ID NO: 11 to SEQ ID NO: 27 and SEQ ID NO: 243 to SEQ ID NO: 333.

[0066] In some embodiments, the antisense strand is selected from or comprises the nucleotide sequence shown in any one of SEQ ID NO: 41 to SEQ ID NO: 57 and SEQ ID NO: 334 to SEQ ID NO: 424.

[0067] In a more specific embodiment, the present disclosure provides an siRNA comprising any one group selected from the following:

[0068] The sense strand comprises the polynucleotide set forth in SEQ ID NO: 11, and the antisense strand comprises the polynucleotide set forth in SEQ ID NO: 41;

[0069] The sense strand comprises the polynucleotide shown in SEQ ID NO: 12, and the antisense strand comprises the polynucleotide shown in SEQ ID NO: 42

[0070] The sense strand comprises the polynucleotide set forth in SEQ ID NO: 13, and the antisense strand comprises the polynucleotide set forth in SEQ ID NO: 43;

[0071] The sense strand comprises the polynucleotide set forth in SEQ ID NO: 14, and the antisense strand comprises the polynucleotide set forth in SEQ ID NO: 44;

[0072] The sense strand comprises the polynucleotide set forth in SEQ ID NO: 15, and the antisense strand comprises the polynucleotide set forth in SEQ ID NO: 45;

[0073] The sense strand comprises the polynucleotide set forth in SEQ ID NO: 16, and the antisense strand comprises the polynucleotide set forth in SEQ ID NO: 46;

[0074] The sense strand comprises the polynucleotide set forth in SEQ ID NO: 17, and the antisense strand comprises the polynucleotide set forth in SEQ ID NO: 47;

[0075] The sense strand comprises the polynucleotide set forth in SEQ ID NO: 18, and the antisense strand comprises the polynucleotide set forth in SEQ ID NO: 48;

[0076] The sense strand comprises the polynucleotide set forth in SEQ ID NO: 19, and the antisense strand comprises the polynucleotide set forth in SEQ ID NO: 49;

[0077] The sense strand comprises the polynucleotide shown in SEQ ID NO: 20, and the antisense strand comprises the polynucleotide shown in SEQ ID NO: 50;

[0078] The sense strand comprises the polynucleotide set forth in SEQ ID NO: 21, and the antisense strand comprises the polynucleotide set forth in SEQ ID NO: 51;

[0079] The sense strand comprises the polynucleotide set forth in SEQ ID NO: 22, and the antisense strand comprises the polynucleotide set forth in SEQ ID NO: 52;

[0080] The sense strand comprises the polynucleotide set forth in SEQ ID NO: 23, and the antisense strand comprises the polynucleotide set forth in SEQ ID NO: 53;

[0081] The sense strand comprises the polynucleotide set forth in SEQ ID NO: 24, and the antisense strand comprises the polynucleotide set forth in SEQ ID NO: 54;

[0082] The sense strand comprises the polynucleotide set forth in SEQ ID NO: 25, and the antisense strand comprises the polynucleotide set forth in SEQ ID NO: 55;

[0083] The sense strand comprises the polynucleotide set forth in SEQ ID NO: 26, and the antisense strand comprises the polynucleotide set forth in SEQ ID NO: 56;

[0084] The sense strand comprises the polynucleotide shown in SEQ ID NO: 27, and the antisense strand comprises the polynucleotide shown in SEQ ID NO: 57.

[0085] The present disclosure also provides an siRNA conjugate, which comprises any one of the above siRNAs and a targeting ligand linked to the siRNA.

[0086] "siRNA conjugate" refers to a compound in which one or more chemical moieties are attached to siRNA.

[0087] In some embodiments, the siRNA and the targeting ligand are covalently or non-covalently linked.

[0088] In some embodiments, the targeting ligand is linked to the antisense strand of the siRNA. In some embodiments, the targeting ligand is linked to the 5' end of the antisense strand of the siRNA. In some embodiments, the targeting ligand is linked to the 3' end of the antisense strand of the siRNA.

[0089] In some embodiments, the targeting ligand is attached to the sense strand of the siRNA. In some embodiments, the targeting ligand is attached to the 5' end of the sense strand of the siRNA. In some embodiments, the targeting ligand is attached to the 3' end of the sense strand of the siRNA.

[0090] In some embodiments, the targeting ligand is linked to the end of the siRNA through a phosphodiester group, a phosphorothioate diester group, or a phosphonic acid group; in some embodiments, the targeting ligand is linked to the end of the siRNA through a phosphodiester group.

[0091] In some embodiments, the targeting ligand is indirectly linked to the siRNA end via a phosphodiester group, a phosphorothioate diester group, or a phosphonic acid group. In some embodiments, the targeting ligand is indirectly linked to the siRNA end via a phosphodiester group. In some embodiments, the targeting ligand is directly linked to the siRNA end via a phosphodiester group, a phosphorothioate diester group, or a phosphonic acid group. In some embodiments, the targeting ligand is directly linked to the siRNA end via a phosphodiester group.

[0092] In some embodiments, the targeting ligand is directly linked to the 3' end of the siRNA sense strand via a phosphodiester group or a phosphorothioate diester group. In some embodiments, the targeting ligand is directly linked to the 3' end of the siRNA sense strand via a phosphodiester group.

[0093] In some embodiments, one or more of the targeting ligands is linked to the sense strand of the siRNA.

[0094] In some embodiments, the targeting ligand has affinity for a cellular receptor expressed on an epithelial cell.

[0095] In some embodiments, the targeting ligand comprises an integrin targeting ligand.

[0096] In some embodiments, the targeting ligand comprises an αvβ6 integrin targeting ligand. The αvβ6 integrin targeting ligand can be any targeting ligand that can bind to the αvβ6 integrin. In some embodiments, the targeting ligand has the following structure:

[0097] In some embodiments, the targeting ligand is targeted to the liver.

[0098] In some embodiments, the targeting ligand binds to the asialoglycoprotein receptor (ASGPR).

[0099] In some embodiments, the targeting ligand comprises a galactose cluster or a galactose derivative cluster, wherein the galactose derivative is selected from N-acetyl-galactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine or N-isobutyrylgalactosamine.

[0100] Targeting ligands known to bind to the asialoglycoprotein receptor (ASGPR) are particularly useful for directing the delivery of oligomeric compounds to the liver. The asialoglycoprotein receptor is abundantly expressed on liver cells (hepatocytes). Cell receptor targeting moieties targeting the ASCPR include galactose and galactose derivatives. Specifically, clusters of galactose derivatives, including clusters consisting of 2, 3, 4, or more than 4 N-acetyl-galactosamine (GalNAc or NAG) can promote the uptake of certain compounds in hepatocytes. The GalNAc clusters coupled to the oligomeric compound are used to direct the composition to the liver, where the N-acetyl-galactosamine sugar can bind to the asialoglycoprotein receptor on the surface of liver cells. Binding of the asialoglycoprotein receptor is believed to initiate receptor-mediated endocytosis, thereby promoting the entry of the compound into the cell interior.

[0101] In some embodiments, the targeting ligand provided by the present disclosure is a compound shown below or a pharmaceutically acceptable salt thereof,

[0102] In some embodiments, the N-acetyl-galactosamine moiety in the above targeting ligands can be replaced with N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine, or N-isobutyrylgalactosamine.

[0103] In some embodiments, the sense strand of the siRNA conjugates described herein comprises a nucleotide sequence as shown in any one of SEQ ID NO: 28 to SEQ ID NO: 30; and / or the antisense strand comprises a nucleotide sequence as shown in any one of SEQ ID NO: 58 to SEQ ID NO: 60.

[0104] In some specific embodiments, the siRNA conjugates of the present disclosure are selected from any one of the following groups:

[0105] The sense strand comprises the polynucleotide set forth in SEQ ID NO: 28, and the antisense strand comprises the polynucleotide set forth in SEQ ID NO: 58;

[0106] The sense strand comprises the polynucleotide set forth in SEQ ID NO: 29, and the antisense strand comprises the polynucleotide set forth in SEQ ID NO: 59;

[0107] The sense strand comprises the polynucleotide shown in SEQ ID NO: 30, and the antisense strand comprises the polynucleotide shown in SEQ ID NO: 60.

[0108] On the other hand, the present disclosure provides a pharmaceutical composition comprising the siRNA, siRNA conjugates described herein, and one or more pharmaceutically acceptable excipients, such as a vehicle, a carrier, a diluent, and / or a delivery polymer. The term "pharmaceutical composition" refers to a mixture containing one or more compounds described herein or their physiologically pharmaceutically acceptable salts or prodrugs with other chemical components, as well as other components such as physiologically pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote administration to an organism, facilitate the absorption of the active ingredient, and thus exert biological activity.

[0109] Various delivery systems are known and can be used for the disclosed siRNA or siRNA conjugates, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the siRNA or siRNA conjugate, receptor-mediated endocytosis, constructing the nucleic acid as part of a retroviral or other vector.

[0110] "Pharmaceutically acceptable excipients" include, but are not limited to, any adjuvant, carrier, glidant, sweetener, diluent, preservative, dye / colorant, flavoring agent, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier approved by the U.S. Food and Drug Administration (FDA) for use by humans or domestic animals.

[0111] In another aspect, the present disclosure provides a use of the above-mentioned siRNA, siRNA conjugate or pharmaceutical composition in preparing a drug for treating a disease in a subject.

[0112] In another aspect, the present disclosure provides a method for treating a disease in a subject, comprising administering the above-mentioned siRNA, siRNA conjugate or pharmaceutical composition to the subject.

[0113] In another aspect, the present disclosure provides a method for inhibiting mRNA expression in a subject, comprising administering the above-mentioned siRNA, siRNA conjugate or pharmaceutical composition to the subject.

[0114] In another aspect, the present disclosure provides a method for delivering an expression-inhibitory oligomeric compound in vivo, the method comprising administering the above-mentioned siRNA, siRNA conjugate or pharmaceutical composition to a subject.

[0115] In some embodiments, the oligomeric compound is delivered to the liver.

[0116] In some embodiments, the oligomeric compound is delivered extrahepatically.

[0117] In some embodiments, the oligomeric compound is delivered to the lung.

[0118] The siRNA, siRNA conjugates, or pharmaceutical compositions and methods disclosed herein can reduce the level of a target mRNA in a cell, cell population, tissue, or subject, by administering to the subject a therapeutic siRNA, siRNA conjugate, or pharmaceutical composition of the present disclosure, thereby inhibiting the expression of the target mRNA in the subject. In some embodiments, an effective amount or effective dose of the siRNA, siRNA conjugate, or pharmaceutical composition of the present disclosure is administered to the subject.

[0119] In some embodiments, the subject has been previously identified as having pathological upregulation of the target gene in the targeted cells or tissues.

[0120] The subject described in the present disclosure refers to a subject diagnosed with (or suspected of having, or susceptible to) a disease or disorder that would benefit from reduction or inhibition of target mRNA expression.

[0121] Delivery can be by local administration (e.g., direct injection, implantation, or topical administration), systemic administration, or subcutaneous, intravenous, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), intramuscular, transdermal, airway (aerosol), nasal, oral, rectal, or topical (including buccal and sublingual) administration.

[0122] In alternative embodiments, the pharmaceutical compositions provided herein can be administered by injection, for example, intravenously, intramuscularly, intradermally, subcutaneously, intraduodenally, or intraperitoneally.

[0123] In an alternative embodiment, after the targeting ligand is linked to the siRNA to form a conjugate, the conjugate can be packaged in a kit.

[0124] In another aspect, the present disclosure further provides a pharmaceutical composition comprising the siRNA or siRNA conjugate of the present disclosure.

[0125] In some embodiments, the pharmaceutical composition may further comprise a pharmaceutically acceptable excipient and / or adjuvant, which may be one or more of various preparations or compounds conventionally used in the art. For example, the pharmaceutically acceptable excipient may include at least one of a pH buffer, a protective agent, and an osmotic pressure regulator.

[0126] In some embodiments, the unit dose of the pharmaceutical composition is 0.001 mg-1000 mg.

[0127] In some embodiments, the pharmaceutical composition contains 0.01-99.99% of the aforementioned compound or its pharmaceutically acceptable salt or its isotopic substitution, based on the total weight of the composition. In some embodiments, the pharmaceutical composition contains 0.1-99.9% of the aforementioned compound or its pharmaceutically acceptable salt or its isotopic substitution. In some embodiments, the pharmaceutical composition contains 0.5%-99.5% of the aforementioned compound or its pharmaceutically acceptable salt or its isotopic substitution. In some embodiments, the pharmaceutical composition contains 1%-99% of the aforementioned compound or its pharmaceutically acceptable salt or its isotopic substitution. In some embodiments, the pharmaceutical composition contains 2%-98% of the aforementioned compound or its pharmaceutically acceptable salt or its isotopic substitution.

[0128] The pharmaceutical composition can be conveniently presented in unit dosage form. Generally, the pharmaceutical composition can be formulated into any suitable dosage form, such as, but not limited to, injection, tablet, capsule, gel, etc. Pharmaceutical compositions include, but are not limited to, solutions, emulsions, and liposome-containing formulations.

[0129] In some embodiments, the pharmaceutical composition contains 0.01% to 99.99% of a pharmaceutically acceptable excipient, based on the total weight of the composition. In some embodiments, the pharmaceutical composition contains 0.1% to 99.9% of a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition contains 0.5% to 99.5% of a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition contains 1% to 99% of a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition contains 2% to 98% of a pharmaceutically acceptable excipient.

[0130] In some embodiments, the siRNA, siRNA conjugate or pharmaceutical composition, when contacted with cells expressing the target gene, 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%, as measured by, for example, psiCHECK activity screening and luciferase reporter gene assays, other methods such as PCR or branched DNA (bDNA)-based methods, or protein-based methods such as immunofluorescence analysis, e.g., Western Blot or flow cytometry.

[0131] In some embodiments, when the above-mentioned siRNA, siRNA conjugate or pharmaceutical composition is contacted with cells expressing the target gene, the percentage of residual target gene mRNA expression caused by the above-mentioned siRNA conjugate or pharmaceutical composition is no more than 99%, no more than 95%, no more than 90%, no more than 85%, no more than 80%, no more than 75%, no more than 70%, no more than 65%, no more than 60%, no more than 55%, no more than 50%, no more than 45%, no more than 40%, no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, or no more than 10%, as determined by, for example, psiCHECK activity screening and luciferase reporter gene assay, other methods such as PCR or branched DNA (bDNA)-based methods, or protein-based methods such as immunofluorescence analysis, such as Western Blot or flow cytometry.

[0132] In some embodiments, when the above-mentioned siRNA, siRNA conjugate or pharmaceutical composition is contacted with cells expressing the target gene, the siRNA conjugate reduces 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% while maintaining target activity as determined by, for example, psiCHECK activity screening and luciferase reporter gene assays, other methods such as PCR or branched DNA (bDNA)-based methods, or protein-based methods such as immunofluorescence analysis, such as Western Blot, or flow cytometry.

[0133] In some embodiments, the siRNA, siRNA conjugate or pharmaceutical composition described above, when contacted with cells expressing the target gene, reduces the 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%, while reducing off-target activity by at least 20%, at least 19%, at most 15%, at most 10%, at most 5% or more than 1%, as measured by, for example, psiCHECK activity screening and luciferase reporter gene assays, other methods such as PCR or branched DNA (bDNA)-based methods, or protein-based methods such as immunofluorescence analysis, e.g., Western Blot, or flow cytometry.

[0134] In some embodiments, the siRNA, siRNA conjugate or pharmaceutical composition, when contacted with cells expressing the target gene, 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 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% as measured by, for example, psiCHECK activity screening and luciferase reporter gene assays, other methods such as PCR or branched DNA (bDNA)-based methods, or protein-based methods such as immunofluorescence analysis, e.g., Western Blot, or flow cytometry.

[0135] The present disclosure also provides a cell comprising the siRNA and / or siRNA conjugate of the present disclosure.

[0136] The present disclosure also provides a kit comprising the siRNA and / or siRNA conjugate and / or pharmaceutical composition of the present disclosure. In some embodiments, the kit comprises one or more containers, wherein the one or more containers comprise the siRNA and / or siRNA conjugate and / or pharmaceutical composition of the present disclosure.

[0137] The present disclosure also provides a method for silencing a target gene or mRNA of a target gene in a cell, comprising the step of introducing the siRNA, siRNA conjugate and / or pharmaceutical composition according to the present disclosure into the cell.

[0138] The present disclosure also provides a method for silencing a target gene or mRNA of a target gene in a cell in vivo or in vitro, the method comprising the step of introducing the siRNA, siRNA conjugate and / or pharmaceutical composition according to the present disclosure into the cell.

[0139] The present disclosure also provides a method for reducing target gene or target gene mRNA expression, the method comprising administering the siRNA and / or siRNA conjugate and / or pharmaceutical composition of the present disclosure to a subject in need thereof. In some embodiments, the method comprises administering an effective amount or effective dose of the siRNA and / or siRNA conjugate of the present disclosure to a subject in need thereof.

[0140] In some embodiments, administration is by a route of administration that includes intramuscular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic, intravenous, subcutaneous, cerebrospinal, or a combination thereof.

[0141] In some embodiments, the effective amount or effective dose of siRNA, siRNA conjugate 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.

[0142] In some embodiments, the target gene is matrix metalloproteinase 7 (MMP7).

[0143] The present disclosure provides the aforementioned siRNA and / or pharmaceutical compositions and / or siRNA conjugates for treating and / or preventing a disease associated with matrix metalloproteinase 7 (MMP7) gene expression 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 infectious diseases, acute respiratory distress syndrome (ARDS) or other types of acute lung injury, pulmonary hypertension, cancer, renal fibrosis, and liver fibrosis.

[0144] The present disclosure provides a method for treating and / or preventing a disease associated with MMP7 gene expression in a subject, comprising administering to the subject an siRNA and / or siRNA conjugate and / or the pharmaceutical composition disclosed herein. In some embodiments, the method comprises administering to the subject an effective amount or effective dose of an siRNA and / or siRNA conjugate and / or the pharmaceutical composition disclosed 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 infectious diseases, acute respiratory distress syndrome (ARDS) or other types of acute lung injury, pulmonary hypertension, cancer, renal fibrosis and liver fibrosis.

[0145] The present disclosure provides the use of the aforementioned siRNA and / or pharmaceutical composition and / or siRNA conjugate in the preparation of a medicament for treating and / or preventing a disease associated with MMP7 gene expression. 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 infectious diseases, acute respiratory distress syndrome (ARDS) or other types of acute lung injury, pulmonary hypertension, cancer, renal fibrosis and liver fibrosis.

[0146] The present disclosure provides use of the aforementioned siRNA and / or pharmaceutical composition and / or siRNA conjugate in the preparation of a medicament for inhibiting the expression of MMP7.

[0147] The present disclosure provides a method for inhibiting MMP7 expression, comprising administering to a subject the aforementioned siRNA and / or pharmaceutical composition and / or siRNA conjugate of the present disclosure. 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 siRNA conjugate of the present disclosure.

[0148] The present disclosure provides the aforementioned siRNA and / or pharmaceutical composition and / or siRNA conjugates for treating and / or preventing a disease. 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 infectious diseases, acute respiratory distress syndrome (ARDS) or other types of acute lung injury, pulmonary hypertension, cancer, renal fibrosis and liver fibrosis.

[0149] The present disclosure provides a method for treating and / or preventing a disease, including administering to a subject the aforementioned siRNA and / or pharmaceutical composition and / or siRNA conjugate. In some embodiments, the method includes administering to a subject an effective amount or an effective dose of the siRNA and / or pharmaceutical composition and / or conjugate of the present 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 infectious diseases, acute respiratory distress syndrome (ARDS) or other types of acute lung injury, pulmonary hypertension, cancer, renal fibrosis and liver fibrosis.

[0150] The present disclosure provides the use of the aforementioned siRNA and / or pharmaceutical composition and / or siRNA conjugate in the preparation of a medicament for treating and / or preventing a disease. 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 infectious diseases, acute respiratory distress syndrome (ARDS) or other types of acute lung injury, pulmonary hypertension, cancer, renal fibrosis and liver fibrosis.

[0151] The present disclosure provides a method for in vivo delivery of siRNA that inhibits MMP7 expression and / or replication, comprising administering the aforementioned siRNA and / or pharmaceutical composition and / or siRNA conjugate to a subject.

[0152] In some embodiments, the siRNA is delivered to the liver.

[0153] In some embodiments, the siRNA is delivered extrahepatically.

[0154] In some embodiments, the siRNA is delivered to the lungs.

[0155] The present disclosure also provides a method for preparing siRNA or siRNA conjugate, which comprises: synthesizing the siRNA or siRNA conjugate described in the present disclosure.

[0156] The present disclosure also provides an siRNA or siRNA conjugate, characterized in that one or more bases U in any siRNA or siRNA conjugate of the present disclosure are replaced by base T, for example, 1, 2, 3, 3, 5, 6, 7, 8, 9, or 10.

[0157] The pharmaceutically acceptable salts of the compounds described in the present disclosure are selected from inorganic salts or organic salts. The compounds described in the present disclosure can react with acidic or basic substances to form corresponding salts.

[0158] On the other hand, without specifying a configuration, the compounds of the present disclosure may exist in specific geometric or stereoisomeric forms. The present disclosure contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the present disclosure. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All of these isomers and their mixtures are included within the scope of the present disclosure.

[0159] In addition, without specifying the configuration, the compounds and intermediates of the present disclosure may also exist in different tautomeric forms, and all such forms are included within the scope of the present disclosure. The term "tautomer" or "tautomeric form" refers to structural isomers of different energies that are interconvertible via a low energy barrier.

[0160] All compounds in this disclosure can be drawn as either Form A or Form B. All tautomeric forms are within the scope of this disclosure. The naming of the compounds does not exclude any tautomers.

[0161] The compounds of the present disclosure may be asymmetric, for example, having one or more stereoisomers. Unless otherwise indicated, all stereoisomers are included, such as enantiomers and diastereomers. The compounds of the present disclosure containing asymmetric carbon atoms can be isolated in optically pure forms or racemic forms. Optically pure forms can be resolved from racemic mixtures or synthesized by using chiral starting materials or chiral reagents.

[0162] Optically active (R)- and (S)-isomers, as well as D and L isomers, can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the present disclosure is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide the pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), a diastereomeric salt is formed with an appropriate optically active acid or base, and then the diastereoisomers are separated by conventional methods known in the art, and then the pure enantiomer is recovered. In addition, the separation of enantiomers and diastereomers is typically accomplished using chromatography, which employs a chiral stationary phase and is optionally combined with a chemical derivatization method (e.g., carbamate formation from an amine).

[0163] The present disclosure also includes isotopically labeled compounds of the present disclosure that are identical to those described herein, but where one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into the compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as 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 et al.

[0164] Unless otherwise stated, when a position is specifically designated as deuterium (D), the position is understood to have deuterium (i.e., at least 10% deuterium incorporation) at least 1000 times greater than the natural abundance of deuterium (which is 0.015%). In the example, the compound has a natural abundance greater than deuterium of at least 1000 times the abundance of deuterium, at least 2000 times the abundance of deuterium, at least 3000 times the abundance of deuterium, at least 4000 times the abundance of deuterium, at least 5000 times the abundance of deuterium, at least 6000 times the abundance of deuterium, or more. The present disclosure also includes compounds in various deuterated forms. Each available hydrogen atom connected to a carbon atom can be independently replaced by a deuterium atom. Those skilled in the art can synthesize compounds in deuterated forms with reference to relevant literature. In preparing deuterated forms of the compounds, commercially available deuterated starting materials may be used, or they may be synthesized using conventional techniques with deuterated reagents including, but not limited to, deuterated borane, trideuterated borane in tetrahydrofuran, deuterated lithium aluminum hydride, deuterated iodoethane, deuterated iodomethane, and the like.

[0165] In the chemical structures of the compounds disclosed herein, the bonds Indicates that the configuration is not specified, that is, if chiral isomers exist in the chemical structure, the bond Can be or include both Although all the above structural formulas are drawn as certain isomers for the sake of simplicity, the present disclosure may include all isomers, such as tautomers, rotational isomers, geometric isomers, diastereomers, racemates and enantiomers. In the chemical structures of the compounds described in the present disclosure, the bonds No configuration is specified, i.e. the bond The configuration can be E-type or Z-type, or include both E and Z configurations.

[0166] The present disclosure incorporates the full texts of WO2022028462A1, WO2023274395A1, and WO2023208023A1.

[0167] Explanation of terms

[0168] In order to make the present disclosure more easily understood, some technical and scientific terms are specifically defined below. Unless otherwise clearly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present disclosure belongs.

[0169] Unless otherwise specified, the terms "matrix metalloproteinase 7" and "MMP7" are used interchangeably in this disclosure. MMP7 includes, but is not limited to, human MMP7, cynomolgus macaque MMP7, mouse MMP7, and rat MMP7. Their amino acid sequences, complete coding sequences, and mRNA sequences are readily available using publicly available databases, such as GenBank, UniProt, OMIM, and the Macaca mulatta genome project website.

[0170] The term "MMP7" also refers to naturally occurring DNA sequence variations of the MMP7 gene, such as single nucleotide polymorphisms (SNPs) in the MMP7 gene. Exemplary SNPs can be found in the dbSNP database.

[0171] The term "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during MMP7 transcription, including mRNAs that are processed as the primary transcription product. The targeted portion of the target sequence should be sufficiently long to serve as a substrate for iRNA-directed cleavage. In one embodiment, the target sequence is within the protein-coding region of MMP7. In the context of this disclosure, "target sequence" and "target mRNA" have the same meaning and are interchangeable.

[0172] The targeting groups described herein may include cell receptor ligands, such as integrin targeting ligands. Integrins are a family of transmembrane receptors that promote cell-extracellular matrix (ECM) adhesion. Among them, integrin alpha-v-beta-6 (αvβ6) is an epithelial-specific integrin. αvβ6 is known to be a receptor for ECM proteins and TGF-β latency-associated peptide (LAP) and is expressed in various cells and tissues. In addition, integrin αvβ6 is highly upregulated in damaged lung epithelial cells.

[0173] The MMP7 siRNA described herein can be linked to an integrin targeting ligand having affinity for the integrin αvβ6. As referred to herein, an "αvβ6 integrin targeting ligand" is a compound having affinity for the integrin αvβ6 that can be used as a ligand to facilitate the targeting and delivery of the siRNA to which it is linked to the desired cells and / or tissues (i.e., cells expressing integrin αvβ6).

[0174] As used herein, in the context of RNA-mediated gene silencing, the positive strand (also known as SS, SS strand or sense strand) refers to the strand comprising a sequence identical or substantially identical to the target mRNA sequence; the antisense strand (also known as AS or AS strand) refers to the strand having a sequence complementary to the target mRNA sequence.

[0175] In the context of describing the sense strand of an siRNA described herein, the term "differs from any of the nucleotide sequences of SEQ ID NO: 1 to SEQ ID NO: 10 by no more than 3 nucleotide sequences and comprises at least 15 consecutive nucleotides" is intended to mean that the sense strand of the siRNA described herein comprises at least 15 consecutive nucleotides of the sense strand of any of SEQ ID NO: 1 to SEQ ID NO: 10, or differs from at least 15 consecutive nucleotides of the sense strand of any of SEQ ID NO: 1 to SEQ ID NO: 10 by no more than 3 nucleotide sequences (optionally, differs from at most 2 nucleotide sequences; optionally, differs from at least 1 nucleotide sequence). Optionally, the sense strand of the siRNA described herein comprises at least 16 consecutive nucleotides of the sense strand of any of SEQ ID NO: 1 to SEQ ID NO: 10, or differs from at least 16 consecutive nucleotides of the sense strand of any of SEQ ID NO: 1 to SEQ ID NO: 10 by no more than 3 nucleotide sequences (optionally, differs from at most 2 nucleotide sequences, optionally, differs from at least 1 nucleotide sequence).

[0176] In the context of describing the antisense strand of the siRNA described herein, the term "a sequence that differs from the antisense strand of any one of SEQ ID NOs: 31 to SEQ ID NOs: 40 by no more than 3 nucleotides and comprises at least 15 consecutive nucleotides" is intended to mean that the antisense strand of the siRNA described herein comprises at least 15 consecutive nucleotides of the antisense strand of any one of SEQ ID NOs: 31 to SEQ ID NOs: 40, or a sequence that differs from at least 15 consecutive nucleotides of the antisense strand of any one of SEQ ID NOs: 31 to SEQ ID NOs: 40 by no more than 3 nucleotides (optionally, a sequence that differs by no more than 2 nucleotides, optionally, a sequence that differs by 1 nucleotide).

[0177] In this disclosure, the "5' region," also known as the "5' end," or "5' terminus" of the sense or antisense strand may be used interchangeably. For example, nucleotides 2 through 8 in the 5' region of the antisense strand may be replaced with nucleotides 2 through 8 at the 5' terminus of the antisense strand. Similarly, the "3' region," "3' terminus," and "3' terminus" of the sense or antisense strand may be used interchangeably.

[0178] As used in the present disclosure, the term "2'-fluoro-modified nucleotide / nucleoside" refers to a nucleotide / nucleoside in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by fluorine, and "non-2'-fluoro-modified nucleotide / nucleoside" refers to a nucleotide / nucleoside or nucleotide analog in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by a non-fluorine group.

[0179] As used herein, the term "2'-methoxy modified nucleotide / nucleoside" refers to a nucleotide / nucleoside in which the 2'-hydroxyl group of the ribose group is replaced by a methoxy group.

[0180] Unless otherwise specified, in the context of the present disclosure, "G", "C", "A", "T" and "U" represent nucleotides, respectively, which include the bases guanine, cytosine, adenine, thymidine and uracil, respectively. It is well known to those skilled in the art that the replacement of bases T and U will not significantly affect the properties of the siRNA sequence. In the sequence of the present disclosure, U can be arbitrarily replaced by T, and the sequence obtained after the replacement is also within the scope of protection of the present disclosure. In the sequence of the present disclosure, for the same nucleic acid chain, with the direction from the 5' end to the 3' end as the left to right direction, the lowercase letter m indicates that the nucleoside adjacent to the left of the letter m is a 2'-methoxy-modified nucleoside; the lowercase letter f indicates that the nucleoside adjacent to the left of the letter f is a 2'-fluoro-modified nucleoside; the lowercase letter s indicates that the two nucleosides adjacent to the letter s are connected by a phosphorothioate diester group. Unless otherwise specified, the "RNAi agent", "nucleotide", "compound", "chemical modification", "oligonucleotide", "double-stranded RNAi inhibitor molecule", "siRNA", "siRNA conjugate", "dsRNA", "nucleic acid" and "RNAi" of the present disclosure can independently exist in the form of salt, mixed salt or non-salt (such as free acid or free base). When present in the form of a salt or a mixed salt, it may be a pharmaceutically acceptable salt. The term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts. When it exists in the form of a salt, some groups may be ionized to form anions / cations. For example, phosphodiester groups and thiophosphonic acid diester groups can exist in the form of anions. The salt forms corresponding to the following structures are also within the scope of protection of the present disclosure. Unless otherwise specified, the 3' position of the first nucleotide at the 3' end of each chain is a hydroxyl group; the 5' position of the first nucleotide at the 5' end of each chain is a hydroxyl group.

[0181] The above-mentioned modification and linking groups have the structures shown in the following table, where Base represents a base:

[0182] Table 1A

[0183] As used herein, the terms "complementary" and "reverse complement" are used interchangeably and have the meanings known to those skilled in the art, i.e., in a double-stranded nucleic acid molecule, the bases of one strand are paired with bases on the other strand in a complementary manner. In DNA, the purine base adenine is always paired with the pyrimidine base thymine (or uracil in RNA); the purine base guanine is always paired with the pyrimidine base cytosine. Each base pair consists of a purine and a pyrimidine. When adenine on one strand is always paired with thymine (or uracil) on the other strand, and guanine is always paired with cytosine, the two strands are considered to be complementary to each other, and the sequence of the strand can be inferred from the sequence of its complementary strand. Accordingly, "mismatch" is used in the art to mean that in a double-stranded nucleic acid, the bases at corresponding positions are not paired in a complementary manner.

[0184] As used herein, the term "inhibit" can be used interchangeably with "reduce", "silence", "downregulate", "suppress" and other similar terms, and includes any level of inhibition. Inhibition can be assessed by the reduction of the absolute or relative level of one or more of these variables compared to the control level. The control level can be any type of control level used in the art, such as baseline level before administration or the level determined from an untreated or control-treated subject, cell, or sample (such as a buffer control or an inert agent control). For example, the remaining expression of mRNA can be used to characterize the degree of inhibition of siRNA on target gene expression, such as the remaining expression of mRNA being no more than 99%, no more than 95%, no more than 90%, no more than 85%, no more than 80%, no more than 75%, no more than 70%, no more than 65%, no more than 60%, no more than 55%, no more than 50%, no more than 45%, no more than 40%, no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, or no more than 10%. The inhibition rate of target gene expression can be measured using The Luciferase Assay System measures firefly chemiluminescence (Fir) and Renilla chemiluminescence (Ren) values, respectively, and calculates the relative value (Ratio = Ren / Fir). In this disclosure, the ratio of remaining mRNA expression (or remaining activity %) = Ratio (siRNA-treated group) / Ratio (no siRNA control group), and the inhibition rate (%) = 100% - remaining mRNA expression (%).

[0185] Unless otherwise specified, the "compound", "ligand", "nucleic acid ligand conjugate", "siRNA conjugate", "nucleic acid", "conjugate", "chemical modification", "targeting ligand", "dsRNA", and "RNAi" of the present disclosure can independently exist in the form of a salt, a mixed salt, or a non-salt (e.g., a free acid or a free base). When present in the form of a salt or a mixed salt, it can be a pharmaceutically acceptable salt.

[0186] The term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0187] "Pharmaceutically acceptable acid addition salts" refer to salts formed with inorganic or organic acids that retain the biological effectiveness of the free base without other side effects. Inorganic acid salts include, but are not limited to, hydrochlorides, hydrobromides, sulfates, nitrates, and phosphates; organic acid salts include, but are not limited to, formates, acetates, 2,2-dichloroacetates, trifluoroacetates, propionates, caproates, octanoates, decanoates, undecylenates, glycolates, gluconates, lactates, sebacates, adipates, glutarates, malonates, oxalates, maleates, succinates, fumarates, tartrates, citrates, palmitates, stearates, oleates, cinnamates, laurates, malates, glutamate, pyroglutamate, aspartate, benzoates, methanesulfonates, benzenesulfonates, p-toluenesulfonates, alginate, ascorbate, salicylates, 4-aminosalicylates, and naphthalene disulfonates. These salts can be prepared by methods known in the art.

[0188] "Pharmaceutically acceptable base addition salts" refer to salts formed with inorganic or organic bases that retain the biological effectiveness 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, aluminum salts, and the like. 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, following salts: primary amines, secondary amines and tertiary amines, substituted amines, including natural 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, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine. These salts can be prepared by methods known in the art.

[0189] "Effective amount" or "effective dose" refers to the amount of a drug, compound, or pharmaceutical composition necessary to achieve any one or more beneficial or desired therapeutic results. For prophylactic uses, beneficial or desired results include eliminating or reducing the risk, lessening the severity, or delaying the onset of a condition, including the biochemical, histological, and / or behavioral symptoms of the condition, its complications, and intermediate pathological phenotypes that present during the progression of the condition. For therapeutic applications, beneficial or desired results include clinical results, such as reducing the incidence of various conditions associated with the disclosed target genes, target mRNAs, or target proteins, or ameliorating one or more symptoms of the condition, reducing the dose of another agent required to treat the condition, enhancing the efficacy of another agent, and / or delaying the progression of a condition associated with the disclosed target genes, target mRNAs, or target proteins in a patient.

[0190] As used herein, "patient," "subject," or "individual" are used interchangeably and include humans or non-human animals, such as mammals, eg, humans or monkeys.

[0191] The siRNA provided herein can be obtained by conventional preparation methods in the art (e.g., solid phase synthesis and liquid phase synthesis methods). Among them, solid phase synthesis already has commercial customization services. Modified nucleotide groups can be introduced into the siRNA described in the present disclosure by using nucleoside monomers with corresponding modifications. Methods for preparing nucleoside monomers with corresponding modifications and methods for introducing modified nucleotide groups into siRNA are also well known to those skilled in the art.

[0192] The term "chemically modified" or "modification" includes all changes in a nucleotide by chemical means, such as the addition or removal of a chemical moiety, or the substitution of one chemical moiety for another.

[0193] The term "base" encompasses any known DNA and RNA base, base analogues such as purines or pyrimidines, and also includes the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogues.

[0194] The terms "blunt end" or "blunt end" are used interchangeably to refer to a given end of an siRNA having no unpaired nucleotides or nucleotide analogs, i.e., no nucleotide overhangs. In most cases, an siRNA having both ends blunt-ended will be double-stranded throughout its entire length.

[0195] The terms "about" and "approximately" refer to values ​​that are within an acceptable error range for a specific value as determined by one of ordinary skill in the art, which depends in part on how it is measured or determined (i.e., the limits of the measurement system). For example, "about" can mean a standard deviation within or exceeding 1. Alternatively, "about" or "substantially comprising" can mean a range of up to 20%, such as 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, each instance in which a number or numerical range is preceded by the term "about" also includes embodiments of the given number. Unless otherwise stated, when a specific value appears in the application and claims, the meaning of "about" or "substantially comprising" should be assumed to be within an acceptable error range for that specific value.

[0196] Unless otherwise indicated, "optionally," "optional," "optional," or "optional" means that the subsequently described event or circumstance may but need not occur, and the description includes situations in which the event or circumstance occurs or does not occur. For example, "optionally, R1 and R2 are directly linked to form a ring" means that R1 and R2 may but need not be directly linked to form a ring, and the description includes situations in which R1 and R2 are directly linked to form a ring and situations in which R1 and R2 do not form a ring.

[0197] In the chemical structural formula disclosed herein, It may be linked to one or more of any groups in accordance with the scope of the invention described herein.

[0198] In the context of this disclosure, the group in It can be replaced by any group that can achieve linkage with adjacent nucleotides.

[0199] The term "linked" when referring to a connection between two molecules means that the two molecules are connected by a covalent bond or the two molecules are associated via a non-covalent bond (eg, hydrogen bond or ionic bond), including direct connection and indirect connection.

[0200] The term "directly linked" refers to a first compound or group being linked to a second compound or group without any intervening atoms or groups of atoms.

[0201] The term "indirectly linked" means that a first compound or group is linked to a second compound or group through an intermediate group, compound or molecule (eg, a linking group).

[0202] The term "substituted" means that any one or more hydrogen atoms on a designated atom (typically a carbon, oxygen, and nitrogen atom) are replaced by any group as defined herein, provided that the normal valence of the designated atom is not exceeded and the substitution produces a stable compound. Non-limiting examples of substituents include C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, cyano, hydroxyl, oxo, carboxyl, cycloalkyl, cycloalkenyl, heterocyclyl, heteroaryl, aryl, ketone, alkoxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, or halogen (e.g., F, Cl, Br, I). When a substituent is ketone or oxo (i.e., =O), then two (2) hydrogen atoms on the atom are replaced.

[0203] "Substituted by one or more..." means that the compound may be substituted by a single or multiple substituents. When substituted by multiple substituents, the substituents may be multiple identical substituents or a combination of one or more different substituents. DETAILED DESCRIPTION

[0204] The present disclosure is further described below with reference to the following examples, but these examples are not intended to limit the scope of this disclosure. Experimental methods in the examples herein where specific conditions are not specified generally follow conventional conditions, such as those in the Cold Spring Harbor Laboratory Manual of Antibody Techniques and the Molecular Cloning Manual, or the conditions recommended by the raw material or product manufacturer. Reagents where the specific source is not specified are commercially available.

[0205] Example 1. Design of human MMP7 siRNA

[0206] Taking human MMP7 gene (NM_002423.5) as the target gene, Table 1B shows the unmodified or modified 19 / 21 nt siRNAs and siRNA conjugates of the present disclosure designed to meet the general rules of active siRNA.

[0207] Table 1B. siRNAs and siRNA conjugates targeting human MMP7

[0208] In Table 1B above, the sequence represents the direction from 5' to 3' from left to right, the lowercase letter m indicates that the nucleoside adjacent to the left of the letter m is a 2'-methoxy-modified nucleoside; the lowercase letter f indicates that the nucleoside adjacent to the left of the letter f is a 2'-fluoro-modified nucleoside; the lowercase letter s indicates that the connection between the two nucleosides adjacent to the left and right of the letter s or the connected nucleosides and the delivery group I-14 is a thiophosphate diester connection; unless otherwise specified, two adjacent nucleosides are connected through a phosphodiester group; unless otherwise specified, the 3' position of the first nucleotide at the 3' end of each chain is a hydroxyl group; the 5' position of the first nucleotide at the 5' end of each chain is a hydroxyl group.

[0209] The structures of the 2'-methoxy modified nucleoside, 2'-fluoro modified nucleoside, phosphorothioate diester group, phosphodiester group, and I-14 are shown in the following table. When the siRNA or siRNA conjugate of the present disclosure exists in the form of a salt, for example, in the form of a sodium salt, the salt forms corresponding to the structures in Table 2 below are also within the scope of protection of the present disclosure:

[0210] Table 2

[0211] Here, Base represents a base.

[0212] Example 2. Synthesis of siRNA of the present disclosure

[0213] siRNA synthesis is similar to conventional phosphoramidite solid-phase synthesis. The synthesis process is briefly described as follows: Nucleoside phosphoramidite monomers were ligated one by one using a universal CPG vector on a Dr. Oligo 48 synthesizer (Biolytic). Nucleoside phosphoramidite monomers, such as 2'-F RNA and 2'-O-methyl RNA, were purchased from Shanghai Zhaowei or Suzhou Jima. 5-Ethylthio-1H-tetrazole (ETT) was used as an activator (0.6 M in acetonitrile), a 0.22 M PADS solution in a 1:1 volume ratio of acetonitrile to collidine (Suzhou Kelema) was used as a sulfurization reagent, and an iodine pyridine / water solution (Kelema) was used as an oxidant.

[0214] After solid-phase synthesis, the oligonucleotides were cleaved from the solid support and soaked in a 3:1 solution of 28% ammonia and ethanol at 50°C for 16 hours. The mixture was then centrifuged, and the supernatant was transferred to another centrifuge tube. After evaporation, the supernatant was purified using C18 reverse-phase chromatography with a mobile phase of 0.1 M TEAA and acetonitrile. DMTr was removed using a 3% trifluoroacetic acid solution. The target oligonucleotides were collected, lyophilized, identified as the target product by LC-MS, and quantified by nanodrop UV light (260 nm).

[0215] The resulting single-stranded oligonucleotides were annealed according to complementary pairing in an equimolar ratio. The resulting double-stranded siRNA was dissolved in 1×PBS and adjusted to the desired concentration for use. The synthesis of oligonucleotides containing I-14 is described in Examples 3 and 4 below.

[0216] Example 3. Synthesis of ligand compound I-12

[0217] The synthetic route of compound I-12 is as follows:

[0218] 3-1 Synthesis of Compound I-2

[0219] To a solution of N-tert-butyloxycarbonylglycine (28.00 g, 159.8 mmol, commercially available) in N-dimethylformamide (200 mL) were added 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), and the mixture was purged with nitrogen three times. Under a nitrogen atmosphere, the reaction was stirred at room temperature for 3 hours. LCMS monitoring indicated the reaction was complete. The reaction solution was quenched with water and 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 on a normal phase silica gel column to obtain compound I-2 (60.0 g, 66% yield). LCMS:MS(ESI)m / z=513.6[M+H-56] + .

[0220] 3-2 Synthesis of Compound I-3

[0221] To a solution of compound I-2 (2.30 g, 4.05 mmol) in methanol (30.0 mL) was added 10% palladium on carbon (0.23 g) under a hydrogen atmosphere. The reaction was stirred at room temperature for 8 hours. LCMS monitoring indicated the reaction was complete. The reaction solution was filtered and concentrated to afford 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).

[0222] 3-3 Synthesis of Compound I-4

[0223] To a solution of compound I-3 (1.00 g, 2.09 mmol) in N,N-dimethylformamide (10.0 mL) were added azide-pentaethylene glycol-p-toluenesulfonyl ester (1.74 g, 4.18 mmol) and potassium carbonate (0.58 g, 4.18 mmol). The reaction was heated to 80 degrees and stirred overnight. LCMS monitoring showed that the reaction was complete. The reaction solution was quenched with saturated sodium bicarbonate solution and extracted with ethyl acetate (3 times, 40 mL each). The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. The concentrated crude product was purified on a normal phase silica gel column to obtain compound I-4 (1.30 g, 85% yield). LCMS: MS (ESI) m / z = 724.5 [M+H] + .

[0224] 3-4 Synthesis of Compound I-5

[0225] To a solution of compound I-4 (1.30 g, 1.79 mmol) in dichloromethane (20.0 mL) was added a solution of 1,4-dioxane hydrochloride (20.0 mL). The reaction was stirred at room temperature overnight. LCMS monitoring indicated the reaction was complete. The reaction solution was concentrated to give compound I-5 (1.10 g, 98% yield). LCMS: MS (ESI) m / z = 624.4 [M+H] + .

[0226] 3-5 Synthesis of Compound I-7

[0227] To a solution of compound I-6 (2.90 g, 20.11 mmol, commercially available) in tert-butyl alcohol (50.0 mL) was added di-tert-butyl dicarbonate (6.58 g, 30.17 mmol). The reaction was stirred at room temperature overnight. LCMS analysis indicated the reaction was complete. The crude product obtained by concentration of the reaction solution was purified on a normal phase silica gel column to obtain compound I-7 (3.90 g, 79% yield). LCMS: MS (ESI) m / z = 245.4 [M+H] + .

[0228] 3-6 Synthesis of Compound I-8

[0229] To a solution of compound I-7 (0.98 g, 4.00 mmol) in N,N-dimethylformamide (10.0 mL) at zero degrees Celsius, sodium hydride (0.19 g, 4.80 mmol) was added. After stirring at room temperature for 30 minutes, ethyl 4-bromobutyrate (0.93 mL, 4.81 mmol) was added. The reaction was stirred at room temperature overnight. LCMS monitoring showed that the reaction was complete. The reaction solution was quenched with water and extracted with ethyl acetate (3 times, 50 mL each). The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. The concentrated crude product was purified on a normal phase silica gel column to obtain compound I-8 (1.30 g, 91% yield). LCMS: MS (ESI) m / z = 359.5 [M+H] + .

[0230] 3-7 Synthesis of Compound I-9

[0231] A 1N lithium hydroxide solution (20.0 mL) was added to a solution of compound I-8 (1.30 g, 3.63 mmol) in tetrahydrofuran (20.0 mL). The reaction was stirred at room temperature for 2 hours. LCMS monitoring showed that the reaction was complete. The reaction solution was adjusted to pH 2 with 1N HCl and extracted with ethyl acetate (3 times, 20 mL each). The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. The filtrate was concentrated to give compound I-9 (1.18 g, 98% yield). LCMS: MS (ESI) m / z = 331.1 [M+H] + .

[0232] 3-8 Synthesis of Compound I-10

[0233] To a solution of compound I-9 (0.20 g, 0.61 mmol) in N-dimethylformamide (5.0 mL) were added 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. LCMS monitoring indicated the reaction was complete. The reaction solution was quenched with water and extracted with ethyl acetate (3 times, 40 mL each). The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. The concentrated crude product was purified on a normal phase silica gel column to afford compound I-10 (0.30 g, 53% yield). LCMS: MS (ESI) m / z = 936.7 [M+H] + .

[0234] 3-9 Synthesis of Compound I-11

[0235] A 1N lithium hydroxide solution (2.0 mL) was added to a solution of compound I-10 (0.07 g, 0.08 mmol) in tetrahydrofuran (2.0 mL). The reaction was stirred at room temperature for 2 hours. LCMS monitoring showed that the reaction was complete. The reaction solution was adjusted to pH 2 with 1N HCl and extracted with ethyl acetate (3 times, 20 mL each). The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. The filtrate was concentrated to give compound I-11 (0.27 g, 91% yield). LCMS: MS (ESI) m / z = 922.1 [M+H] + .

[0236] 3-10 Synthesis of Compound I-12

[0237] Trifluoroacetic acid (4.0 mL) was added to a solution of compound I-11 (0.27 g, 0.29 mmol) in dichloromethane (4.0 mL). The reaction was stirred at room temperature for 3 hours. LCMS analysis indicated the reaction was complete. The reaction solution was concentrated to obtain a crude product which was purified by preparative HPLC to yield compound I-12 (0.09 g, 37% yield). LCMS: 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).

[0238] The binding ability of compound I-12 to αvβ6 protein was determined by fluorescence polarization (FP) method conventional in the art, and the IC value of compound I-12 binding to human recombinant αvβ6 protein was determined. 50 The value is 79.86 nM, indicating that compound I-12 is a ligand that can bind to the αvβ6 protein. The specific detection method is:

[0239] Add 3.5 μL of test compound I-12 to a 384-well black plate. The test compound was set at 12 concentration points, with the highest concentration being 10 μM and a 3-fold gradient dilution. The DMSO control well was used to determine the maximum signal, and the 500 nM compound CWHM-12 (MCE, HY-18644) control well was used as the minimum signal. Then add 3.5 μL of 4X human recombinant αvβ6 protein working solution. After incubation at room temperature for 15 minutes, add 7 μL of 2X fluorescent RGD peptide solution to each well. Incubate at room temperature for 1 hour, and then read the FP signal using Envision. Use GraphPad Prism to fit the inhibition curve and calculate the IC 50 value,

[0240] Calculate IC using the following formula 50 Value (Z prime>0.5):

[0241] Curve fitting formula: Y = Bottom + (Top - Bottom) / (1 + 10^((LogIC50 - X) * Hill Slope))

[0242] X: Log inhibitor concentration; Y: inhibition rate (%).

[0243] Example 4. Synthesis of siRNA conjugates of the present disclosure

[0244] 4-1 Synthesis of the positive chain connected with I-13'

[0245] The sense chain containing I-13' in Table 3 was synthesized in the same manner as in Example 2, except that the alkyne-containing phosphoramidite monomer I-13 prepared according to the method described in patent application WO2019161213A1 was used in the solid phase synthesis at the position corresponding to I-13' to obtain the sense chain containing I-13'. The structures of I-13 and I-13' are shown below:

[0246] Table 3. Sense chain sequences linked to I-13' or I-14

[0247] 4-2 Synthesis of the Sense Strand Containing I-14: Prepare 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 in deionized water. Separately, prepare a 2 M TEAA solution (10,000 ng / µL) of I-13-28 (2.5 µmol) prepared in step 4-1 and a 25 mmol / L DMSO solution of I-12 (35 µmol). The DMSO solution containing I-12 was added to the 2M TEAA solution containing I-13-28 and shaken to mix. Then, 75uL of 0.5M Cu(II) (Cu(II)SO4·5H2O), 75uL of 0.5M THPTA, and 18uL of 2M ascorbate were added to the centrifuge tube in sequence. After shaking in a constant temperature reactor for 1 hour, the reaction was detected by LCMS. After centrifugation, the supernatant was purified by hydrophobic chromatography (A: ammonium sulfate buffer B: pure water) to obtain the positive chain containing I-14 as shown in SEQ ID NO: 28.

[0248] The same method as above was used to prepare the positive chains shown as SEQ ID NO: 29 and SEQ ID NO: 30, respectively, except that I-13-28 in the above step was replaced by I-13-29 and I-13-30, respectively.

[0249] 4-3 Synthesis of siRNA Conjugates Containing I-14

[0250] In the same manner as in Example 2, the sense strand containing I-14 and the corresponding antisense strand obtained in step 4-2 were annealed in an equimolar ratio according to complementary pairing to obtain an siRNA conjugate containing I-14.

[0251] Example 5. siRNA psiCHECK Target Activity at Three Concentrations

[0252] The siRNA of the present disclosure was screened for on-target activity at the in vitro molecular level using three concentration gradients (10 nM, 1 nM, 0.1 nM) in HEK293A cells.

[0253] HEK293A cells were cultured in DMEM high-glucose medium supplemented with 10% fetal bovine serum at 37°C in 5% CO2. 24 h before transfection, HEK293A cells were seeded in 96-well plates at a density of 8 × 103 cells per well in 100 μL of culture medium per well.

[0254] According to the instructions, cells were co-transfected with siRNA and the corresponding MMP7 full-length plasmid using Lipofectamine 2000 (ThermoFisher, 11668019), using 0.3 μL of Lipofectamine 2000 per well. The plasmid transfection amount was 40 ng per well. For the target sequence plasmid, siRNA was set at three concentration points: 10 nM, 1 nM, and 0.1 nM, with duplicate wells for each concentration. 24 hours after transfection, the target level was detected using the Dual-Luciferase Reporter Assay System (Promega, E2940). The activity results are recorded in Table 4. As shown in Table 4, the siRNA disclosed herein has good MMP7 mRNA inhibitory activity in the psi-CHECK system.

[0255] Table 4. siRNA activity results at three concentration points in the psi-Check system

[0256] Example 6. siRNA psiCHECK Target Activity at 9 Concentrations

[0257] The siRNAs disclosed herein were screened for on-target activity at the in vitro molecular level using nine concentration gradients in HEK293A cells.

[0258] HEK293A cells were cultured in DMEM high-glucose medium supplemented with 10% fetal bovine serum at 37°C in 5% CO2. 24 h before transfection, HEK293A cells were seeded in 96-well plates at a density of 1 × 104 cells per well in 100 μL of culture medium per well.

[0259] According to the instructions, cells were co-transfected with siRNA and the corresponding plasmid using Lipofectamine 2000 (ThermoFisher, 11668019), using 0.3 μL Lipofectamine 2000 per well. The plasmid transfection amount was 20 ng per well. For the target sequence plasmid, siRNA was set at 9 concentration points, with the highest concentration point having a final concentration of 20 nM, and a 3-fold gradient dilution: 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 h after transfection, the target level was detected using the Dual-Luciferase Reporter Assay System (Promega, E2940). The results are shown in Table 5. As shown in Table 5, the siRNA disclosed herein has good MMP7 mRNA on-target activity under the 9-point concentration conditions in the psi-CHECK system.

[0260] Table 5. siRNA activity results at 9 points in the psiCHECK system

[0261] Example 7. Target activity of siRNA at seven endogenous concentrations

[0262] Seven concentration gradients were used to screen the in vitro molecular activity of siRNA in HCC38 cells.

[0263] HCC38 cells were cultured in 1640 medium supplemented with 10% fetal bovine serum at 37°C in 5% CO2. 24 h before transfection, HCC38 cells were seeded in 96-well plates at a density of 1.5 × 104 cells per well in 100 μL of culture medium per well.

[0264] Cells were transfected with siRNA using Lipofectamine RNAi Max (Invitrogen, 13778-150) according to the manufacturer's instructions, with 0.3 μL of Lipofectamine RNAi Max used per well. Seven siRNA concentrations were set: 20.0000 nM, 10.0000 nM, 2.0000 nM, 0.4000 nM, 0.0800 nM, 0.0160 nM, and 0.0032 nM. 24 hours after transfection, RNA was extracted and Q-PCR was performed to detect siRNA activity. The results are shown in Table 6. The siRNAs disclosed herein exhibited significant on-target activity against MMP7 mRNA in HCC38 cells.

[0265] Table 6. siRNA activity results at 7 points in HCC38 cells

[0266] Example 8. Target activity of siRNA at seven endogenous concentrations

[0267] Seven concentration gradients were used to screen the in vitro molecular activity of siRNA in HCC38 cells.

[0268] HCC38 cells were cultured in 1640 medium supplemented with 10% fetal bovine serum at 37°C in 5% CO2. 24 h before transfection, HCC38 cells were seeded in 96-well plates at a density of 1.5 × 104 cells per well in 100 μL of culture medium per well.

[0269] Cells were transfected with siRNA using Lipofectamine RNAi Max (Invitrogen, 13778-150) according to the manufacturer's instructions, with 0.3 μL of Lipofectamine RNAi Max used per well. Seven siRNA concentrations were set: 20.0000 nM, 4.0000 nM, 0.8000 nM, 0.1600 nM, 0.0320 nM, 0.0064 nM, and 0.0013 nM. 24 hours after transfection, RNA was extracted and Q-PCR was performed to detect siRNA activity. The results are shown in Table 7. The siRNAs disclosed herein exhibited significant on-target activity against MMP7 mRNA in HCC38 cells.

[0270] Table 7. siRNA activity results at 7 points in HCC38 cells

[0271] Example 9. siRNA psiCHECK Target Activity at 9 Concentrations

[0272] siRNA was screened for on-target activity at the in vitro molecular level using nine concentration gradients in HEK293A cells.

[0273] HEK293A cells were cultured in DMEM high-glucose medium supplemented with 10% fetal bovine serum at 37°C in 5% CO2. 24 h before transfection, HEK293A cells were seeded in 96-well plates at a density of 1 × 104 cells per well in 100 μL of culture medium per well.

[0274] According to the instructions, cells were co-transfected with siRNA and the corresponding plasmid using Lipofectamine 2000 (ThermoFisher, 11668019), using 0.3 μL Lipofectamine 2000 per well. The amount of plasmid transfection was 20 ng per well. For the target sequence plasmid, siRNA was set at 9 concentration points, with the highest concentration point final concentration being 20 nM, 3-fold gradient dilution, 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 h after transfection, the target level was detected using the Dual-Luciferase Reporter Assay System (Promega, E2940). The results are shown in Table 8, where NA indicates not detected. As shown in Table 8, the siRNAs of the present disclosure exhibited good on-target activity against MMP7 mRNA in the psiCHECK system.

[0275] Table 8. siRNA psiCHECK on-target activity at nine concentrations

[0276] Example 10. Target activity of siRNA conjugates at two endogenous concentrations

[0277] The siRNA conjugates were screened for in vitro molecular activity in HCC38 cells using two concentration gradients.

[0278] HCC38 cells were cultured in 1640 medium supplemented with 10% fetal bovine serum at 37°C in 5% CO2. 24 h before transfection, HCC38 cells were seeded in 96-well plates at a density of 1.5 × 104 cells per well in 100 μL of culture medium per well.

[0279] Cells were transfected with siRNA conjugates using Lipofectamine RNAi Max (Invitrogen, 13778-150) according to the manufacturer's instructions, with 0.3 μL of Lipofectamine RNAi Max used per well. Two concentrations of siRNA conjugates were set: 10 nM and 2 nM. 24 hours after transfection, RNA was extracted and Q-PCR was performed to analyze the activity of the siRNA conjugates. The results, shown in Table 9, demonstrate that the siRNA conjugates of the present disclosure exhibited significant on-target activity against MMP7 mRNA in HCC38 cells.

[0280] Table 9. Results of 2-point activity of siRNA conjugates in HCC38 cells

Claims

1. An siRNA comprising a sense strand and an antisense strand forming a double-stranded region; The sense strand comprises at least 15 consecutive nucleotides and differs from any nucleotide sequence in SEQ ID NO: 1 to SEQ ID NO: 10 by no more than 3 nucleotides; and The antisense strand comprises at least 15 consecutive nucleotides and differs from any nucleotide sequence of SEQ ID NO: 31 to SEQ ID NO: 40 by no more than 3 nucleotides.

2. The siRNA according to claim 1, wherein: The sense strand comprises at least 17 consecutive nucleotides that differ from any one of the nucleotide sequences of SEQ ID NO: 1 to SEQ ID NO: 10 by no more than 3 nucleotides; The antisense strand comprises at least 17 consecutive nucleotides that differ from any one of SEQ ID NO: 31 to SEQ ID NO: 40 by no more than 3 nucleotides; Preferably, the sense strand comprises at least 19 consecutive nucleotides that differ from any one of SEQ ID NO: 1 to SEQ ID NO: 10 by no more than 3 nucleotides; preferably, the nucleotide sequence differs by no more than 1 nucleotide, and / or, The antisense strand comprises at least 21 consecutive nucleotides that differ from any one of SEQ ID NO: 31 to SEQ ID NO: 40 by no more than 3 nucleotides; preferably, the nucleotide sequence differs by no more than 1 nucleotide.

3. The siRNA according to claim 1 or 2, comprising or selected from the sense strand and antisense strand of any one of the following groups: Group 1), the sense strand as shown in SEQ ID NO: 1 and the antisense strand as shown in SEQ ID NO: 31; Group 2), the sense strand as shown in SEQ ID NO: 2 and the antisense strand as shown in SEQ ID NO: 32; Group 3), the sense strand as shown in SEQ ID NO: 3 and the antisense strand as shown in SEQ ID NO: 33; Group 4), the sense strand as shown in SEQ ID NO: 4 and the antisense strand as shown in SEQ ID NO: 34; Group 5), the sense strand as shown in SEQ ID NO: 5 and the antisense strand as shown in SEQ ID NO: 35; Group 6), the sense strand as shown in SEQ ID NO: 6 and the antisense strand as shown in SEQ ID NO: 36; Group 7), the sense strand as shown in SEQ ID NO: 7 and the antisense strand as shown in SEQ ID NO: 37; Group 8), the sense strand as shown in SEQ ID NO: 8 and the antisense strand as shown in SEQ ID NO: 38; Group 9), the sense strand as shown in SEQ ID NO: 9 and the antisense strand as shown in SEQ ID NO: 39; and Group 10), such as the sense strand shown in SEQ ID NO: 10 and the antisense strand shown in SEQ ID NO:

40.

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

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

6. The siRNA according to any one of claims 1 to 5, wherein at least one phosphodiester group in the sense strand and / or the antisense strand is a phosphodiester group having a modified group, preferably a thiophosphate diester group.

7. The siRNA according to claim 6, wherein the phosphodiester group having a modification group is present at one or more positions selected from the following positions: Between the first nucleotide and the second nucleotide at the 5' end of the sense strand; Between the second nucleotide and the third nucleotide at the 5' end of the sense strand; Between the first nucleotide and the second nucleotide at the 5' end of the antisense strand; Between the second nucleotide and the third nucleotide at the 5' end of the antisense strand; Between the first nucleotide and the second nucleotide at the 3' end of the antisense strand; Between the second nucleotide and the third nucleotide at the 3' end of the antisense strand; Preferably, the sense strand and / or antisense strand comprises a plurality of phosphodiester groups having modification groups.

8. The siRNA according to any one of claims 1 to 7, wherein The sense strand comprises the nucleotide sequence shown in any one of SEQ ID NO: 11 to SEQ ID NO: 27; and / or, The antisense strand comprises the nucleotide sequence shown in any one of SEQ ID NO: 41 to SEQ ID NO:

57.

9. A siRNA conjugate comprising: The siRNA according to any one of claims 1 to 8, and a targeting ligand attached to the siRNA; The targeting ligand has affinity for a cell receptor expressed on an epithelial cell; Preferably, the targeting ligand comprises an integrin targeting ligand; More preferably, the targeting ligand comprises an αvβ6 integrin targeting ligand; Further preferably, the targeting ligand has a structure as shown in I-14:

10. The siRNA conjugate according to claim 9, wherein The targeting ligand is linked to the sense strand of the siRNA; Preferably, the targeting ligand is linked to the 5' end of the sense strand of the siRNA.

11. The siRNA conjugate according to claim 9 or 10, wherein The sense strand comprises a nucleotide sequence as shown in any one of SEQ ID NO: 28 to SEQ ID NO: 30; and / or The antisense strand comprises a nucleotide sequence as shown in any one of SEQ ID NO: 58 to SEQ ID NO:

60.

12. A pharmaceutical composition comprising: The siRNA according to any one of claims 1 to 8 or the siRNA conjugate according to any one of claims 9 to 11, and a pharmaceutically acceptable carrier.

13. A cell comprising: The siRNA according to any one of claims 1 to 8, or The siRNA conjugate according to any one of claims 9 to 11.

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

15. A method for reducing the expression of matrix metalloproteinase 7 (MMP7), comprising: The siRNA according to any one of claims 1 to 8, the siRNA conjugate according to any one of claims 9 to 11, or the pharmaceutical composition according to claim 12 is administered to a subject.

16. A method for treating and / or preventing a disease, comprising: Administering to a subject the siRNA according to any one of claims 1 to 8, the siRNA conjugate according to any one of claims 9 to 11, or the pharmaceutical composition according to claim 12; Preferably, the disease is selected from asthma, fibrosis, chronic inflammation, interstitial lung disease, infectious disease, acute lung injury, pulmonary hypertension, cancer; Preferably, the infectious disease is SARS-COV-2; Preferably, the acute lung injury is acute respiratory distress syndrome; Preferably, the fibrosis is idiopathic pulmonary fibrosis, renal fibrosis or liver fibrosis.

17. A method for in vivo delivery of siRNA that inhibits MMP7 expression and / or replication, the method comprising: Administering the siRNA according to any one of claims 1 to 8, the siRNA conjugate according to any one of claims 9 to 11, or the pharmaceutical composition according to claim 12 to a subject; Preferably, the siRNA is delivered outside the liver; More preferably, the siRNA is delivered to the lungs.

18. A method for preparing siRNA or siRNA conjugate, comprising: Synthesize the siRNA according to any one of claims 1-8 or the siRNA conjugate according to any one of claims 9-11.

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