RNAi agents targeting RAGE and medical uses thereof

Abstract

Relates to an RNAi agent targeting RAGE and a medical application thereof. Specifically, the invention relates to an RAGE-targeted RNAi agent, a pharmaceutical composition and a medical application of the RAGE-targeted RNAi agent. The invention also relates to a pharmaceutical composition, a cell or a kit comprising the RNAi agent, and a method of using the RNAi agent for treating and / or preventing a subject suffering from a related disorder.

Description

RNAi agents targeting RAGE and their medical uses

[0001] The present disclosure claims the priority of Chinese patent application 202311070972.3 with an application date of August 24, 2023 and the priority of Chinese patent application 202410074707.0 with an application date of January 18, 2024, and the present disclosure cites the full text of the above-mentioned Chinese patent applications. Technical Field

[0002] The present disclosure belongs to the field of biomedicine, and specifically relates to an RNAi agent targeting the receptor for advanced glycosylation end-products (RAGE or AGER or "advanced glycosylation end-product specific receptor"), a pharmaceutical composition, and medical uses thereof. Background Art

[0003] The receptor for advanced glycation end products (RAGE or AGER) is a pro-inflammatory receptor with a short intracellular domain, a transmembrane domain, and an extracellular domain composed of three immunoglobulin-like domains. RAGE is widely distributed in many tissues and cell types, expressed during embryonic development, downregulated in adulthood, and then upregulated again in old age. Under normal circumstances, RAGE is expressed at low levels in most healthy cells. However, in the lungs, it is highly expressed throughout the life cycle. RAGE is associated with chronic pathological inflammation, which leads to many diseases, including lung disease, cardiovascular disease, cancer, diabetes, chronic kidney disease, neurodegenerative diseases, rheumatoid arthritis, non-alcoholic steatohepatitis, damage caused by certain viral infections, etc.

[0004] In the field of lung disease, RAGE knockout mice are completely physiologically and histologically immune to allergic asthma induced by dust mite allergens or ovalbumin. Similarly, RAGE knockout mice are protected from acute lung injury and inflammation induced by hyperoxia or lipopolysaccharide. In addition, RAGE expression levels are significantly upregulated in allergic airway inflammation tissues. Genome-wide association studies (GWAS) have linked the RAGE gain-of-function mutant allele (G82S) to increased inflammation, decreased lung function, and asthma risk, indicating that knockdown of RAGE has a therapeutic effect on diseases such as lung inflammation or asthma.

[0005] Summary of the Invention

[0006] The present disclosure provides an RNAi agent (RNA interference agent) targeting RAGE.

[0007] In some embodiments, the present disclosure provides a RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region;

[0008] The sense strand comprises a nucleotide sequence that differs from any one of SEQ ID NO: 1 to SEQ ID NO: 8 and SEQ ID NO: 53 to SEQ ID NO: 106, 275, and 277 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] The antisense strand comprises a nucleotide sequence that differs from any one of SEQ ID NO: 27 to SEQ ID NO: 34 and SEQ ID NO: 107 to SEQ ID NO: 160, 276, 278 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.

[0010] 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.

[0011] 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.

[0012] In some embodiments, the RNAi agents of the disclosure comprise one or two blunt ends.

[0013] In some specific embodiments, each strand of the RNAi agent independently comprises 1 to 2 unpaired nucleotides.

[0014] In some embodiments, the RNAi agents of the present disclosure comprise an overhang located at the 3' end of the antisense strand of the RNAi agent.

[0015] 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).

[0016] In some embodiments, the sense strand and the antisense strand are the same or different lengths, 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 RNAi agent 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 length ratio of the sense strand to the antisense strand of the RNAi agent is 19 / 21, 21 / 23, or 23 / 25. In some specific embodiments, the length ratio of the sense strand to the antisense strand of the RNAi agent is 19 / 21.

[0017] In some embodiments, the sense strand comprises at least 15 consecutive nucleotides and differs from any one of SEQ ID NO: 1 to SEQ ID NO: 8 and SEQ ID NO: 53 to SEQ ID NO: 106, 275, and 277 by no more than 2 nucleotides. In some embodiments, the nucleotide sequence differs by no more than 1 nucleotide. In some embodiments, the difference is 1 nucleotide.

[0018] In some embodiments, the antisense strand comprises at least 15 contiguous nucleotides and differs by no more than 2 nucleotides from the nucleotide sequences of SEQ ID NOs: 27 to 34 and SEQ ID NOs: 107 to 160, 276, and 278. In some embodiments, the nucleotide sequences differ by no more than 1 nucleotide. In some embodiments, the nucleotide sequences differ 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: 8 and SEQ ID NO: 53 to SEQ ID NO: 106, 275, and 277. In some embodiments, at least 16 consecutive nucleotides. In some embodiments, at least 17 consecutive nucleotides. In some embodiments, at least 19 consecutive nucleotides. In some embodiments, at least 18 consecutive nucleotides.

[0020] In some embodiments, the antisense strand comprises at least 15 consecutive nucleotides of any one of SEQ ID NO: 27 to SEQ ID NO: 34 and SEQ ID NO: 107 to SEQ ID NO: 160, 276, and 278. 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: 8 and SEQ ID NO: 53 to SEQ ID NO: 106, 275, 277.

[0022] In some embodiments, the antisense strand comprises or is selected from any one of the following nucleotide sequences: SEQ ID NO: 27 to SEQ ID NO: 34 and SEQ ID NO: 107 to SEQ ID NO: 160, 276, 278.

[0023] In some embodiments, the RNAi agent comprises a sense strand and an antisense strand, or is 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: 27;

[0025] Group 2), the sense strand set forth in SEQ ID NO: 7 and the antisense strand set forth in SEQ ID NO: 33;

[0026] Group 3), the sense strand set forth in SEQ ID NO: 2 and the antisense strand set forth in SEQ ID NO: 28;

[0027] Group 4), the sense strand set forth in SEQ ID NO: 8 and the antisense strand set forth in SEQ ID NO: 34;

[0028] Group 5), the sense strand set forth in SEQ ID NO: 3 and the antisense strand set forth in SEQ ID NO: 29;

[0029] Group 6), the sense strand set forth in SEQ ID NO: 4 and the antisense strand set forth in SEQ ID NO: 30;

[0030] Group 7), the sense strand set forth in SEQ ID NO: 5 and the antisense strand set forth in SEQ ID NO: 31;

[0031] Group 8), the sense strand shown in SEQ ID NO: 6 and the antisense strand shown in SEQ ID NO: 32.

[0032] In some embodiments, at least one nucleotide in the sense strand and / or the antisense strand is a modified nucleotide.

[0033] In some embodiments, all of the nucleotides in the sense strand and / or the antisense strand are modified nucleotides.

[0034] In some embodiments, three consecutive nucleotides in the sense strand of the RNAi agent are 2'-fluoro-modified nucleotides.

[0035] In some embodiments, three consecutive nucleotides at positions 7-9 of the sense strand, in the 5' to 3' direction, are 2'-fluoro-modified nucleotides.

[0036] In some embodiments, from the 5' end to the 3' end, three consecutive nucleotides at positions 7-9 of the sense strand are 2'-fluoro-modified nucleotides, and the remaining positions of the sense strand are 2'-methoxy-modified nucleotides.

[0037] 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.

[0038] In some embodiments, the 5'-terminal phosphorus-containing group of the antisense strand comprises a phosphorus-containing group, and the phosphorus-containing group is a 5'-vinyl phosphodiester group (5'-VP). When the 5'-terminal phosphorus-containing group is a 5'-vinyl phosphodiester group (5'-VP), the 5'-VP can be a 5'-E-VP isomer (i.e., a trans-vinyl phosphodiester group), a 5'-Z-VP isomer (i.e., a cis-vinyl phosphodiester group), or a mixture thereof.

[0039] In some embodiments, the 5' end of the antisense strand comprises a phosphorus-containing group that is a 5'-E-VP isomer (ie, a trans-vinyl phosphodiester group).

[0040] In some embodiments, the 5' end of the antisense strand comprises a phosphorus-containing group, wherein the phosphorus-containing group is or a mixture thereof.

[0041] In some embodiments, the 5' end of the antisense strand comprises a phosphorus-containing group, wherein the phosphorus-containing group is

[0042] 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 RNAi agent 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. In some embodiments, the phosphodiester group with a modifying group is a 5'-vinyl phosphodiester group.

[0043] In some embodiments, the phosphorothioate diester group is present in at least one of the following positions:

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

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

[0046] between the first and second nucleotides at the 3' end of the sense strand;

[0047] between the second and third nucleotides at the 3' end of the sense strand;

[0048] between the first and second nucleotides at the 5' end of the antisense strand;

[0049] between the second and third nucleotides at the 5' end of the antisense strand;

[0050] between the first nucleotide and the second nucleotide at the 3' end of the antisense strand; and

[0051] between the second and third nucleotides at the 3' end of the antisense strand.

[0052] In some embodiments, the sense strand and / or antisense strand includes multiple phosphorothioate diester groups present in:

[0053] between the first nucleotide and the second nucleotide at the 5' end of the sense strand; and

[0054] between the second and third nucleotides at the 5' end of the sense strand; and

[0055] between the first nucleotide and the second nucleotide at the 5' end of the antisense strand; and

[0056] between the second and third nucleotides from the 5' end of the antisense strand; and

[0057] between the first nucleotide and the second nucleotide at the 3' end of the antisense strand; and

[0058] between the second and third nucleotides at the 3' end of the antisense strand.

[0059] In some embodiments, the sense strand and / or antisense strand includes multiple phosphorothioate diester groups present in:

[0060] between the first nucleotide and the second nucleotide at the 5' end of the sense strand; and

[0061] between the second and third nucleotides at the 5' end of the sense strand; and

[0062] between the first nucleotide and the second nucleotide at the 3' end of the sense strand; and

[0063] between the second and third nucleotides at the 3' end of the sense strand; and

[0064] between the first nucleotide and the second nucleotide at the 5' end of the antisense strand; and

[0065] between the second and third nucleotides from the 5' end of the antisense strand; and

[0066] between the first nucleotide and the second nucleotide at the 3' end of the antisense strand; and

[0067] between the second and third nucleotides at the 3' end of the antisense strand.

[0068] In some embodiments, the 5'-vinyl phosphodiester group is present at the 5' end of the antisense strand.

[0069] In some embodiments, the sense strand is selected from or comprises the nucleotide sequence shown in any one of SEQ ID NO: 9 to SEQ ID NO: 20 and SEQ ID NO: 161 to SEQ ID NO: 214.

[0070] In some embodiments, the antisense strand is selected from or comprises the nucleotide sequence shown in any one of SEQ ID NO: 35 to SEQ ID NO: 46 and SEQ ID NO: 215 to SEQ ID NO: 268.

[0071] In some embodiments, the RNAi agent comprises a sense strand and an antisense strand, or is selected from any one of the following groups:

[0072] Group 1), the sense strand set forth in SEQ ID NO: 9 and the antisense strand set forth in SEQ ID NO: 35;

[0073] Group 2), the sense strand set forth in SEQ ID NO: 10 and the antisense strand set forth in SEQ ID NO: 36;

[0074] Group 3), the sense strand set forth in SEQ ID NO: 11 and the antisense strand set forth in SEQ ID NO: 37;

[0075] Group 4), the sense strand set forth in SEQ ID NO: 12 and the antisense strand set forth in SEQ ID NO: 38;

[0076] Group 5), the sense strand set forth in SEQ ID NO: 13 and the antisense strand set forth in SEQ ID NO: 39;

[0077] Group 6), the sense strand set forth in SEQ ID NO: 14 and the antisense strand set forth in SEQ ID NO: 40;

[0078] Group 7), the sense strand set forth in SEQ ID NO: 15 and the antisense strand set forth in SEQ ID NO: 41;

[0079] Group 8), the sense strand set forth in SEQ ID NO: 16 and the antisense strand set forth in SEQ ID NO: 42;

[0080] Group 9), the sense strand set forth in SEQ ID NO: 17 and the antisense strand set forth in SEQ ID NO: 43;

[0081] Group 10), the sense strand set forth in SEQ ID NO: 18 and the antisense strand set forth in SEQ ID NO: 44;

[0082] Group 11), the sense strand set forth in SEQ ID NO: 19 and the antisense strand set forth in SEQ ID NO: 45;

[0083] Group 12), the sense strand shown in SEQ ID NO: 20 and the antisense strand shown in SEQ ID NO: 46.

[0084] In some embodiments, the RNAi agent further comprises a targeting ligand.

[0085] In some embodiments, the RNAi agent and the targeting ligand are covalently or non-covalently linked.

[0086] In some embodiments, the targeting ligand is linked to the antisense strand of the RNAi agent. In some embodiments, the targeting ligand is linked to the 5' end of the antisense strand of the RNAi agent. In some embodiments, the targeting ligand is linked to the 3' end of the antisense strand of the RNAi agent.

[0087] In some embodiments, the targeting ligand is attached to the sense strand of the RNAi agent. In some embodiments, the targeting ligand is attached to the 5' end of the sense strand of the RNAi agent. In some embodiments, the targeting ligand is attached to the 3' end of the sense strand of the RNAi agent.

[0088] In some embodiments, the targeting ligand is linked to the 3' end or the 5' end of the sense strand through a phosphodiester group, a phosphorothioate diester group, or a phosphonic acid group.

[0089] In some embodiments, the targeting ligand is linked to the 3' end or the 5' end of the sense strand via a phosphodiester group. In some embodiments, the targeting ligand is linked to the 5' end of the sense strand via a phosphodiester group.

[0090] In some embodiments, the targeting ligand is indirectly linked to the 3' end or the 5' end of the sense strand through a phosphodiester group, a phosphorothioate diester group, or a phosphonic acid group.

[0091] In some embodiments, the targeting ligand is indirectly linked to the 3' end or the 5' end of the sense strand through a phosphodiester group.

[0092] In some embodiments, the targeting ligand is indirectly linked to the 5' end of the sense strand via a phosphodiester group.

[0093] In some embodiments, the targeting ligand is directly linked to the 3' end or the 5' end of the sense strand via a phosphodiester group, a phosphorothioate diester group, or a phosphonic acid group.

[0094] In some embodiments, the targeting ligand is directly linked to the 3' end or the 5' end of the sense strand via a phosphodiester group. In some embodiments, the targeting ligand is directly linked to the 5' end of the sense strand via a phosphodiester group.

[0095] In some embodiments, the targeting ligand may include 2, 3, 4 or more targeting groups. In some embodiments, the targeting ligand described in the present disclosure may include 1, 2, 3, 4 or more targeting groups connected to the 3' end or 5' end of the sense strand via a linker.

[0096] In some embodiments, the targeting ligand is targeted to the lung.

[0097] In some embodiments, the targeting ligand has affinity for a cell receptor expressed on epithelial cells. In some embodiments, the targeting ligand comprises an integrin targeting ligand. In some embodiments, the integrin targeting ligand is an αvβ6 integrin targeting ligand.

[0098] In some embodiments, the targeting ligand has the following structure:

[0099] In some embodiments, the targeting ligand is composed of one or more targeting groups, which assist in guiding the delivery of the therapeutic agent to which it is attached to the desired target location. In some cases, the targeting ligand can bind to cells or cell receptors and initiate endocytosis to promote the entry of therapeutic agents into cells. The targeting ligand can include compounds with affinity for cell receptors or cell surface molecules or antibodies. Various targeting ligands containing targeting groups can be linked to therapeutic agents and other compounds to target the agents to cells and specific cell receptors.

[0100] In some embodiments, each targeting group comprises a galactosamine derivative, which is N-acetyl-galactosamine. Other sugars that can be used as targeting groups and have affinity for asialoglycoprotein receptors can be selected from galactose, galactosamine, N-formyl-galactosamine, N-acetyl-galactosamine, N-propionyl-galactosamine, N-n-butyryl-galactosamine, and N-isobutyryl-galactosamine.

[0101] In some embodiments, the targeting ligands of the present disclosure include N-acetylgalactosamine as a targeting group.

[0102] In some embodiments, the targeting ligand comprises three terminal galactosamines or galactosamine derivatives (such as N-acetyl-galactosamine), each of which has affinity for sialoglycoprotein receptors. In some embodiments, the targeting ligand comprises three terminal N-acetyl-galactosamines (GalNAc or NAG) as targeting groups.

[0103] In some embodiments, the targeting ligand comprises four terminal galactosamines or galactosamine derivatives (such as N-acetyl-galactosamine), each of which has affinity for the asialoglycoprotein receptor. In some embodiments, the targeting ligand comprises four terminal N-acetyl-galactosamines (GalNAc or NAG) as targeting groups.

[0104] Terms commonly used in the art when referring to the three terminal N-acetyl-galactosamines include tri-antennary, tri-valent, and trimer.

[0105] Terms commonly used in the art when referring to the four terminal N-acetyl-galactosamines include tetra-antennary, tetra-valent, and tetramer.

[0106] In some embodiments, the targeting ligand provided by the present disclosure is a compound shown below or a pharmaceutically acceptable salt thereof,

[0107] 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.

[0108] In some embodiments, the sense strand of the RNAi agent described herein comprises or is selected from the nucleotide sequence shown in any one of SEQ ID NO: 21 to SEQ ID NO: 26; and / or the antisense strand comprises or is selected from the nucleotide sequence shown in any one of SEQ ID NO: 47 to SEQ ID NO: 52.

[0109] In some embodiments, the RNAi agent of the present disclosure comprises or is selected from the sense strand and antisense strand of any one of the following groups:

[0110] Group 1), the sense strand set forth in SEQ ID NO: 21 and the antisense strand set forth in SEQ ID NO: 47;

[0111] Group 2), the sense strand set forth in SEQ ID NO: 22 and the antisense strand set forth in SEQ ID NO: 48;

[0112] Group 3), the sense strand set forth in SEQ ID NO: 23 and the antisense strand set forth in SEQ ID NO: 49;

[0113] Group 4), the sense strand set forth in SEQ ID NO: 24 and the antisense strand set forth in SEQ ID NO: 50;

[0114] Group 5), the sense strand set forth in SEQ ID NO: 25 and the antisense strand set forth in SEQ ID NO: 51;

[0115] Group 6), the sense strand shown in SEQ ID NO: 26 and the antisense strand shown in SEQ ID NO: 52.

[0116] In another aspect, the present disclosure provides a pharmaceutical composition comprising the RNAi agent described herein, and one or more pharmaceutically acceptable excipients, such as non-limiting examples of excipients including vehicles, carriers, diluents, and / or delivery polymers.

[0117] Various delivery systems are known and can be used for the RNAi agents of the present disclosure, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the RNAi agent, receptor-mediated endocytosis, constructing the nucleic acid as part of a retroviral or other vector.

[0118] In another aspect, the present disclosure provides a use of the RNAi agent or pharmaceutical composition in the preparation of a medicament for treating a disease in a subject. In some embodiments, the disease is selected from a lung disease.

[0119] In another aspect, the present disclosure provides a method for treating a disease in a subject, comprising administering to the subject the RNAi agent or pharmaceutical composition of the present disclosure.

[0120] In another aspect, the present disclosure provides a method for inhibiting mRNA expression in a subject, comprising administering to the subject the RNAi agent or pharmaceutical composition of the present disclosure.

[0121] In another aspect, the present disclosure provides a method for delivering an RNAi agent that inhibits target gene expression and / or replication to the lung in vivo, the method comprising administering the RNAi agent or pharmaceutical composition of the present disclosure to a subject.

[0122] In some embodiments, the target gene is the receptor for advanced glycation end products (RAGE or AGER or "advanced glycosylation end-product specific receptor").

[0123] The RNAi agents or pharmaceutical compositions and methods described in the present disclosure can reduce the level of target mRNA in a cell, a cell population, a cell population, a tissue or a subject, comprising: administering to the subject a therapeutically effective amount of the RNAi agent or pharmaceutical composition described in the present disclosure, wherein the RNAi agent is linked to a targeting ligand, thereby inhibiting the expression of the target mRNA in the subject.

[0124] In some embodiments, the subject has been previously identified as having pathological upregulation of the target gene in the targeted cells or tissues.

[0125] The subject described in this disclosure is a subject diagnosed with (or suspected of having, or susceptible to) a disease or condition. Without being bound by a particular theory, it is expected that the subject will benefit from reducing or inhibiting the expression of the target mRNA.

[0126] 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.

[0127] In alternative embodiments, the pharmaceutical compositions provided herein can be administered by injection, for example, intravenously, intramuscularly, intradermally, subcutaneously, intraduodenally, or intraperitoneally.

[0128] In alternative embodiments, the RNAi agents described herein can be packaged in a kit.

[0129] 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.

[0130] In some embodiments, the unit dose of the pharmaceutical composition is 0.001 mg-1000 mg.

[0131] In certain embodiments, the pharmaceutical composition contains 0.01-99.99% of the RNAi agent of the present disclosure, based on the total weight of the pharmaceutical composition. In certain embodiments, the pharmaceutical composition contains 0.1-99.9% of the RNAi agent of the present disclosure. In certain embodiments, the pharmaceutical composition contains 0.5%-99.5% of the RNAi agent of the present disclosure. In certain embodiments, the pharmaceutical composition contains 1%-99% of the RNAi agent of the present disclosure. In certain embodiments, the pharmaceutical composition contains 2%-98% of the RNAi agent of the present disclosure.

[0132] In certain embodiments, the pharmaceutical composition comprises 0.01% to 99.99% of a pharmaceutically acceptable excipient, based on the total weight of the pharmaceutical composition. In certain embodiments, the pharmaceutical composition comprises 0.1% to 99.9% of a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition comprises 0.5% to 99.5% of a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition comprises 1% to 99% of a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition comprises 2% to 98% of a pharmaceutically acceptable excipient.

[0133] In some embodiments, when the RNAi agent or pharmaceutical composition of the present disclosure is contacted with a cell expressing the target gene, the RNAi agent or pharmaceutical composition of the present disclosure 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.

[0134] In some embodiments, when the RNAi agent or pharmaceutical composition of the present disclosure is contacted with a cell expressing the target gene, the percentage of remaining target gene mRNA expression caused by the RNAi agent or pharmaceutical composition of the present disclosure 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.

[0135] In some embodiments, when the RNAi agent or pharmaceutical composition of the present disclosure is contacted with a cell expressing a target gene, the RNAi agent 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, e.g., Western Blot, or flow cytometry.

[0136] In some embodiments, the RNAi agent or pharmaceutical composition of the present disclosure, 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 the off-target activity by at least 20%, at most 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.

[0137] In some embodiments, the RNAi agent or pharmaceutical composition of the present disclosure, when contacted with a cell expressing a target gene, increases 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.

[0138] The present disclosure also provides a cell comprising the RNAi agent of the present disclosure.

[0139] The present disclosure also provides a kit comprising the RNAi agent or pharmaceutical composition described in the present disclosure.

[0140] 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 RNAi agent and / or pharmaceutical composition according to the present disclosure into the cell.

[0141] 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 RNAi agent and / or pharmaceutical composition according to the present disclosure into the cell.

[0142] The present disclosure also provides a method for inhibiting the expression of a target gene or target gene mRNA, comprising administering an effective amount or effective dose of the RNAi agent and / or pharmaceutical composition according to the present disclosure to a subject in need thereof.

[0143] 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.

[0144] In some embodiments, the effective amount or effective dose of the RNAi agent 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.

[0145] In the present disclosure, RAGE should be interpreted broadly to refer to the RAGE gene itself and its various forms of expression products at various stages, such as but not limited to molecules produced during gene amplification, replication, transcription, splicing, processing, translation, and modification, such as cDNA, mRNA, precursor protein, mature protein, natural variants, modified forms, and fragments thereof.

[0146] In the present disclosure, RAGE is human (Homo sapiens) RAGE.

[0147] In some embodiments, the target gene is the receptor for advanced glycation end products (RAGE or AGER or "advanced glycosylation end-product specific receptor"). As a specific example, the target gene is the mRNA of RAGE.

[0148] The present disclosure provides the RNAi agent and / or pharmaceutical composition, which can inhibit RAGE gene expression and / or replication.

[0149] The present disclosure provides RNAi agents and / or pharmaceutical compositions for treating and / or preventing a disease in a subject associated with RAGE gene expression. In some embodiments, the disease is selected from a disease, condition, or symptom mediated at least in part by RAGE activity and / or AGER gene expression. In some embodiments, the disease is selected from a lung disease. In some embodiments, the lung disease is selected from asthma, acute respiratory distress syndrome, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, cystic fibrosis, and pneumonia.

[0150] The present disclosure provides a method for treating and / or preventing a disease associated with RAGE gene expression in a subject, comprising administering to the subject an effective amount or effective dose of an RNAi agent or pharmaceutical composition as described herein. In some embodiments, the disease is selected from a lung disease associated with the RAGE gene. In some embodiments, the disease is selected from a lung disease; in some embodiments, the lung disease is selected from asthma, acute respiratory distress syndrome, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, cystic fibrosis, and pneumonia.

[0151] The present disclosure provides use of the RNAi agent and / or pharmaceutical composition in preparing a medicament for treating and / or preventing diseases associated with RAGE gene expression.

[0152] The RNAi agents and / or pharmaceutical compositions provided herein can be used to treat lung diseases. In some embodiments, the lung diseases are selected from lung diseases associated with the RAGE gene. In some embodiments, the lung diseases are selected from asthma, acute respiratory distress syndrome, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, cystic fibrosis, and pneumonia.

[0153] The present disclosure provides use of the RNAi agent and / or pharmaceutical composition in preparing a medicament for inhibiting the expression of RAGE.

[0154] The present disclosure provides a method for inhibiting RAGE expression, comprising administering an effective amount or effective dose of the RNAi agent and / or pharmaceutical composition of the present disclosure to a subject.

[0155] The present disclosure provides a method for treating and / or preventing a disease, comprising administering to a subject an effective amount or effective dose of an RNAi agent and / or pharmaceutical composition of the present disclosure. In some embodiments, the disease is selected from a lung disease associated with the RAGE gene. In some embodiments, the disease is selected from a lung disease. In some embodiments, the lung disease is selected from asthma, acute respiratory distress syndrome, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, cystic fibrosis, and pneumonia.

[0156] The present disclosure provides the use of the RNAi agent and / or pharmaceutical composition in the preparation of a medicament for treating and / or preventing a disease. In some embodiments, the disease is selected from a lung disease associated with the RAGE gene. In some embodiments, the disease is selected from a lung disease (asthma, acute respiratory distress syndrome, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, cystic fibrosis, pneumonia).

[0157] The present disclosure provides a method for delivering an RNAi agent that inhibits RAGE expression and / or replication in vivo, the method comprising administering the RNAi agent and / or pharmaceutical composition of the present disclosure to a subject.

[0158] The present disclosure also provides a method for preparing an RNAi agent, comprising: synthesizing the RNAi agent described in the present disclosure.

[0159] 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.

[0160] On the other hand, without specifying configuration, the compounds described herein 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.

[0161] In addition, without specifying the configuration, the compounds and intermediates described in 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 can interconvert via a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) include interconversions via proton migration, such as keto-enol and imine-enamine, lactam-lactim isomerizations.

[0162] The compounds disclosed herein may be asymmetric, for example, having one or more stereoisomers. Unless otherwise indicated, all stereoisomers are included, such as enantiomers and diastereomers. Compounds disclosed herein containing asymmetric carbon atoms can be isolated in optically pure or racemic forms. Optically pure forms can be resolved from racemic mixtures or synthesized using chiral starting materials or chiral reagents.

[0163] 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).

[0164] 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 structural formulas described in this disclosure are drawn as certain isomers for the sake of simplicity, the 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 this disclosure, the bond No configuration is specified, i.e. the bond The configuration can be E-type or Z-type, or contain both E and Z configurations.

[0165] The present disclosure incorporates the full texts of WO2022028462A, WO2023274395A, and WO2023208023A.

[0166] Explanation of terms

[0167] 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.

[0168] As used herein, "RNAi agent" (also referred to as "RNAi trigger") means an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule that is capable of degrading or inhibiting (e.g., degrading or inhibiting) a target mRNA in a sequence-specific manner. The RNAi agent used herein can act through an RNA interference mechanism (i.e., by inducing RNA interference through interaction with the RNA interference pathway machinery of mammalian cells (RNA-induced silencing complex or RISC)) or through any alternative mechanism or pathway. Although it is believed that the term RNAi agent used herein acts primarily through an RNA interference mechanism, the disclosed RNAi agents are not bound or limited by any particular pathway or mechanism of action. The RNAi agents disclosed herein comprise a sense strand and an antisense strand, and include, but are not limited to, short (or small) interfering RNA (siRNA). The antisense strand of the RNAi agent described herein is at least partially complementary to the targeted mRNA. The RNAi agent may comprise one or more modified nucleotides and / or one or more phosphodiester groups having a modifying group.

[0169] Unless otherwise specified, the terms "receptor for advanced glycation end products," "RAGE," "AGER," and "advanced glycosylation end-product specific receptor" are used interchangeably herein. RAGE includes, but is not limited to, human RAGE, cynomolgus monkey RAGE, mouse RAGE, and rat RAGE, and their amino acid and complete coding sequences and mRNA sequences are readily available using publicly available databases, such as GenBank, UniProt, OMIM, and the Macaca genome project website.

[0170] The term "RAGE" also refers to naturally occurring DNA sequence variations of the RAGE gene, such as single nucleotide polymorphisms (SNPs) in the RAGE 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 transcription of RAGE, including mRNAs that are RNA processing products of the primary transcription product. In one embodiment, the target sequence is within the protein coding region of RAGE.

[0172] The term "pulmonary disease associated with RAGE gene" includes any disease or condition associated with decreased RAGE expression and / or activity. Typical "pulmonary diseases associated with RAGE gene" include lung diseases (asthma, acute respiratory distress syndrome, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, cystic fibrosis, pneumonia).

[0173] The term "αvβ6" refers to an epithelial-specific integrin that is a known receptor for ECM proteins and TGF-β latency-associated peptide (LAP) and is expressed in various cells and tissues. Integrin αvβ6 is known to be significantly upregulated in damaged lung epithelium.

[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 a strand comprising a sequence that is identical or substantially identical (differences in no more than 3 nucleotide sequences) to the target mRNA sequence; the antisense strand (also known as AS or AS strand) refers to a strand having a sequence that is complementary or partially complementary (mismatches in no more than 3 nucleotide sequences) to the target mRNA sequence.

[0175] In the context of describing the sense strand of an RNAi agent described herein, the term "differs from any one of SEQ ID NO: 1 to SEQ ID NO: 8 by no more than 3 nucleotide sequences and comprises at least 15 consecutive nucleotides" is intended to mean that the sense strand of the RNAi agent described herein comprises at least 15 consecutive nucleotides of the sense strand of any one of SEQ ID NO: 1 to SEQ ID NO: 8, or differs from at least 15 consecutive nucleotides of the sense strand of any one of SEQ ID NO: 1 to SEQ ID NO: 8 by no more than 3 nucleotide sequences (exemplarily, differs from at most 2 nucleotide sequences; exemplary, differs from 1 nucleotide sequence). Exemplarily, the sense strand of the RNAi agent described herein comprises at least 16 consecutive nucleotides of the sense strand of any one of SEQ ID NO: 1 to SEQ ID NO: 8, or differs from at least 16 consecutive nucleotides of the sense strand of any one of SEQ ID NO: 1 to SEQ ID NO: 8 by no more than 3 nucleotide sequences (exemplarily, differs from at most 2 nucleotide sequences; exemplary, differs from 1 nucleotide sequence).

[0176] In the context of describing the antisense strand of the RNAi agent described herein, the term "differences from the antisense strand of any one of SEQ ID NO: 27 to SEQ ID NO: 34 by no more than 3 nucleotide sequences and comprises at least 15 consecutive nucleotides" is intended to mean that the antisense strand of the RNAi agent described herein comprises at least 15 consecutive nucleotides of the antisense strand of any one of SEQ ID NO: 27 to SEQ ID NO: 34, or differs from at least 15 consecutive nucleotides of the antisense strand of any one of SEQ ID NO: 27 to SEQ ID NO: 34 by no more than 3 nucleotide sequences (exemplarily, differs by no more than 2 nucleotide sequences, exemplary, differs by 1 nucleotide sequence).

[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] Unless otherwise specified, in the context of this disclosure, "G," "C," "A," "T," and "U" represent nucleotides, including guanine, cytosine, adenine, thymidine, and uracil, respectively. A lowercase letter "m" indicates that the nucleotide immediately upstream of the letter "m" is a methoxy-modified nucleotide; a lowercase letter "f" indicates that the nucleotide immediately upstream of the letter "f" is a fluorinated nucleotide; and a lowercase letter "s" indicates that the two nucleotides immediately adjacent to the letter "s" are linked by a phosphorothioate diester linkage.

[0179] As used in the present disclosure, the term "2'-fluoro-modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2'-ribose group of the nucleotide is substituted with fluorine.

[0180] As used in the present disclosure, the term "2'-methoxy-modified nucleotide" refers to a nucleotide in which the 2'-hydroxyl group of the ribose group is replaced by a methoxy group.

[0181] 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.

[0182] 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 a baseline level before administration or a level determined from an untreated or control-treated subject, cell, or sample (e.g., only 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 RNAi agent expression of the target gene, 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 (RNAi-treated group) / Ratio (no-RNAi control group), and the inhibition rate (%) = 100% - remaining mRNA expression (%).

[0183] Unless otherwise specified, the "compound", "ligand", "nucleic acid ligand conjugate", "nucleic acid", "conjugate", "chemical modification", "targeting ligand", "RNAi agent", "siRNA", and "dsRNA" of the present disclosure may 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 may be a pharmaceutically acceptable salt.

[0184] The term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0185] "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.

[0186] "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, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like. In some embodiments, the inorganic salt is ammonium, sodium, potassium, calcium, and magnesium, and in some embodiments, it is sodium. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines, including naturally substituted amines, cyclic amines, and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like. Organic bases in some embodiments include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. These salts can be prepared by methods known in the art.

[0187] "Effective amount" or "effective dose" refers to the amount of RNAi agent, compound, or pharmaceutical composition necessary to achieve any one or more beneficial or desired prophylactic or therapeutic results. For prophylactic uses, beneficial or desired results include eliminating or reducing the risk, reducing 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.

[0188] As used herein, "patient," "subject," or "individual" are used interchangeably and include humans or non-human animals, such as mammals, eg, humans or monkeys.

[0189] The RNAi agents 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 RNAi agents 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 RNAi agents are also well known to those skilled in the art.

[0190] 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.

[0191] 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.

[0192] The terms "blunt end" or "blunt end" are used interchangeably and refer to the absence of unpaired nucleotides or nucleotide analogs at a given end of an RNAi agent, i.e., no nucleotide overhangs. In most cases, an RNAi agent having both ends blunt-ended will be double-stranded throughout its entire length.

[0193] 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 the value is measured (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 case 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.

[0194] 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.

[0195] 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.

[0196] 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.

[0197] 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.

[0198] 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).

[0199] 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.

[0200] "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. Specific implementation method:

[0201] 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.

[0202] Example 1. Design of human RAGE RNAi agent

[0203] Using the human RAGE gene (NM-001136.5) as the target gene, 19 / 21 nt RNAi agents were designed to meet the general criteria for active RNAi agents. The modified and unmodified sense and antisense strand sequences are shown in Tables 1, 2, and 3.

[0204] Table 1. Modified or unmodified sense and antisense strands of human RAGE RNAi agents

[0205] Table 2. Unmodified sense and antisense strands of human RAGE RNAi agents

[0206] Table 3. Modified sense and antisense strands of human RAGE RNAi agents

[0207] In Table 1, Table 2, and Table 3, the sequence represents the direction from the 5' end to the 3' end from left to right, and the lowercase letter m indicates that the nucleotide adjacent to the upstream of the letter m is a 2'-methoxy-modified nucleotide; the lowercase letter f indicates that the nucleotide adjacent to the upstream of the letter f is a 2'-fluoro-modified nucleotide; the lowercase letter s indicates that the connection between the two nucleosides adjacent to the letter s or the connected nucleosides and the targeting ligand I-14 is a thiophosphate diester connection; unless otherwise specified, two adjacent nucleosides are connected by 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. The structures of the 2'-methoxy-modified nucleosides, 2'-fluoro-modified nucleosides, thiophosphate diester groups, phosphodiester groups, I-14, and VPUm are shown in the following table. When the RNAi agent of the present disclosure exists in salt form, for example, in the form of a sodium salt, the structure of the salt form corresponding to the structure in Table 4 below is also within the scope of protection of the present disclosure:

[0208] Table 4

[0209] Example 2. Synthesis of RNAi Agents

[0210] The synthesis of RNAi agents follows the conventional phosphoramidite solid-phase synthesis method. The synthesis process is briefly described as follows: Nucleoside phosphoramidite monomers were ligated one by one on a Dr. Oligo 48 synthesizer (Biolytic) starting with a universal CPG vector. Nucleoside phosphoramidite monomers, such as 2'-F RNA and 2'-O-methyl RNA, were purchased from Shanghai Zhaowei or Suzhou Jima. VPUm nucleoside phosphoramidite monomers were purchased from Jiangsu Shenji. 5-Ethylthio-1H-tetrazole (ETT) was used as the activating agent (0.6 M in acetonitrile), a 0.22 M PADS solution in a 1:1 volume ratio of acetonitrile and collidine (Suzhou Kelema) was used as the sulfurizing agent, and an iodine pyridine / water solution (Kelema) was used as the oxidizing agent.

[0211] After solid-phase synthesis, the oligoribonucleotides 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 oligonucleotide was collected, lyophilized, identified as the target product by LC-MS, and quantified by UV (260 nm).

[0212] The obtained single-stranded oligonucleotides were annealed according to the complementary pairing in an equal molar ratio, and the resulting RNAi agent was dissolved in 1× PBS and adjusted to the required concentration for the experiment.

[0213] Example 3. Synthesis of ligand compound I-12

[0214] The synthetic route of compound I-12 is as follows:

[0215] 3-1 Synthesis of Compound I-2

[0216] 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).

[0217] LCMS:MS(ESI)m / z=513.6[M+H-56] + .

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

[0219] 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).

[0220] 1 H 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).

[0221] 3-3 Synthesis of Compound I-4

[0222] 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] + .

[0223] 3-4 Synthesis of Compound I-5

[0224] 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] + .

[0225] 3-5 Synthesis of Compound I-7

[0226] 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] + .

[0227] 3-6 Synthesis of Compound I-8

[0228] 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] + .

[0229] 3-7 Synthesis of Compound I-9

[0230] 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] + .

[0231] 3-8 Synthesis of Compound I-10

[0232] 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] + .

[0233] 3-9 Synthesis of Compound I-11

[0234] 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] + .

[0235] 3-10 Synthesis of Compound I-12

[0236] To a solution of compound I-11 (0.27 g, 0.29 mmol) in dichloromethane (4.0 mL) was added trifluoroacetic acid (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 afford compound I-12 (0.09 g, 37% yield).

[0237] LCMS: MS (ESI) m / z = 822.1 [M+H] + .

[0238] 1 H 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).

[0239] 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:

[0240] 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, use the following formula to calculate IC 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 RNAi Agents with Ligand I-14

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

[0245] The sense chain containing I-13' in Table 5 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 WO2019161213A was used in 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] 4-2 Synthesis of the positive chain containing I-14

[0247] Prepare stock solutions of 0.5 M tris(3-hydroxypropyltriazolylmethyl)amine (THPTA), 0.5 M copper(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-13-21 (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-13-21 and shaken to mix. Then, 75 μL of 0.5M Cu(II) (Cu(II)SO4·5H2O), 75 μL of 0.5M THPTA and 18 μL 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: 21.

[0248] The same method as above was used to prepare the positive chains shown in SEQ ID NO: 22 to SEQ ID NO: 26, respectively, except that the positive chains shown in I-13-22 to I-13-26 were used to replace I-13-21 in the above steps.

[0249] Table 5. Sense chain sequences linked to I-13' or I-14

[0250] 4-3 Synthesis of RNAi Agents Containing I-14

[0251] 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 RNAi agent containing I-14.

[0252] Example 5. RNAi agent psiCHECK Target Activity at 9 concentrations

[0253] RNAi agents were screened for on-target activity at the in vitro molecular level using nine concentration gradients in HEK293A cells.

[0254] HEK293A cells were cultured in DMEM high glucose medium containing 10% fetal bovine serum at 37°C and 5% CO2. 24 h before transfection, HEK293A cells were seeded in 96-well plates at a seeding density of 1 × 10 cells per well. 4 cells, 100 μL culture medium per well.

[0255] According to the instructions, cells were co-transfected with RNAi agents and corresponding plasmids 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, the RNAi agent was set at a total of 9 concentration points, with the highest concentration point having a final concentration of 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 Dual-Luciferase Reporter Assay System (Promega, E2940) was used to detect target levels. The results are shown in Table 6.

[0256] Table 6. RNA activity results at 9 points in the psiCHECK system

[0257] In Table 6, NA means not detected.

[0258] Example 6. RNAi agent psiCHECK Target Activity at 9 concentrations

[0259] RNAi agents were screened for on-target activity at the in vitro molecular level using nine concentration gradients in HEK293A cells.

[0260] HEK293A cells were cultured in DMEM high glucose medium containing 10% fetal bovine serum at 37°C and 5% CO2. 24 h before transfection, HEK293A cells were seeded in 96-well plates at a seeding density of 1 × 10 cells per well. 4 cells, 100 μL culture medium per well.

[0261] According to the instructions, cells were co-transfected with RNAi agents and corresponding plasmids 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, the RNAi agent was set at a total of 9 concentration points, with the highest concentration point having a final concentration of 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 7.

[0262] Table 7. RNA activity results at 9 points in the psiCHECK system

[0263] Example 7. Screening of endogenous cellular activity of RNAi agents

[0264] For some sequences, three concentration gradients were used to screen the molecular level of endogenous cell activity of RNAi agents in BEAS-2B cells, and for other sequences, seven concentration gradients were used to screen the molecular level of endogenous cell activity of RNAi agents in BEAS-2B cells.

[0265] BEAS-2B cells were cultured in BEAS-2B complete medium (Nanjing Kebai, CC-3170) at 37°C and 5% CO2. 24 h before transfection, BEAS-2B cells were seeded in 96-well plates at a seeding density of 1 × 10 cells per well. 4 cells, 100 μL culture medium per well.

[0266] Cells were transfected with RNAi agents using RNAi MAX transfection reagent (ThermoFisher, 13778150) according to the manufacturer's instructions. 0.3 μL of RNAi MAX transfection reagent was used per well. Three concentration points were set for some RNAi agents, with a final concentration of 20 nM at the highest point and a five-fold serial dilution sequence of 20 nM, 4 nM, and 0.8 nM. Seven concentration points were set for other RNAi agents, with a final concentration of 100 nM at the highest point and a five-fold serial dilution sequence of 100.0000 nM, 20 nM, 4 nM, 0.8 nM, 0.16 nM, 0.032 nM, and 0.0064 nM. 48 h after transfection, total RNA was extracted from cells using a high-throughput RNA extraction kit (ThermoFisher, A27828), RNA reverse transcription was performed (Takara, RR037B), and quantitative real-time PCR was performed (ThermoFisher, 4444557) to measure the mRNA level of human RAGE. The mRNA level of human RAGE was corrected according to the level of the GAPDH internal reference gene.

[0267] After the Q-PCR test is completed, the corresponding Ct value is obtained according to the threshold value automatically set by the system. The expression of a gene can be relatively quantified by comparing the Ct value: Comparative Ct refers to calculating the gene expression difference by the difference between the Ct value and the internal reference gene, also known as 2 -△△Ct , ΔΔCt = [(Ct experimental group target gene - Ct experimental group internal reference) - (Ct control group target gene - Ct control group internal reference)]. Inhibition rate (%) = (1 - target gene expression remaining amount) * 100%. The results are expressed as the percentage of human RAGE expression remaining in cells treated with dsRNA. The IC of inhibition rate 50 The results are shown in Table 8:

[0268] Table 8. IC of RNAi agents in BEAS-2B cells 50 result

[0269] In Table 8, NA means not detected.

[0270] Example 8. RNAi agent psiCHECK Target Activity at 9 concentrations

[0271] RNAi agents were screened for on-target activity at the in vitro molecular level using nine concentration gradients in HEK293A cells.

[0272] HEK293A cells were cultured in DMEM high glucose medium containing 10% fetal bovine serum at 37°C and 5% CO2. 24 h before transfection, HEK293A cells were seeded in 96-well plates at a seeding density of 1 × 10 cells per well. 4cells, 100 μL culture medium per well.

[0273] According to the instructions, cells were co-transfected with RNAi agents and corresponding plasmids 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, the RNAi agent was set at a total of 9 concentration points, with the highest concentration point having a final concentration of 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 9.

[0274] Table 9. IC50 activity results of RNAi agents in the psiCHECK system

[0275] Example 9. Screening of endogenous cellular activity of RNAi agents

[0276] The sequences were screened in BEAS-2B cells using 7 concentration gradients to simulate the endogenous cell activity of RNAi agents at the molecular level.

[0277] BEAS-2B cells were cultured in BEAS-2B complete medium (Nanjing Kebai, CC-3170) at 37°C in 5% CO2. RNAi agents were transfected into cells using RNAi MAX transfection reagent (ThermoFisher, 13778150) according to the manufacturer's instructions, with 0.3 μL of RNAi MAX transfection reagent used per well. Seven concentration points were set for the RNAi agent, with the highest concentration point being 100 nM. Five-fold serial dilutions were performed: 100.0000 nM, 20 nM, 4 nM, 0.8 nM, 0.16 nM, 0.032 nM, and 0.0064 nM. 48 h after transfection, total RNA was extracted from cells using a high-throughput RNA extraction kit (ThermoFisher, A27828), RNA reverse transcription was performed (Takara, RR037B), and quantitative real-time PCR was performed (ThermoFisher, 4444557) to measure the mRNA level of human RAGE. The mRNA level of human RAGE was corrected according to the level of the GAPDH internal reference gene.

[0278] After the Q-PCR test is completed, the corresponding Ct value is obtained according to the threshold value automatically set by the system. The expression of a gene can be relatively quantified by comparing the Ct value: Comparative Ct refers to calculating the gene expression difference by the difference between the Ct value and the internal reference gene, also known as 2 -△△Ct , ΔΔCt = [(Ct experimental group target gene - Ct experimental group internal reference) - (Ct control group target gene - Ct control group internal reference)]. Inhibition rate (%) = (1 - target gene expression remaining amount) * 100%. The results are expressed as the percentage of human RAGE expression remaining in cells treated with dsRNA. The IC of inhibition rate 50 The results are shown in Table 10:

[0279] Table 10. IC of RNAi agents in BEAS-2B cells 50 result

Claims

1. A RNAi agent 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 of the nucleotide sequences of SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 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:27, SEQ ID NO:33, SEQ ID NO:28, SEQ ID NO:34, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32 by no more than 3 nucleotides.

2. The RNAi agent according to claim 1, The sense strand comprises at least 17 consecutive nucleotides that differ from any one of SEQ ID NO: 1 to SEQ ID NO: 8 by no more than 3 nucleotide sequences; and The antisense strand comprises at least 17 consecutive nucleotides that differ from any one of SEQ ID NO: 27 to SEQ ID NO: 34 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: 8 by no more than 3 nucleotides; preferably, the nucleotide sequence differs by no more than 1 nucleotide, and, The antisense strand comprises at least 21 consecutive nucleotides that differ from any one of SEQ ID NO: 27 to SEQ ID NO: 34 by no more than 3 nucleotides; preferably, the nucleotide sequence differs by no more than 1 nucleotide.

3. The RNAi agent according to any one of claims 1-2, comprising a sense strand containing at least 18 consecutive nucleotides as shown in any of the following groups; and comprising an antisense strand containing at least 20 consecutive nucleotides as shown in any 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: 27; Group 2), the sense strand as shown in SEQ ID NO: 7 and the antisense strand as shown in SEQ ID NO: 33; Group 3), the sense strand as shown in SEQ ID NO: 2 and the antisense strand as shown in SEQ ID NO: 28; Group 4), the sense strand as shown in SEQ ID NO: 8 and the antisense strand as shown in SEQ ID NO: 34; Group 5), the sense strand as shown in SEQ ID NO: 3 and the antisense strand as shown in SEQ ID NO: 29; Group 6), the sense strand as shown in SEQ ID NO: 4 and the antisense strand as shown in SEQ ID NO: 30; Group 7), the sense strand as shown in SEQ ID NO: 5 and the antisense strand as shown in SEQ ID NO: 31; Group 8), the sense strand as shown in SEQ ID NO: 6 and the antisense strand as shown in SEQ ID NO: 32; Preferably, it comprises a sense strand and an antisense strand as shown in any one group.

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

5. The RNAi agent according to any one of claims 1 to 4, wherein: The three consecutive nucleotides in the sense strand are 2'-fluoro-modified nucleotides; preferably, in the direction from the 5' end to the 3' end, the 7th, 8th and 9th 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; 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; The nucleotides at the remaining positions in the sense strand and the antisense strand are 2'-methoxy-modified nucleotides.

6. The RNAi agent 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 RNAi agent according to claim 6, wherein the phosphorothioate diester group is present in at least one of 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 3' end of the sense strand; Between the second nucleotide and the third nucleotide at the 3' 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 phosphorothioate diester groups, wherein the phosphorothioate diester groups are present in: between the first nucleotide and the second nucleotide at the 5' end of the sense strand; and between the second nucleotide and the third nucleotide at the 5' end of the sense strand; and between the first nucleotide and the second nucleotide at the 5' end of the antisense strand; and between the second nucleotide and the third nucleotide at the 5' end of the antisense strand; and between the first nucleotide and the second nucleotide at the 3' end of the antisense strand; and Between the second nucleotide and the third nucleotide at the 3' end of the antisense strand; or, The phosphorothioate diester group is present in: between the first nucleotide and the second nucleotide at the 5' end of the sense strand; and between the second nucleotide and the third nucleotide at the 5' end of the sense strand; and between the first nucleotide and the second nucleotide at the 3' end of the sense strand; and between the second nucleotide and the third nucleotide at the 3' end of the sense strand; and between the first nucleotide and the second nucleotide at the 5' end of the antisense strand; and between the second nucleotide and the third nucleotide at the 5' end of the antisense strand; and between the first nucleotide and the second nucleotide at the 3' end of the antisense strand; and The 3' end of the antisense strand is between the second nucleotide and the third nucleotide.

8. The RNAi agent according to any one of claims 1 to 7, wherein The 5' end of the antisense strand contains a phosphorus-containing group, which is a 5'-vinyl phosphodiester group.

9. The RNAi agent according to any one of claims 1 to 8, wherein The sense strand comprises the nucleotide sequence shown in any one of SEQ ID NO: 9 to SEQ ID NO: 20; and, The antisense strand comprises the nucleotide sequence shown in any one of SEQ ID NO: 35 to SEQ ID NO:

46.

10. The RNAi agent according to any one of claims 1 to 9, wherein The end of the sense strand is connected to the targeting ligand; Preferably, the 5' end of the sense strand is linked to a targeting ligand.

11. The RNAi agent according to claim 10, wherein The targeting ligand has affinity for a cell receptor expressed on an epithelial cell; Preferably, the targeting ligand is an integrin targeting ligand; More preferably, the integrin targeting ligand is an αvβ6 integrin targeting ligand.

12. The RNAi agent according to claim 10 or 11, wherein The sense strand comprises a nucleotide sequence as shown in any one of SEQ ID NO: 21 to SEQ ID NO: 26; and The antisense strand comprises a nucleotide sequence as shown in any one of SEQ ID NO: 47 to SEQ ID NO:

52.

13. A pharmaceutical composition comprising the RNAi agent according to any one of claims 1 to 12, and a pharmaceutically acceptable excipient.

14. A cell comprising the RNAi agent of any one of claims 1-12.

15. A kit comprising the RNAi agent according to any one of claims 1 to 12 or the pharmaceutical composition according to claim 13.

16. A method for reducing the expression of the receptor for advanced glycation end products (RAGE), comprising administering to a subject an effective amount or an effective dose of the RNAi agent according to any one of claims 1 to 12 or the pharmaceutical composition according to claim 13.

17. A method for treating and / or preventing a disease associated with RAGE gene expression in a subject, comprising administering to the subject an effective amount or an effective dose of the RNAi agent according to any one of claims 1 to 12, or the pharmaceutical composition according to claim 13.

18. A method for treating and / or preventing a lung disease, comprising administering to a subject an effective amount or an effective dose of the RNAi agent according to any one of claims 1 to 12, or the pharmaceutical composition according to claim 13; Preferably, the pulmonary disease is selected from asthma, acute respiratory distress syndrome, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, cystic fibrosis, pneumonia.

19. A method for delivering a RNAi agent that inhibits RAGE expression and / or replication to the lung in vivo, comprising administering to a subject the RNAi agent according to any one of claims 1 to 12, or the pharmaceutical composition according to claim 13.

20. A method for preparing a RNAi agent, comprising: Synthesize the RNAi agent of any one of claims 1-12.