SiRNA for inhibiting MUC5AC and modifier and application thereof

Abstract

The invention relates to siRNA for inhibiting MUC5AC and a modifier and application thereof, and belongs to the technical field of biology. The invention provides siRNA (small interfering Ribonucleic Acid) for inhibiting MUC5AC. Experiments prove that the siRNA can be used for inhibiting the expression of MUC5AC genes. The invention also provides modified siRNA for inhibiting MUC5AC, the modification comprises methoxy modification, fluoro modification and phosphorothioate group connection, and experiments prove that the siRNA can inhibit the expression of the MUC5AC gene under the concentration of 1nM, and the siRNA also has good inhibitory activity under the concentration of 0.1 nM.

Description

[0001] This application is based on and claims priority to a Chinese patent application with the application number CN202311741497.8 and the filing date of December 18, 2023. The entire content of the above-mentioned patent application is hereby incorporated into this application by reference. Technical Field

[0002] The present invention relates to siRNAs and their modifications for inhibiting MUC5AC and their applications, belonging to the field of biotechnology. Background Art

[0003] There is a thin layer of mucus on the airway surface, which is composed of water (98%), inorganic salts (0.9%), protein (0.8%), and high molecular polymers (0.3%), and is mainly secreted by airway goblet cells, submucosal glands, and Clara cells. Airway mucus is divided into a mucus layer and a sol layer. The sol layer contains more water, and its thickness is roughly the same as the height of cilia, with a low viscosity, which is of great significance for maintaining the effective beating of cilia; the mucus layer (about 2 μm thick) contains less water and contains mucin, lysozyme, and various polypeptides, which can adhere to foreign particles and pathogenic microorganisms invading the airway, slide directionally on the surface of epithelial cells with the beating of cilia, and then be discharged by coughing.

[0004] Currently, 21 types of mucins have been identified in the human genome, which can be classified into membrane-associated mucins and gel-forming mucins according to their characteristics. Among them, MUC5AC and MUC5B are the main respiratory gel-forming mucins. MUC5AC is mainly produced by airway epithelial goblet cells in the proximal airway, while MUC5B is produced by surface secretory cells in the entire airway and submucosal glands.

[0005] MUC5AC is the main increased type of airway mucin under pathological conditions. The transcriptional level and protein content of MUC5AC in airway epithelium can represent the intensity of airway mucus secretion. In respiratory epithelial cells, the MUC5AC gene is induced by various stimuli including smoking, viral infection, oxidative stress, and air pollutants, and can produce a large amount of secretagogues and inflammatory mediators such as interleukin-13, tumor necrosis factor α (TNF-α), and IL-9, which directly or indirectly act on goblet cells and submucosal gland cells to cause excessive production of airway mucus.

[0006] Small interfering RNA (siRNA), usually double-stranded RNA with a length of 20 to 25 nucleotides, mainly regulates gene expression in a specific manner through the RNA interference (RNAi) mechanism to achieve the purpose of treating diseases. Therefore, developing a siRNA that inhibits the production of MUC5AC will be an effective way to treat asthma and its various complications. Summary of the Invention

[0007] To solve the above problems, the present invention provides a siRNA for inhibiting MUC5AC, the siRNA contains a sense strand and an antisense strand, and the sense strand can at least partially reverse complement with the antisense strand to form a double-stranded region.

[0008] Wherein, the sense strand contains the nucleotide sequence shown in SEQ ID NO.9, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.10, or,

[0009] Wherein, the sense strand contains the nucleotide sequence shown in SEQ ID NO.21, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.22, or,

[0010] Wherein, the sense strand contains the nucleotide sequence shown in SEQ ID NO.35, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.36, or,

[0011] Wherein, the sense strand contains the nucleotide sequence shown in SEQ ID NO.43, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.44, or,

[0012] Wherein, the sense strand contains the nucleotide sequence shown in SEQ ID NO.47, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.48, or,

[0013] Wherein, the sense strand contains the nucleotide sequence shown in SEQ ID NO.63, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.64, or,

[0014] Wherein, the sense strand contains the nucleotide sequence shown in SEQ ID NO.83, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.84, or,

[0015] Wherein, the sense strand contains the nucleotide sequence shown in SEQ ID NO.85, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.86, or,

[0016] Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO. 91, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO. 92.

[0017] In one embodiment of the present invention, taking the sense strand and antisense strand shown in SEQ ID NO. 9 and SEQ ID NO. 10 as examples, the sense strand contains: the nucleotide sequence shown in SEQ ID NO. 9 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity with the nucleotide sequence shown in SEQ ID NO. 9 and retaining the biological function of the sequence from which it is derived, and the antisense strand contains: the nucleotide sequence shown in SEQ ID NO. 10 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity with the nucleotide sequence shown in SEQ ID NO. 10 and retaining the biological function of the sequence from which it is derived. The nucleotide sequences of the remaining sense strand and antisense strand are nucleotide sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity and retaining the biological function of the sequence from which they are derived.

[0018] In one embodiment of the present invention, the siRNA is prepared by solid-phase synthesis or liquid-phase synthesis.

[0019] In one embodiment of the present invention, the nucleotides in the siRNA are each independently modified or unmodified nucleotides.

[0020] In one embodiment of the present invention, each nucleotide in the siRNA is an unmodified nucleotide.

[0021] In one embodiment of the present invention, some or all of the nucleotides in the siRNA are modified nucleotides, and these modifications on the nucleotide groups do not cause the function of the siRNA disclosed in the present application to significantly weaken or lose its ability to inhibit the expression of the MUC5AC gene.

[0022] In one embodiment of the present invention, at least one nucleotide in the sense strand or antisense strand of the siRNA is a modified nucleotide.

[0023] In one embodiment of the present invention, at least one phosphate group in the sense strand or antisense strand of the siRNA is a phosphate group with a modifying group.

[0024] In one embodiment of the present invention, at least a part of the phosphate ester group and / or ribose group in the phospho-sugar backbone of at least one single strand of the sense strand and the antisense strand of the siRNA is a phosphate ester group with a modifying group and / or a ribose group with a modifying group.

[0025] In one embodiment of the present invention, all nucleotides in the sense strand and / or antisense strand of the siRNA are modified nucleotides, and these modifications on the nucleotide groups do not significantly weaken or lose the function of the siRNA of the present disclosure in inhibiting MUC5AC gene expression.

[0026] In one embodiment of the present invention, each nucleotide in the sense strand and antisense strand of the siRNA is independently a fluorinated modified nucleotide or a non-fluorinated modified nucleotide.

[0027] In one embodiment of the present invention, the modification is a chemical modification, and the chemical modification is selected from one or more of methoxy modification, fluorination modification or phosphorothioate linkage.

[0028] In one embodiment of the present invention, a "fluorinated modified nucleotide" refers to a nucleotide formed by substituting the hydroxyl group at the 2'-position of the ribose group of the nucleotide with fluorine, and it has the structure shown in the following formula (1). The non-fluorinated modified nucleotide is independently selected from a nucleotide formed by substituting the hydroxyl group at the 2'-position of the ribose group of the nucleotide with a non-fluorine group or a nucleotide analogue.

[0029] In one embodiment of the present invention, the nucleotides formed by substituting the hydroxyl group at the 2'-position of the ribose group with a non-fluorine group are well-known to those skilled in the art, and these nucleotides can be selected from one of 2'-alkoxy modified nucleotides, 2'-substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2'-amino modified nucleotides, 2'-substituted amino modified nucleotides, 2'-deoxynucleotides.

[0030] In one embodiment of the present invention, the 2'-alkoxy modified nucleotide is a 2'-methoxy (2'-OMe) modified nucleotide, as shown in formula (2); the 2'-substituted alkoxy modified nucleotide can be, for example, a 2'-O-methoxyethyl (2'-MOE) modified nucleotide, as shown in formula (3), the 2'-amino (2'-NH2) modified nucleotide as shown in formula (4), and the 2'-deoxynucleotide (DNA) as shown in formula (5):

[0031]

[0032] In one embodiment of the present invention, the fluorine-modified nucleotides are located in the antisense strand and the sense strand of the nucleotide sequence. And, in the direction from the 5'-end to the 3'-end, at least the 7th, 8th, and 9th nucleotides of the sense strand are fluorine-modified nucleotides, and at least the 2nd, 6th, 14th, and 16th nucleotides of the antisense strand are fluorine-modified nucleotides.

[0033] In one embodiment of the present invention, the fluorine-modified nucleotides are located in the sense strand and the antisense strand. The number of fluorine-modified nucleotides in the sense strand is no more than 5. And, in the direction from the 5'-end to the 3'-end, at least the 7th, 8th, and 9th nucleotides of the sense strand are fluorine-modified nucleotides. The number of fluorine-modified nucleotides in the antisense strand is no more than 7, and at least the 2nd, 6th, 14th, and 16th nucleotides of the antisense strand are fluorine-modified nucleotides.

[0034] In one embodiment of the present invention, in the direction from the 5'-end to the 3'-end, in the sense strand, the 7th, 8th, and 9th nucleotides of the sense strand are fluorine-modified nucleotides, and the nucleotides at the remaining positions in the sense strand are non-fluorine-modified nucleotides. In the antisense strand, the 2nd, 6th, 14th, and 16th nucleotides of the antisense strand are fluorine-modified nucleotides, and the nucleotides at the remaining positions in the antisense strand are non-fluorine-modified nucleotides.

[0035] In one embodiment of the present invention, the methoxy-modified nucleotides are located in the antisense strand and the sense strand of the nucleotide sequence. And, in the direction from the 5'-end to the 3'-end, at least the 1st, 2nd, 3rd, 4th, 5th, 6th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, and 19th nucleotides of the sense strand are methoxy-modified nucleotides, and at least the 1st, 3rd, 4th, 5th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 15th, 17th, 18th, 19th, 20th, and 21st nucleotides of the antisense strand are methoxy-modified nucleotides.

[0036] In one embodiment of the present invention, the nucleotide analog refers to a group that can replace nucleotides in nucleic acids but has a structure different from that of adenosine ribonucleotide, guanosine ribonucleotide, cytidine ribonucleotide, uridine ribonucleotide, or thymidine deoxyribonucleotide.

[0037] In one embodiment of the present invention, the nucleotide analog can be a heteronucleotide, a bridged nucleotide, or an acyclic nucleotide.

[0038] In one embodiment of the present invention, the bridged nucleic acid (BNA) refers to a constrained or inaccessible nucleic acid. BNA can contain a bridged structure with a "fixed" C3'-endo sugar puckering in a five-membered ring, six-membered ring, or seven-membered ring. Usually, this bridge is incorporated at the 2'- and 4'-positions of the ribose to provide a 2',4'-BNA nucleotide.

[0039] In one embodiment of the present invention, the BNA can be LNA, ENA, cET BNA, etc. Among them, LNA is shown in formula (6), ENA is shown in formula (7), and cET BNA is shown in formula (8):

[0040]

[0041] In one embodiment of the present invention, at least a part of the phosphate ester groups in the phospho-sugar backbone of at least one single strand of the sense strand and the antisense strand of the siRNA is a phosphate ester group with a modifying group.

[0042] In one embodiment of the present invention, the phosphate ester group with a modifying group is a phosphorothioate group formed by replacing at least one oxygen atom in the phosphodiester bond of the phosphate ester group with a sulfur atom.

[0043] In one embodiment of the present invention, the phosphate ester group with a modifying group is a phosphorothioate group having the structure shown in formula (9):

[0044]

[0045] In one embodiment of the present invention, the connection of the phosphorothioate group exists in at least one of the positions consisting of the following groups: between the first and second nucleotides at either end of the sense strand or the antisense strand; between the second and third nucleotides at either end of the sense strand or the antisense strand; or any combination of the above.

[0046] In one embodiment of the present invention, the connection of the phosphorothioate group exists at all the above positions except the 5'-end of the sense strand.

[0047] In one embodiment of the present invention, the connection of the phosphorothioate group exists at all the above positions except the 3'-end of the sense strand.

[0048] In one embodiment of the present invention, the connection of the phosphorothioate group exists in at least one of the following positions:

[0049] between the 1st and 2nd nucleotides at the 5'-end of the sense strand;

[0050] between the second and third nucleotides at the 5'-end of the sense strand;

[0051] between the first and second nucleotides at the 3'-end of the sense strand;

[0052] between the second and third nucleotides at the 3'-end of the sense strand;

[0053] between the first and second nucleotides at the 5'-end of the antisense strand;

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

[0055] between the first and second nucleotides at the 3'-end of the antisense strand; and

[0056] between the second and third nucleotides at the 3'-end of the antisense strand.

[0057] In one embodiment of the present invention, the phosphorothioate-linked nucleotides are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5'-end to the 3'-end, at least the first and second positions, and the second and third positions of the sense strand are nucleotides linked by a phosphorothioate group; in the direction from the 5'-end to the 3'-end, at least the first and second positions, the second and third positions, the 19th and 20th positions, and the 20th and 21st positions of the antisense strand are nucleotides linked by a phosphorothioate group.

[0058] In one embodiment of the present invention, the modification further includes nucleotides selected from 2'-deoxynucleotides, 2'3'-seco nucleotide analogs, locked nucleotides, unlocked nucleic acid nucleotides (UNA), glycol nucleic acid nucleotides (GNA), 2'-F-arabinonucleotides, 2'-methoxyethyl nucleotides, abasic nucleotides, ribitol, inverted nucleotides, inverted abasic nucleotides, inverted 2'-OMe nucleotides, inverted 2'-deoxynucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides and 3'-OMe nucleotides, nucleotides containing a 5'-thiophosphate group, or terminal nucleotides linked to a cholesterol derivative or a didodecylamide group, 2'-amino modified nucleotides, aminophosphates, or unnatural bases containing nucleotides.

[0059] In one embodiment of the present invention, two nucleotides complementary to the 3'-end of the antisense strand are further linked to the 5'-end of the siRNA sense strand;

[0060] The fluorine-modified nucleotides are located in the antisense strand and the sense strand of the nucleotide sequence. And, in the direction from the 5'-end to the 3'-end, the nucleotides at least at the 9th, 10th, and 11th positions of the sense strand are fluorine-modified nucleotides, and the nucleotides at least at the 2nd, 10th, 12th, 14th, 16th, 18th, and 20th positions of the antisense strand are fluorine-modified nucleotides;

[0061] The methoxy-modified nucleotides are located in the antisense strand and the sense strand of the nucleotide sequence. And, in the direction from the 5'-end to the 3'-end, the nucleotides at least at the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, and 21st positions of the sense strand are methoxy-modified nucleotides, and the nucleotides at least at the 1st, 3rd, 4th, 6th, 8th, 9th, 11th, 13th, 15th, 17th, 19th, 21st positions of the antisense strand are methoxy-modified nucleotides;

[0062] The phosphorothioate-linked nucleotides are located in the antisense strand and the sense strand of the nucleotide sequence. And, in the direction from the 5'-end to the 3'-end, the nucleotides between at least the 1st and 2nd positions, the 19th and 20th positions, and the 20th and 21st positions of the sense strand are linked by phosphorothioate groups, and the nucleotides between at least the 1st and 2nd positions, the 2nd and 3rd positions, the 19th and 20th positions, and the 20th and 21st positions of the antisense strand are linked by phosphorothioate groups;

[0063] The 2'-deoxynucleotides are located in the antisense strand of the nucleotide sequence. And, in the direction from the 5'-end to the 3'-end, the nucleotides at least at the 5th and 7th positions of the antisense strand are 2'-deoxynucleotides.

[0064] In one embodiment of the present invention, 2'-O-hexadecyl is linked to the 6th position of the siRNA sense strand in the direction from the 5'-end to the 3'-end.

[0065] In one embodiment of the present invention, the 5'-end and / or 3'-end of the siRNA sense strand contains more than one inverted abasic residue.

[0066] In one embodiment of the present invention, the modified nucleotides are introduced into the siRNA by using nucleotide monomers with corresponding modifications.

[0067] The present invention also provides a product for inhibiting MUC5AC, the product comprising an active ingredient and a pharmaceutically acceptable carrier, and the active ingredient is the above siRNA or the above modified siRNA.

[0068] In one embodiment of the present invention, the product is a pharmaceutical composition or a kit.

[0069] In one embodiment of the present invention, the product is a pharmaceutical composition, and the pharmaceutically acceptable carrier may be a carrier commonly used in the field of siRNA administration, such as but not limited to magnetic nanoparticles (such as nanoparticles based on Fe3O4 or Fe2O3), carbon nanotubes, mesoporous silicon, calcium phosphate nanoparticles, polyethyleneimine (PEI), polyamidoamine (PAMAM) dendrimer, poly(L-lysine) (PLL), chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), poly(D&L-lactic / glycolic acid) copolymer (PLGA), poly(2-aminoethyl ethylene phosphate) (PPEEA), and poly(2-dimethylaminoethyl methacrylate) (PDMAEMA) and one or more of their derivatives.

[0070] In one embodiment of the present invention, there is no particular requirement for the contents of siRNA and the pharmaceutically acceptable carrier, and they may be the conventional contents of each component.

[0071] In one embodiment of the present invention, in the pharmaceutical composition, the weight ratio of the active ingredient to the pharmaceutically acceptable carrier is 1:(1 - 500).

[0072] In one embodiment of the present invention, in the pharmaceutical composition, the weight ratio of the active ingredient to the pharmaceutically acceptable carrier is 1:(1 - 50).

[0073] In one embodiment of the present invention, the pharmaceutical composition may further contain other pharmaceutically acceptable excipients, and the excipient may be one or more of various preparations or compounds conventionally used in the art.

[0074] In one embodiment of the present invention, the other pharmaceutically acceptable excipients may include at least one of a pH buffer, a protective agent, and an osmotic pressure regulator.

[0075] In one embodiment of the present invention, the pH buffer can be tris(hydroxymethyl)aminomethane hydrochloride buffer with a pH value of 7.5 - 8.5 and / or phosphate buffer with a pH value of 5.5 - 8.5.

[0076] In one embodiment of the present invention, the cryoprotectant can be at least one of inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose, and glucose.

[0077] In one embodiment of the present invention, based on the total weight of the pharmaceutical composition, the content of the cryoprotectant can be 0.01 - 30% by weight.

[0078] In one embodiment of the present invention, the osmotic pressure regulator can be sodium chloride and / or potassium chloride.

[0079] In one embodiment of the present invention, the content of the osmotic pressure regulator makes the osmotic pressure of the pharmaceutical composition 200 - 700 milliosmoles per liter (mOSM / L). Those skilled in the art can easily determine the content of the osmotic pressure regulator according to the required osmotic pressure.

[0080] In one embodiment of the present invention, the pharmaceutical composition can be a liquid preparation, such as an injection; or it can be a freeze-dried powder injection, which is mixed with liquid excipients during administration to prepare a liquid preparation.

[0081] In one embodiment of the present invention, the liquid preparation can be used for subcutaneous, intramuscular, or intravenous injection, and can also be used for administration to the lungs by spraying, or for administration to other organ tissues (such as the liver) through the lungs by spraying.

[0082] In one embodiment of the present invention, the pharmaceutical composition is used for intravenous injection.

[0083] In one embodiment of the present invention, the pharmaceutical composition can be in the form of a liposomal preparation.

[0084] In one embodiment of the present invention, the pharmaceutically acceptable carrier used in the liposomal preparation includes an amine-containing transfection compound (hereinafter also referred to as an organic amine), a co-lipid, and / or a polyethylene glycolylated lipid.

[0085] In one embodiment of the present invention, the organic amine, co-lipid, and polyethylene glycolylated lipid can be selected from one or more of the amine-containing transfection compounds or their pharmaceutically acceptable salts or derivatives, co-lipids, and polyethylene glycolylated lipids described in CN108220295B (which is incorporated herein by reference in its entirety).

[0086] In one embodiment of the present invention, the pharmaceutically acceptable targeting group in the siRNA conjugate can be galactose or N-acetylgalactosamine (GalNAc). N-acetylgalactosamine is a ligand that binds to the asialoglycoprotein receptor (ASGPR) on the liver surface. The asialoglycoprotein receptor is an endocytic receptor specifically expressed in hepatocytes. N-acetylgalactosamine is used as a targeting molecule to deliver small RNAs to the liver.

[0087] In one embodiment of the present invention, the galactose or N-acetylgalactosamine molecule can be monovalent, divalent, trivalent, or tetravalent. The monovalent, divalent, trivalent, and tetravalent refer to the molar ratio of the siRNA molecule to the galactose or N-acetylgalactosamine molecule containing the coupling group as the targeting group in the siRNA conjugate being 1:1, 1:2, 1:3, or 1:4 after the formation of the siRNA conjugate.

[0088] In one embodiment of the present invention, when the siRNA is conjugated to a coupling group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent or tetravalent.

[0089] In one embodiment of the present invention, when the siRNA is conjugated to a coupling group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent.

[0090] In one embodiment of the present invention, the targeting group can be linked to the siRNA molecule via a suitable linker, and those skilled in the art can select a suitable linker according to the specific type of the targeting group.

[0091] In one embodiment of the present invention, for the types of the linker, the targeting group, and the connection mode with the siRNA, reference can be made to the disclosure of WO2015006740A2, and its entire content is incorporated herein by reference.

[0092] In one embodiment of the present invention, the siRNA conjugate formed by GalNAc and the siRNA molecule has the structure shown in the following formula (10):

[0093]

[0094] In one embodiment of the present invention, the kit further comprises a pharmaceutically acceptable carrier and / or excipient.

[0095] In one embodiment of the present invention, the siRNA, pharmaceutically acceptable carrier and / or excipient in the kit can exist alone, exist in the form of a mixture of two or more of them, or exist in the form of a final pharmaceutical composition.

[0096] In one embodiment of the present invention, in the kit, the pharmaceutically acceptable carrier is an amine-containing compound, a helper lipid and a polyethylene glycolylated lipid.

[0097] In one embodiment of the present invention, in the kit, the pharmaceutically acceptable carrier is a mixture or exists independently.

[0098] In one embodiment of the present invention, the siRNA, pharmaceutically acceptable carrier and / or excipient in the kit are provided in liquid form, dry form or lyophilized form.

[0099] In one embodiment of the present invention, the siRNA, pharmaceutically acceptable carrier and / or excipient in the kit are substantially pure and / or sterile.

[0100] In one embodiment of the present invention, in the kit, it includes a container for providing siRNA, one or more containers for providing an amine-containing compound, a helper lipid and a polyethylene glycolylated lipid, and optionally, a container for providing excipients.

[0101] In one embodiment of the present invention, the kit further includes one or more components necessary or beneficial for a specific application, and the components are selected from:

[0102] One or more components for achieving the desired cell transfection;

[0103] One or more components for achieving the diagnosis, treatment or prevention of a specific disease or physical disorder;

[0104] One or more buffers;

[0105] Positive or negative control samples;

[0106] Excipients, stabilizers or preservatives.

[0107] In one embodiment of the present invention, the one or more components for achieving the diagnosis, treatment or prevention of a specific disease or physical disorder are one or more additional therapeutic compounds or compositions, one or more diagnostic reagents.

[0108] In one embodiment of the present invention, the kit further includes one or more of sterile water, physiological saline and PBS.

[0109] The present invention also provides the use of the above siRNA or the above product in the preparation of a product for preventing, diagnosing and / or treating a pathological condition or disease caused by MUC5AC.

[0110] In one embodiment of the present invention, the disease caused by MUC5AC is asthma.

[0111] The technical solution of the present invention has the following advantages:

[0112] The present invention provides a modified siRNA for inhibiting MUC5AC, and the modifications include methoxy modification, fluoro modification and phosphorothioate linkage. Activity detection was carried out in A549 cells. At a concentration of 10 nM, most of them could significantly inhibit the expression of the MUC5AC gene. SiRNAs with an inhibition rate > 80% were screened out. Activity detection was carried out again in A549 cells at concentrations of 1 nM and 0.1 nM. Among them, the siRNAs represented by MUC-20M, MUC-40M, MUC-69M, MUC-78M, MUC-87M, MUC-125M, MUC-182M, MUC-185M and MUC-190M could significantly inhibit the expression of the MUC5AC gene at a concentration of 1 nM, with an inhibition rate > 70%. And the siRNAs at a concentration of 0.1 nM also had good inhibitory activity. The inhibition rates of the siRNAs represented by MUC-20M, MUC-40M, MUC-69M, MUC-125M, MUC-182M and MUC-185M were all > 40%. The inhibition rate of the siRNA represented by MUC-182M was > 60% at a concentration of 0.1 nM. Description of the Drawings

[0113] Figure 1 : Structural formula of C16. Detailed Embodiments

[0114] The following examples are provided to better understand the present invention further. It is not limited to the best embodiment, and does not limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior art features falls within the protection scope of the present invention.

[0115] In the following examples, MUC5AC mRNA refers to the mRNA with the sequences shown in GeneBank accession numbers NM_001304359.2, XM_045372409.1, and MUC5AC NM_010844.3. Further, unless otherwise stated, the term "target gene" used in the present disclosure refers to the gene that transcribes the above MUC5AC mRNA, and the term "target mRNA" refers to the above MUC5AC mRNA.

[0116] For those without specific experimental steps or conditions noted in the following examples, the operations or conditions of the conventional experimental steps described in the literature in this field can be followed. For reagents or instruments without the manufacturer noted, they are all conventional reagent products that can be obtained through commercial purchase.

[0117] In the following examples, the capital letters C, G, U, and A represent ribonucleotides; the lowercase letter m indicates that the nucleotide adjacent to the left of the letter m is a nucleotide with a methoxy modification; the lowercase letter f indicates that the nucleotide adjacent to the left of the letter f is a nucleotide with a fluoro modification; the lowercase letter s indicates that there is a phosphorothioate modification between the two nucleotides adjacent to the left and right of the letter s.

[0118] In the following examples, "modified nucleotide" refers to a nucleotide or nucleotide analogue formed by replacing the hydroxyl group at the 2'-position of the ribose group of a nucleotide with another group, or a nucleotide whose base on the nucleotide is a modified base. "Fluoro-modified nucleotide" refers to a nucleotide formed by replacing the hydroxyl group at the 2'-position of the ribose group of a nucleotide with fluorine, and "non-fluoro-modified nucleotide" refers to a nucleotide or nucleotide analogue formed by replacing the hydroxyl group at the 2'-position of the ribose group of a nucleotide with a non-fluorine group. "Nucleotide analogue" refers to a group that can replace a nucleotide in a nucleic acid but has a structure different from adenosine ribonucleotide, guanosine ribonucleotide, cytidine ribonucleotide, uridine ribonucleotide, or thymidine deoxyribonucleotide. Such as an isonucleotide, a bridged nucleic acid (abbreviated as BNA), or an acyclic nucleotide. "Methoxy-modified nucleotide" refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribose group with a methoxy group.

[0119] In the following examples, the terms "complementary" or "reverse complementary" can be used interchangeably and have the meanings well-known to those skilled in the art, that is, in a double-stranded nucleic acid molecule, the bases of one strand pair with the bases of the other strand in a complementary manner. In DNA, the purine base adenine (A) always pairs with the pyrimidine base thymine (T) (or uracil (U) in RNA); the purine base guanine (C) always pairs with the pyrimidine base cytosine (G). Each base pair includes a purine and a pyrimidine. When adenine on one strand always pairs with thymine (or uracil) on the other strand, and guanine always pairs with cytosine, the two strands are considered to be complementary to each other, and the sequence of one strand can be inferred from the sequence of its complementary strand.

[0120] In the following examples, especially when describing the preparation methods of the siRNAs, pharmaceutical compositions or siRNA conjugates of the present disclosure, unless otherwise specified, a nucleoside monomer refers to an unmodified or modified RNA phosphoramidite monomer (sometimes RNA phosphoramidites are also referred to as Nucleoside phosphoramidites) used in phosphoramidite solid-phase synthesis according to the types and sequences of nucleotides in the siRNA or siRNA conjugate to be prepared. Phosphoramidite solid-phase synthesis is a method used in RNA synthesis well-known to those skilled in the art. The nucleoside monomers used in the present disclosure are all commercially available.

[0121] In the following examples, "coupling" means that two or more chemical moieties each having a specific function are connected to each other in a covalent linkage; correspondingly, a "conjugate" means a compound formed by covalent connection between these individual chemical moieties. Further, an "siRNA conjugate" means a compound formed by covalently connecting one or more chemical moieties having specific functions to an siRNA. An siRNA conjugate should be understood, according to the context, as the general term for a plurality of siRNA conjugates or an siRNA conjugate represented by a certain chemical formula. In the context of the present disclosure, a "coupling molecule" should be understood as a specific compound that can be coupled to an siRNA through a reaction to ultimately form the siRNA conjugate of the present disclosure.

[0122] In the following examples, "optional" or "optionally" means that the event or condition described thereafter may or may not occur, and the description includes both the case where the event or condition occurs and the case where it does not occur. For example, an "alkyl group" that is "optionally substituted" includes an "alkyl group" and a "substituted alkyl group" as defined below. Those skilled in the art will understand that for any group containing one or more substituents, these groups are not intended to introduce any substitutions or substitution patterns that are spatially impracticable, synthetically infeasible, and / or inherently unstable.

[0123] In the following examples, "treat", "alleviate" or "ameliorate" can be used interchangeably herein. These terms refer to methods of obtaining a beneficial or desired result, including but not limited to a therapeutic benefit. A "therapeutic benefit" means eradicating or ameliorating the underlying disorder being treated. In addition, a therapeutic benefit is obtained by eradicating or ameliorating one or more physiological symptoms associated with the underlying disorder, such that an improvement is observed in the subject, although the subject may still be afflicted with the underlying disorder.

[0124] In the following examples, "prevent" and "prevention" are used interchangeably. These terms refer to methods of obtaining beneficial or desired results, including but not limited to prophylactic benefits. To obtain "prophylactic benefits", the composition can be administered to a subject at risk of developing a particular disease, or to a subject reporting one or more pathological symptoms of a disease, even if a diagnosis of the disease may not have been made.

[0125] Unless otherwise specified, the reagents and culture media used in the following examples are commercially available products, and the operations such as nucleic acid electrophoresis and real-time PCR are carried out according to the methods described in "Molecular Biology (Fourth Edition)" (edited by Alexander McLennan et al., 2019).

[0126] The experimental cells involved in the following examples are A549, purchased from the Cell Bank of the Chinese Academy of Sciences;

[0127] The siRNA involved in the following examples is the siRNA sequence synthesized by phosphoramidite solid-phase synthesis.

[0128] When the siRNA, siRNA conjugate targeting the MUC5AC gene or the siRNA, siRNA conjugate used as a negative control involved in the following examples are transfected into cells, Lipofectamine 2000 (purchased from Invitrogen) is used as the transfection reagent, and the specific operation is referred to the instruction manual provided by the manufacturer. When performing qPCR detection, HiScript III RT SuperMix for qPCR (purchased from Vazyme) is used as the reverse transcription reagent, and the specific operation is referred to the instruction manual provided by the manufacturer.

[0129] Unless otherwise stated, the reagent ratios provided below are calculated by volume ratio (v / v).

[0130] Example 1: An siRNA for inhibiting MUC5AC

[0131] This example provides an siRNA for inhibiting MUC5AC. The nucleotide sequence of the siRNA is designed based on the target mRNA, as shown in Table 1. The siRNA molecule with the following sequence was synthesized by Suzhou GenePharma Co., Ltd.

[0132] Table 1. Nucleotide sequence of siRNA for inhibiting MUC5AC

[0133]

[0134]

[0135]

[0136] Example 2: A modified siRNA for inhibiting MUC5AC

[0137] This embodiment provides a modified siRNA for inhibiting MUC5AC. The modified siRNA includes MUC5AC-4M to MUC5AC-420M. The sense strands of MUC5AC-4M to MUC5AC-420M are respectively obtained by chemically modifying the sequences shown in SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19, SEQ ID NO.21, SEQ ID NO.23, SEQ ID NO.25, SEQ ID NO.27, SEQ ID NO.29, SEQ ID NO.31, SEQ ID NO.33, SEQ ID NO.35, SEQ ID NO.37, SEQ ID NO.39, SEQ ID NO.41, SEQ ID NO.43, SEQ ID NO.45, SEQ ID NO.47, SEQ ID NO.49, SEQ ID NO.51, SEQ ID NO.53, SEQ ID NO.55, SEQ ID NO.57, SEQ ID NO.59, SEQ ID NO.61, SEQ ID NO.63, SEQ ID NO.65, SEQ ID NO.67, SEQ ID NO.69, SEQ ID NO.71, SEQ ID NO.73, SEQ ID NO.75, SEQ ID NO.77, SEQ ID NO.79, SEQ ID NO.81, SEQ ID NO.83, SEQ ID NO.85, SEQ ID NO.87, SEQ ID NO.89, SEQ ID NO.91, SEQ ID NO.93 and SEQ ID NO.95. In the direction from the 5'-end to the 3'-end, the nucleotides at the 1st and 2nd positions, and the 2nd and 3rd positions are linked by phosphorothioate groups. The nucleotides at the 1st, 2nd, 3rd, 4th, 5th, 6th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th positions are nucleotides modified with methoxy groups, and the nucleotides at the 7th, 8th, 9th positions are nucleotides modified with fluorine groups.The antisense strands of MUC5AC-4M to MUC5AC-566M are respectively obtained by chemically modifying the sequences shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26, SEQ ID NO.28, SEQ ID NO.30, SEQ ID NO.32, SEQ ID NO.34, SEQ ID NO.36, SEQ ID NO.38, SEQ ID NO.40, SEQ ID NO.42, SEQ ID NO.44, SEQ ID NO.46, SEQ ID NO.48, SEQ ID NO.50, SEQ ID NO.52, SEQ ID NO.54, SEQ ID NO.56, SEQ ID NO.58, SEQ ID NO.60, SEQ ID NO.62, SEQ ID NO.64, SEQ ID NO.66, SEQ ID NO.68, SEQ ID NO.70, SEQ ID NO.72, SEQ ID NO.74, SEQ ID NO.76, SEQ ID NO.78, SEQ ID NO.80, SEQ ID NO.82, SEQ ID NO.84, SEQ ID NO.86, SEQ ID NO.88, SEQ ID NO.90, SEQ ID NO.92, SEQ ID NO.94 and SEQ ID NO.96. In the direction from the 5'-end to the 3'-end, the nucleotides at the 1st and 2nd positions, the 2nd and 3rd positions, the 19th and 20th positions, and the 20th and 21st positions are linked by phosphorothioate groups. The nucleotides at the 1st, 3rd, 4th, 5th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 15th, 17th, 18th, 19th, 20th, and 21st positions are nucleotides modified with methoxy groups, and the nucleotides at the 2nd, 6th, 14th, and 16th positions are nucleotides modified with fluoro groups.

[0138] Experimental Example 1: Activity Detection of Modified siRNA for Inhibiting MUC5AC in Cell A549

[0139] This experimental example provides the determination of the relative inhibition level of the siRNA compound of the present invention on MUC5AC mRNA in cell A549 by quantitative real-time PCR (hereinafter referred to as qPCR).

[0140] Experiment for detecting the activity of the modified siRNA for inhibiting MUC5AC in A549 cells was carried out as follows:

[0141] 1. A549 cells were cultured in F12K complete medium (Hyclone) containing 10% fetal bovine serum (FBS, Hyclone) and 0.2% penicillin-streptomycin (Gibco, Invitrogen). The cultured cells were diluted with F-12K complete medium to 5.55×10 4 / ml to obtain an A549 cell suspension, which was plated into a 96-well plate with 90 μL of the cell suspension added to each well.

[0142] 2. The dry powder of the siRNA to be tested prepared in Example 2 was centrifuged at low temperature and high speed, and then dissolved in ultrapure distilled water to prepare a 100 μM siRNA mother solution.

[0143] 3. Prepare a 10 nM siRNA transfection dilution

[0144] (1) Preparation of 10 μM siRNA stock solution: Take 2 μl of the 100 μM siRNA mother solution described above and add 18 μl of ultrapure distilled water to obtain a 10 μM siRNA stock solution;

[0145] (2) Preparation of 10 nM siRNA transfection dilution: Take 2 μl of the 10 μM siRNA stock solution and add 98 μl of ultrapure distilled water to obtain a 0.2 μM siRNA dilution; then add an equal volume of Lipofectaine RNAiMax transfection reagent dilution (3 μl of Lipofectaine RNAiMax transfection reagent was added to 97 μl of Opti-medium to obtain Lipofectaine RNAiMax transfection reagent dilution) to obtain a 0.1 μM siRNA dilution; take 10 μl of the above 0.1 μM siRNA dilution and add it to the transfected and cultured A549 cells in the 96-well plate to obtain a final siRNA concentration of 10 nM.

[0146] 4. Incubate the transfected cells for 48 hours, and set up three replicate wells. Additionally, set up NC and MUC-Positive as controls; the NC group uses siRNA that is a negative control siRNA unrelated to MUC5AC, with its sense strand (5’-3’): CfsAmsCfUmUfAmCfGmCfUmGfAmGfUmAfCmUfUmCfGmAf, and the antisense strand (5’-3’): UmsCfsGmAfAmGfUmAfCmUfCmAfGmCfGmUfAmAfGmUfGmsAfsUm. The MUC-Positive group uses the Arrowhead patent sequence as a control group.

[0147] 5. Use a magnetic bead method cell total RNA extraction kit (purchased from Suzhou GenePharma Co., Ltd., product number E31008) to extract the total RNA from the cells in each well according to the method described in the kit instructions.

[0148] 6. RNA reverse transcription

[0149] Use HiScript III RT SuperMix for qPCR (purchased from Vazyme Biotech Co., Ltd., product number R323-01), and the experimental steps refer to the product instruction manual; prepare a 20 μL reverse transcription reaction system according to the reverse transcription operation steps in the kit instructions to perform reverse transcription on the total RNA of the cells; the reverse transcription conditions are: incubate the reverse transcription reaction system at 37 °C for 15 min, then incubate at 85 °C for 5 s, and add 80 μL of DEPC water to each reverse transcription reaction system to obtain a solution containing cDNA;

[0150] 7. Configuration of qPCR reaction system

[0151] For each reverse transcription reaction system, 4 μL of the above cDNA-containing solution was taken as a template, and the reagents provided by the AceQ Universal SYBR qPCR MasterMix kit (purchased from Vazyme, catalog number Q511-02) were used to prepare a 20-μL qPCR reaction system on an ice box according to Table 2. Among them, Primer1 and Primer2 are the PCR primer sequences for amplifying the target gene MUC5AC and the internal reference gene GAPDH, respectively (as shown in Table 3). Each qPCR reaction system was placed on an ABI StepOnePlus Real-Time PCR instrument and amplified using the three-step method. The amplification program was pre-denaturation at 95 °C for 10 min, then denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s. After repeating the above denaturation, annealing, and extension processes 40 times, a product W containing the amplified target gene MUC5AC and internal reference gene GAPDH was obtained. The product W was then incubated at 95 °C for 15 s, 60 °C for 1 min, and 95 °C for 15 s in sequence. The real-time fluorescence quantitative PCR instrument collected the melting curves of the target gene MUC5AC and the internal reference gene GAPDH in the product W respectively, and the Ct values of the target gene MUC5AC and the internal reference gene GAPDH were obtained.

[0152] Table 2 RNA amplification reaction system

[0153] Reagent Name Volume per well (μL) 2×AceQUniversalSYBR qPCR Master Mix 10 Primer1 (10 μM) 0.4 Primer2 (10 μM) 0.4 Template DNA / cDNA 4 <![CDATA[ddH2O]]> 5.2 Total Volume 20ul

[0154] Table 3 Primer information

[0155]

[0156] The comparative Ct (ΔΔCt) method was used to perform relative quantitative calculations on the target gene MUC5AC in each test group. The calculation method was as follows:

[0157] ΔCt (test group) = Ct (test group target gene) – Ct (test group internal reference gene)

[0158] ΔCt (control group) = Ct (control group target gene) – Ct (control group internal reference gene)

[0159] ΔΔCt (test group) = ΔCt (test group) - ΔCt (control group average)

[0160] ΔΔCt (control group) = ΔCt (control group) - ΔCt (control group average)

[0161] Among them, ΔCt (control group average) is the arithmetic mean of the ΔCt (control group) of the 4 samples in the control group. Thus, each sample in the test group and the control group corresponds to a ΔΔCt value.

[0162] Normalize the expression level of MUC5AC mRNA in the test group based on the control group, and define the expression level of MUC5AC mRNA in the control group as 100%.

[0163] Relative expression level of MUC5AC mRNA in the test group = 2 -ΔΔCt(测试组) × 100%

[0164] Compare the MUC5AC mRNA level with the internal reference gene GAPDH, normalize this value to the average of the normal saline control group, represent the data as a percentage relative to the normal saline control group, and present it as the average plus the standard deviation.

[0165] The activity results of 48 siRNAs in cell A549 are shown in Table 4. Among them, the siRNAs represented by MUC-4M, MUC-13M, MUC-16M, MUC-20M, MUC-28M, MUC-29M, MUC-38M, MUC-39M, MUC-40M, MUC-42M, MUC-49M, MUC-62M, MUC-69M, MUC-78M, MUC-87M, MUC-90M, MUC-120M, MUC-125M, MUC-138M, MUC-164M, MUC-175M, MUC-182M, MUC-185M, MUC-186M and MUC-190M all have high inhibitory activity, with an inhibition rate > 80%. Among them, the inhibition rates of the siRNAs represented by MUC-16M, MUC-20M, MUC-28M, MUC-29M, MUC-39M, MUC-40M, MUC-69M, MUC-87M, MUC-120M, and MUC-125M are > 90%.

[0166] Table 4 Activity of modified siRNAs in cell A549

[0167]

[0168]

[0169] Experimental Example 2: Detection of the activity of modified siRNAs for inhibiting MUC5AC in cell A549

[0170] This experimental example provides the determination of the relative inhibitory level of the siRNA compound of the present invention on MUC5AC mRNA in cell A549 by real-time fluorescence quantitative PCR (Quantitative Real-Time PCR, hereinafter referred to as qPCR).

[0171] Use the sequences of MUC-4M, MUC-13M, MUC-16M, MUC-20M, MUC-28M, MUC-29M, MUC-38M, MUC-39M, MUC-40M, MUC-42M, MUC-49M, MUC-62M, MUC-69M, MUC-78M, MUC-87M, MUC-90M, MUC-123M, MUC-120M, MUC-125M, MUC-138M, MUC-164M, MUC-175M, MUC-182M, MUC-185M, MUC-186M, and MUC-190M with high inhibition rates screened in Experimental Example 1 to detect the activity in A549 cells at lower concentrations.

[0172] Refer to Experimental Example 1 for the experimental procedure. On the basis of Experimental Example 1, retain Steps 1 to 2. In Step 3, prepare 1 nM siRNA transfection dilution and 0.1 nM siRNA transfection dilution. The preparation steps are as follows:

[0173] The preparation steps of 1 nM siRNA transfection dilution are as follows:

[0174] (1) Preparation of 10 μM siRNA stock solution: Take 2 μl of the above 100 μM siRNA mother solution and add 18 μl of ultrapure distilled water to obtain a 10 μM siRNA stock solution;

[0175] (2) Preparation of 1 nM siRNA transfection dilution: Take 2 μl of 10 μM siRNA stock solution and add 18 μl of ultrapure distilled water to obtain a 1 μM siRNA dilution; then take 2 μl of 1 μM siRNA dilution and add 98 μl of ultrapure distilled water to obtain a 20 nM siRNA dilution; then add an equal volume of Lipofectaine RNAiMax transfection reagent dilution (add 3 μl of Lipofectaine RNAiMax transfection reagent to 97 μl of Opti-medium to obtain Lipofectaine RNAiMax transfection reagent dilution) to obtain a 10 nM siRNA dilution; take 10 μl of the above 10 nM siRNA dilution and add it to the A549 cells transfected and cultured in a 96-well plate to obtain a final siRNA concentration of 1 nM.

[0176] The preparation steps of 0.1 nM siRNA transfection dilution are as follows:

[0177] (1) Preparation of 10 μM siRNA stock solution: Take 2 μl of the above 100 μM siRNA mother solution and add 18 μl of ultrapure distilled water to obtain a 10 μM siRNA stock solution;

[0178] (2) Preparation of 0.1 nM siRNA transfection diluent: Take 2 μl of 10 μM siRNA stock solution, add 18 μl of ultrapure distilled water to obtain a 1 μM siRNA diluent; take 2 μl of 1 μM siRNA diluent, add 18 μl of ultrapure distilled water to obtain a 0.1 μM siRNA diluent; then take 2 μl of 0.1 μM siRNA diluent, add 98 μl of ultrapure distilled water to obtain a 2 nM siRNA diluent; then add an equal volume of Lipofectaine RNAiMax transfection reagent diluent (add 3 μl of Lipofectaine RNAiMax transfection reagent to 97 μl of Opti-medium to obtain Lipofectaine RNAiMax transfection reagent diluent) to obtain a 1 nM siRNA diluent; take 10 μl of the above 1 nM siRNA diluent and add it to the A549 cells transfected and cultured in a 96-well plate to obtain a final siRNA concentration of 0.1 nM.

[0179] Steps 4 to 7 refer to Experimental Example 1 to detect the activities of 1 nM and 0.1 nM siRNAs in A549 cells. In addition to the NC group, a MOCK group is set as a control. The MOCK group is the group that only adds Lipofectamine RNAiMAX reagent without adding any siRNA.

[0180] The activity results of 26 siRNAs in A549 cells are shown in Table 5. Among them, the siRNAs shown as MUC-20M, MUC-40M, MUC-69M, MUC-78M, MUC-87M, MUC-125M, MUC-182M, MUC-185M, and MUC-190M all have high inhibitory activities at the 1 nM concentration, with an inhibition rate > 70%, and the siRNAs at the 0.1 nM concentration also have good inhibitory activities. The inhibition rates of the siRNAs shown as MUC-20M, MUC-40M, MUC-69M, MUC-125M, MUC-182M, and MUC-185M are all > 40%, and the inhibition rate of the siRNA shown as MUC-182M at the 0.1 nM concentration is > 60%.

[0181] Table 5 Results of 1 nM and 0.1 nM transfection experiments

[0182]

[0183]

[0184] Example 3: A modified siRNA for inhibiting MUC5AC

[0185] This example provides a modified siRNA for inhibiting MUC5AC. The siRNA is modified based on Example 1, and the modified sequences are shown in Table 6 and Table 7. Taking MUC5AC-13CAM1 as an example, it is modified based on the sequence MUC5AC-13. Taking MUC5AC-13CAM2 as an example, it is modified based on the sequence MUC5AC-13.

[0186] Table 6 Nucleotide Sequences of siRNAs for Inhibiting MUC5AC

[0187]

[0188]

[0189] Table 7 Nucleotide Sequences of siRNAs for Inhibiting MUC5AC

[0190]

[0191]

[0192] Among them, A(d) represents 2'-deoxyadenosine-3'-phosphate, C(d) represents 2'-deoxycytidine-3'-phosphate, G(d) represents 2'-deoxyguanosine-3'-phosphate, T(d) represents 2'-deoxythymidine-3'-phosphate, invAb is reverse abasic deoxyribonucleotide-5'-phosphate, and the structure of C16 is shown in Figure 1 .

[0193] Experimental Example 3: Activity Detection of Modified siRNA for Inhibiting MUC5AC in Cells A549

[0194] This experimental example provides the determination of the relative inhibitory level of the siRNA compound of the present invention on MUC5AC mRNA in cells A549 by quantitative real-time PCR (hereinafter referred to as qPCR).

[0195] The siRNA sequences of Example 3 were used for activity detection in A549 cells.

[0196] The experimental procedure referred to Experimental Example 2, and the activity results of 40 siRNAs in cells A549 are shown in Table 8 and Table 9.

[0197] Table 8 Results of Transfection Experiments at 10 nM, 1 nM, and 0.1 nM

[0198]

[0199] Table 9 Results of Transfection Experiments at 10 nM, 1 nM, and 0.1 nM

[0200]

[0201]

[0202] Obviously, the above embodiments are merely examples for clear illustration and not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation manners here. And the obvious changes or modifications derived therefrom still fall within the protection scope of the present invention.

Claims

1. An siRNA for inhibiting MUC5AC, characterized in that: The siRNA contains a sense strand and an antisense strand, wherein the sense strand can be at least partially reverse-complemented with the antisense strand to form a double-stranded region. Wherein, the sense strand comprises the nucleotide sequence shown in SEQ ID NO.9, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.10, or, Wherein, the sense strand comprises the nucleotide sequence shown in SEQ ID NO.21, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.22, or, Wherein, the sense strand comprises the nucleotide sequence shown in SEQ ID NO.35, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.36, or, Wherein, the sense strand comprises the nucleotide sequence shown in SEQ ID NO.43, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.44, or, Wherein, the sense strand comprises the nucleotide sequence shown in SEQ ID NO.47, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.48, or, Wherein, the sense strand comprises the nucleotide sequence shown in SEQ ID NO.63, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.64, or, Wherein, the sense strand comprises the nucleotide sequence shown in SEQ ID NO.83, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.84, or, Wherein, the sense strand comprises the nucleotide sequence shown in SEQ ID NO.85, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.86, or, Wherein, the sense strand comprises the nucleotide sequence shown in SEQ ID NO.91, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.

92.

2. The siRNA according to claim 1, wherein At least one nucleotide in the sense strand or the antisense strand of the siRNA is a modified nucleotide.

3. The siRNA according to claim 2, characterized in that The modification is a chemical modification, and the chemical modification is selected from one or more of methoxy modification, fluorination modification and thiophosphate modification.

4. The siRNA according to any one of claims 2 to 3, wherein The fluorinated modified nucleotides are located in the antisense strand and the sense strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the 7th, 8th, and 9th nucleotides of the sense strand are fluorinated modified nucleotides, and at least the 2nd, 6th, 14th, and 16th nucleotides of the antisense strand are fluorinated modified nucleotides.

5. The siRNA according to any one of claims 2 to 3, characterized in that The methoxy-modified nucleotides are located in the antisense strand and the sense strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the 1st, 2nd, 3rd, 4th, 5th, 6th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, and 19th nucleotides of the sense strand are methoxy-modified nucleotides, and at least the 1st, 3rd, 4th, 5th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 15th, 17th, 18th, 19th nucleotides of the antisense strand are methoxy-modified nucleotides.

6. The siRNA according to any one of claims 2 to 3, characterized in that The nucleotides linked by the thiophosphate groups are located in the antisense strand and the sense strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the 1st and 2nd, and the 2nd and 3rd nucleotides of the sense strand are linked by the thiophosphate groups, and at least the 1st and 2nd, the 2nd and 3rd, the 19th and 20th, and the 20th and 21st nucleotides of the antisense strand are linked by the thiophosphate groups.

7. The siRNA according to claim 2 to 3, characterized in that The 3' end of the sense strand of the siRNA is coupled with a ligand.

8. The siRNA according to claim 2, characterized in that The modifications also include 2′-deoxynucleotides, 2′3′-seco nucleotide mimetics, locked nucleotides, open-loop nucleic acid nucleotides (UNA), glycol nucleic acid nucleotides (GNA), 2′-F-arabino nucleotides, 2′-methoxyethyl nucleotides, abasic nucleotides, ribitol, reverse nucleotides, reverse abasic nucleotides, reverse 2′-OMe nucleotides, reverse 2′-deoxynucleotides, 2′-amino modified nucleotides, 2′-alkyl modified nucleotides, morpholino nucleotides and 3′-OMe nucleotides, nucleotides containing 5′-thiophosphate groups, or terminal nucleotides connected to cholesterol derivatives or dodecanoic acid bisdecylamide groups, 2′-amino modified nucleotides, aminophosphorylates, or non-natural bases containing nucleotides.

9. The siRNA according to any one of claims 2 or 8, characterized in that The 5' end of the siRNA sense strand is also connected to two nucleotides complementary to the 3' end of the antisense strand; The fluorinated modified nucleotides are located in the antisense strand and the sense strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the 9th, 10th, and 11th nucleotides of the sense strand are fluorinated modified nucleotides, and at least the 2nd, 10th, 12th, 14th, 16th, 18th, and 20th nucleotides of the antisense strand are fluorinated modified nucleotides; The methoxy-modified nucleotides are located in the antisense strand and the sense strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th and 21st nucleotides of the sense strand are methoxy-modified nucleotides, and at least the 1st, 3rd, 4th, 6th, 8th, 9th, 11th, 13th, 15th, 17th, 19th, 21st nucleotides of the antisense strand are methoxy-modified nucleotides; The nucleotides linked by the phosphorothioate groups are located in the antisense strand and the sense strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 1 and 2, positions 19 and 20, and positions 20 and 21 of the sense strand are linked by the phosphorothioate groups, and at least the nucleotides at positions 1 and 2, positions 2 and 3, positions 19 and 20, and positions 20 and 21 of the antisense strand are linked by the phosphorothioate groups; The 2'-deoxynucleotides are located in the antisense strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the 5th and 7th nucleotides of the antisense strand are 2'-deoxynucleotides.

10. The siRNA according to any one of claims 9, characterized in that In the direction from the 5' end to the 3' end, the 6th position of the siRNA sense strand is connected with a 2'-O-hexadecyl group.

11. The siRNA according to any one of claims 10, characterized in that The 5' end and / or the 3' end of the sense strand of the siRNA comprises more than one inverted abasic residue.

12. A product for inhibiting MUC5AC, characterized in that The product comprises an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is the siRNA according to any one of claims 1 to 7.

13. The product according to claim 12, characterized in that The product is a pharmaceutical composition or a kit.

14. Use of the siRNA of any one of claims 1 to 7 or the product of any one of claims 12 to 13 in the preparation of a product for preventing, diagnosing and / or treating a pathological condition or disease caused by MUC5AC.

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