Application of RNAi agent in treatment of periodontitis and related diseases

Abstract

The present disclosure provides an application of an RNAi agent in the preparation of a drug for treatment and / or prevention of periodontitis and periodontitis-related diseases, the RNAi agent containing an siRNA conjugate and / or a pharmaceutically acceptable salt thereof wherein the siRNA conjugate comprises an siRNA group and a conjugation group covalently linked to the siRNA group, the siRNA group is formed by removing one or more atoms or functional groups from siRNA capable of inhibiting CC3 mRNA, and the conjugation group contains one or more targeting groups having affinity for mammalian asialoglycoprotein receptors (ASGPR). The disclosure also provides a method of treating and / or preventing periodontitis and periodontitis-related diseases comprising administering to a subject in need thereof an effective amount of the RNAi agent described above.

Description

Technical Field

[0001] The present disclosure relates to the use of an RNAi agent in treating and / or preventing periodontitis and related diseases. The present disclosure also relates to a method for treating periodontitis and related diseases using an RNAi agent. Background Art

[0002] Periodontitis is an inflammatory disease that affects the periodontal tissues. It is caused by the toxins secreted by bacteria in dental plaque that accumulate on the gums and the tooth surfaces and cervical margins nearby, which cause inflammation of the periodontal tissues. Common periodontal pathogens are Gram-negative anaerobes such as Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis. Their toxins cause the host to release cytokines such as IL-1β, IL-6, TNF-α, and PGE2, leading to the formation of periodontal pockets and inflammation of the pocket walls, alveolar bone absorption, and separation of the gums from the teeth, and gradual loosening or loss of teeth. Periodontitis is the main cause of tooth loss in adults. Literature reports that periodontitis is associated with an increased risk of diabetes, stroke, myocardial infarction, atherosclerosis, hypertension, osteoporosis, and premature birth, and is also associated with the occurrence of colorectal cancer and pancreatic cancer.

[0003] Porphyromonas gingivalis, the major pathogen implicated in periodontitis, inhibits the complement cascade, which normally converges on the third complement component (complement C3, or CC3) and leads to the production of effector molecules that mediate the recruitment and activation of inflammatory cells through the anaphylatoxins C3a and C5a, microbial opsonization and cellular phagocytosis through opsonins such as C3b, and direct lysis of target microorganisms through the C5b-9 membrane attack complex.

[0004] CN104640567A discloses a method for treating or preventing periodontitis or a disease associated with periodontitis in an individual, the method comprising administering to the individual a compound that inhibits or blocks the expression, activity or activation of C3. However, the method uses a local injection method in the gums, and the administration frequency is high, which may cause greater pain to the subject and a decrease in the bioavailability of the drug in the oral cavity due to dilution by saliva. Summary of the invention

[0005] The present disclosure provides an application of an RNAi agent in the preparation of a drug for treating and / or preventing periodontitis and periodontitis-related diseases, wherein the RNAi agent contains an siRNA conjugate and / or a pharmaceutically acceptable salt thereof, wherein the siRNA conjugate comprises an siRNA group and a conjugated group covalently linked to the siRNA group, the siRNA group is formed by removing one or more atoms or functional groups from an siRNA capable of inhibiting CC3 mRNA, and the conjugated group contains one or more targeting groups having affinity for mammalian asialoglycoprotein receptor (ASGPR).

[0006] In another aspect, the present disclosure also provides a method for treating and / or preventing periodontitis and periodontitis-related diseases, comprising administering an effective amount of the above-mentioned RNAi agent to a subject in need thereof.

[0007] Incorporated by Reference

[0008] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0009] Beneficial Effects

[0010] The RNAi agent described in the application or method provided by the present disclosure has a significant inhibitory efficiency on CC3 mRNA in liver tissue and CC3 protein expression in vivo. By administering the RNAi agent described in the application or method provided by the present disclosure, periodontitis and its symptoms can be effectively prevented, treated or alleviated, and diseases related to periodontitis can be treated and / or prevented.

[0011] First, the RNAi agent described in the application or method provided by the present disclosure can effectively inhibit CC3 mRNA and exhibit excellent inhibitory activity.

[0012] For example, on the 7th day after a single subcutaneous administration, under the condition of a dosage of 3 mg / kg, the siRNA conjugate described in the application or method provided by the present disclosure can achieve an inhibition rate of 74.09% on CC3 mRNA in rats, or even 87.44%. Under the condition of a dosage of 9 mg / kg, the siRNA conjugate described in the application or method provided by the present disclosure can achieve an inhibition rate of 92.19% on CC3 mRNA in rats, or even 97.19%.

[0013] For another example, on the 7th day after a single subcutaneous administration, under the condition of a dosage of 3 mg / kg, the siRNA conjugate described in the application or method provided by the present disclosure can achieve an inhibition rate of CC3 mRNA in mice by 68.25%, or even 85.67%. Under the condition of a dosage of 9 mg / kg, the siRNA conjugate described in the application or method provided by the present disclosure can achieve an inhibition rate of CC3 mRNA in mice by 81.80%, or even 93.40%.

[0014] For another example, for hC3 transgenic mice, the inhibition rates of CC3 mRNA in mice can reach 79.5% and 93.5% respectively by administering the siRNA conjugates described in the applications or methods provided by the present disclosure at doses of 1 mg / kg and 3 mg / kg, and the expression level of hCC3 protein in mice can be reduced to less than 30% on the 22nd day of administration compared to before administration.

[0015] Second, the application or method may not be limited to a method of administration, and may be administered subcutaneously, and show an efficient and excellent therapeutic effect on periodontitis symptoms. For example, in periodontitis model mice, compared with the negative control group, only subcutaneous administration of the siRNA conjugate described in the application or method provided by the present disclosure can significantly inhibit the alveolar bone resorption of periodontitis model mice, and can inhibit the expression of inflammatory factors in the periodontitis model mice to the same level as normal mice, indicating that the application or method provided by the present disclosure can effectively treat periodontitis.

[0016] Third, the RNAi agent described in the application or method provided by the present disclosure shows lower hepatotoxic reactions due to off-target effects. For rats given high-dose siRNA conjugates up to 100 mg / kg, no obvious liver inflammation was found in all rats, indicating that the siRNA conjugate described in the application or method provided by the present disclosure hardly causes hepatotoxic reactions due to off-target effects, and therefore shows high safety in the preparation of drugs for the treatment and / or prevention of periodontitis and periodontitis-related diseases or symptoms. Therefore, the application or method provided by the present disclosure has excellent development prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figures 1A-1E The figure is a stereomicroscope photograph showing the alveolar bone resorption of mice in a blank control group, a negative control group and after administration of different doses of the siRNA conjugates described in the application or method provided by the present disclosure.

[0018] Figures 2A-2D The bar graphs show the relative expression levels of inflammatory factors IL-17a, IL6, Tnf-a in lymph node tissues and Tnfsf11 mRNA in periodontal tissues of each group of mice. DETAILED DESCRIPTION

[0019] The specific embodiments of the present disclosure are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present disclosure, and are not used to limit the present disclosure.

[0020] In the present disclosure, unless otherwise specified, CC3 mRNA or "mRNA expressed by CC3 gene" refers to mRNA having a sequence as shown in Genbank Accession No. NM_000064.4, and CC3 gene refers to a gene that transcribes the above CC3 mRNA.

[0021] definition

[0022] In the above and below, unless otherwise specified, capital letters C, G, U, and A represent the base composition of nucleotides; lowercase letter m represents that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide; lowercase letter f represents that the nucleotide adjacent to the left of the letter f is a fluorinated-modified nucleotide; lowercase letter s represents that the two nucleotides adjacent to the left and right of the letter s are connected by phosphorothioate subunits; P1 represents that the nucleotide adjacent to the right of P1 is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide, and in some embodiments, P1 is VP, Ps or P representing a specific modification, wherein the letter combination VP represents that the nucleotide adjacent to the right of the letter combination VP is a vinyl phosphate (5'-(E)-vinylphosphonate, E-VP) modified nucleotide, the letter combination Ps represents that the nucleotide adjacent to the right of the letter combination Ps is a phosphorothioate modified nucleotide, and the capital letter P represents that the nucleotide adjacent to the right of the letter P is a 5'-phosphate nucleotide.

[0023] In the above and below, the "fluorinated modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by fluorine, and the "non-fluorinated modified nucleotide" refers to a nucleotide or nucleotide analog in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by a non-fluorinated group. "Nucleotide analog" refers to a group that can replace nucleotides in nucleic acids but has a structure different from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide or thymine deoxyribonucleotide. Such as isonucleotides, bridged nucleic acids (BNA for short) or acyclic nucleotides. The "methoxy-modified nucleotide" refers to a nucleotide in which the 2'-hydroxyl group of the ribose group is replaced by a methoxy group.

[0024] In the above and below, the expressions "complementary" or "reverse complementary" may be 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 chain are paired in the 5'-3' direction with the bases of the other chain in the 3'-5' direction in a manner consistent with the principle of base complementary pairing. In DNA, the purine base adenine (A) is always paired with the pyrimidine base thymine (T) (or uracil (U) in RNA); the purine base guanine (C) is always paired with the pyrimidine base cytosine (G). Each base pair includes a purine and a pyrimidine. When adenine on one chain is always paired with thymine (or uracil) on the other chain, and guanine is always paired with cytosine, the two chains are considered to be complementary to each other, and the sequence of the chain can be inferred from the sequence of its complementary chain. Correspondingly, "mismatch" in the art means that in a double-stranded nucleic acid, bases exist at corresponding positions, but the two bases are not paired in a complementary form.

[0025] In the above and below, unless otherwise specified, "substantially reverse complementary" means that there are no more than 3 base mismatches between the two nucleotide sequences involved; "substantially reverse complementary" means that there are no more than 1 base mismatch between the two nucleotide sequences; and "completely reverse complementary" means that there are no base mismatches between the two nucleotide sequences.

[0026] In the above and below, especially when describing the preparation method of the siRNA conjugate described in the application or method provided by the present disclosure, unless otherwise specified, the nucleoside monomer (nucleoside monomer) refers to the modified or unmodified nucleoside phosphoramidite monomer (unmodified or modified RNA phosphoramidites, sometimes RNA phosphoramidites are also referred to as Nucleoside phosphoramidites) used in phosphoramidite solid phase synthesis according to the type and order of nucleotides in the siRNA conjugate to be prepared. Phosphoramidite solid phase synthesis is a method used in RNA synthesis known to those skilled in the art. Unless otherwise specified, the nucleoside monomers used in the present disclosure are all commercially available.

[0027] In the above or below, "substituted" or "substituted" groups, such as substituted alkyl, substituted alkoxy, substituted amino, substituted aliphatic, substituted heteroaliphatic, substituted acyl, substituted aryl or substituted heteroaryl. Wherein, unless otherwise specified, a "substituted" or "substituted" group refers to a group formed by replacing the hydrogen atoms in the group with one or more substituents. For example, "substituted alkoxy" refers to a group formed by replacing one or more hydrogen atoms in an alkoxy group with a substituent. Those skilled in the art will understand that various substituents may be included in the compounds that can be used in the present disclosure, as long as the introduction of the substituent does not affect the function of the present disclosure and can achieve the purpose of the present disclosure, it can be used in the present disclosure. In some embodiments, the substituent is selected from the group consisting of the following groups: C1-C 10 Alkyl, C6-C 10 Aryl, C5-C 10 Heteroaryl, C1-C 10 Haloalkyl, -OC1-C 10 Alkyl, -OC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-OH, -OC1-C 10 Halogenated alkyl, -SC1-C 10 Alkyl, -SC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-SH, -SC1-C 10 Haloalkyl, halogen substituent, -OH, -SH, -NH2, -C1-C 10 Alkyl-NH2, -N(C1-C 10 Alkyl)(C1-C 10 Alkyl), -NH(C1-C 10 Alkyl), -N(C1-C 10 Alkyl)(C1-C 10 Alkylphenyl), -NH(C1-C 10 alkylphenyl), -CN, -NO2, -CO2H, -C(O)O(C1-C 10 Alkyl), -CON(C1-C 10 Alkyl)(C1-C 10 Alkyl), -CONH(C1-C 10 alkyl), -CONH2, -NHC(O)(C1-C 10 alkyl), -NHC(O)(phenyl), -N(C1-C 10 Alkyl)C(O)(C1-C 10 Alkyl), -N(C1-C 10 alkyl)C(O)(phenyl), -C(O)C1-C10 Alkyl, -C(O)C1-C 10 Alkylphenyl, -C(O)C1-C 10 Haloalkyl, -OC(O)C1-C 10 Alkyl, -SO2(C1-C 10 Alkyl), -SO2(phenyl), -SO2(C1-C 10 haloalkyl), -SO2NH2, -SO2NH(C1-C 10 Alkyl), -SO2NH(phenyl), -NHSO2(C1-C 10 alkyl), -NHSO2(phenyl) and -NHSO2(C1-C 10 In some embodiments, the substituent is one of C1-C3 alkyl, C6-C8 aryl, -OC1-C3 alkyl, -OC1-C3 alkylphenyl, halogen, -OH, -NH2, -CN or -NO2. It will be understood by those skilled in the art that for any group containing one or more substituents, these groups are not intended to introduce any substitution or substitution pattern that is sterically impractical, synthetically infeasible and / or inherently unstable.

[0028] As used herein and context, the terms "alkyl", "aryl", "heteroaryl", "halo", "alkoxy" and the like have meanings well known to those skilled in the art, and those skilled in the art are able to select appropriate corresponding groups as needed, and expect that the siRNA conjugate containing the group has corresponding physical and chemical properties.

[0029] Various hydroxyl protecting groups can be used in the present disclosure. In general, the protecting group makes the chemical functional group insensitive to specific reaction conditions, and can be added and removed on the functional group in the molecule without substantially damaging the rest of the molecule. Representative hydroxyl protecting groups are disclosed in Beaucage et al., Tetrahedron 1992, 48, 2223-2311, and Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2ded, John Wiley & Sons, New York, 1991, and the above-mentioned documents are incorporated herein by reference in their entirety. In some embodiments, the protecting group is stable under alkaline conditions, but can be removed under acidic conditions. In some embodiments, non-exclusive examples of hydroxyl protecting groups that can be used herein include dimethoxytrityl (DMT), monomethoxytrityl, 9-phenyloxanthene-9-yl (Pixyl) and 9-(p-methoxyphenyl)oxanthene-9-yl (Mox). In some embodiments, non-exclusive examples of hydroxy protecting groups that may be used herein include Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4'-dimethoxytrityl), and TMTr (4,4',4"-trimethoxytrityl).

[0030] The term "subject", as used herein, refers to any animal, such as a mammal or marsupial. Subjects of the present disclosure include, but are not limited to, humans, non-human primates (e.g., rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cows, rabbits, sheep, rats, and poultry of any kind.

[0031] As used herein, "treatment" refers to an approach to obtaining beneficial or desired results, including but not limited to a therapeutic benefit. "Therapeutic benefit" means eradication or amelioration of the underlying disorder being treated. In addition, a therapeutic benefit is obtained by eradication or amelioration of 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.

[0032] As used herein, "prevention" refers to a method of obtaining a beneficial or desired result, including but not limited to a preventive benefit. In order to obtain a "preventive benefit", the RNAi agent can be administered to a subject at risk for periodontitis or a periodontitis-related disease, or to a subject reporting one or more physiological symptoms of periodontitis or a periodontitis-related disease, even though a diagnosis of the disease may not have been made. In some embodiments, the physiological symptoms include but are not limited to red and swollen gums, tooth sensitivity to hot and cold, bad breath, bleeding gums, etc.

[0033] In the applications or methods provided by the present disclosure, in some embodiments, all of the pharmaceutically active components are the siRNA conjugates, and in some embodiments, part or all of the pharmaceutically active components are pharmaceutically acceptable salts of the siRNA conjugates. In the siRNA conjugates, each adjacent nucleotide is connected by a phosphodiester bond or a phosphorothioate diester bond, and the non-bridging oxygen atoms or sulfur atoms in the phosphodiester bond or the phosphorothioate diester bond exist in the form of hydroxyl groups or sulfhydryl groups. The pharmaceutically acceptable salt of the siRNA conjugate refers to a salt or partial salt compound formed by partially or completely replacing the hydrogen ions in the hydroxyl groups or sulfhydryl groups connected to the non-bridging oxygen atoms or sulfur atoms in the above-mentioned phosphodiester bonds or phosphorothioate diester bonds with cations. The cation can be any pharmaceutically acceptable cation, such as a metal cation, an ammonium ion NH4 + , one of the organic ammonium cations. In order to improve solubility, in some embodiments, the cation is selected from one or more of an alkali metal ion, an ammonium cation formed by a tertiary amine, and a quaternary ammonium cation. The alkali metal cation can be K + and / or Na + , the cation formed by the tertiary amine can be an ammonium ion formed by triethylamine and / or an ammonium ion formed by N,N-diisopropylethylamine. Therefore, in the RNA agent provided by the present disclosure, part or all of the pharmaceutically active components are water-soluble salts of siRNA conjugates, such as one of alkali metal salts or ammonium salts. In some embodiments, the pharmaceutically acceptable salt of the siRNA conjugate refers to the sodium salt or partial sodium salt of the siRNA conjugate. Unless otherwise specified, in the context, when the siRNA conjugate described in the application or method provided by the present disclosure is mentioned separately, including but not limited to the siRNA conjugate represented by any structural formula described in the application or method provided by the present disclosure, it also refers to the pharmaceutically acceptable salt of the siRNA conjugate according to the context.

[0034] Applications of RNAi Agents

[0035] In one aspect, the present disclosure provides an application of an RNAi agent in the preparation of a drug for treating and / or preventing periodontitis and periodontitis-related diseases, wherein the RNAi agent contains a pharmaceutically active component, which is an siRNA conjugate and / or a pharmaceutically acceptable salt thereof, wherein the siRNA conjugate comprises an siRNA group and a conjugated group covalently linked to the siRNA group, wherein the siRNA group is formed by removing one or more atoms or functional groups from an siRNA capable of inhibiting CC3 mRNA, and the conjugated group contains one or more targeting groups having affinity for mammalian asialoglycoprotein receptor (ASGPR).

[0036] The siRNA group in the siRNA conjugate described in the application or method provided by the present disclosure contains a nucleotide group as a basic structural unit, and the nucleotide group contains a phosphate group, a ribose group and a base, and the ribose group forms a ribose ring, and is connected to the adjacent nucleotide group in the oligonucleotide single strand (sense strand or antisense strand) through a phosphate group at the 5' and 3' positions of the ribose ring, respectively, and is connected to the base at the 1' position of the ribose ring. The above structure is well known to those skilled in the art and will not be repeated here.

[0037] siRNA conjugates

[0038] The RNAi agent suitable for the application of the present disclosure contains a pharmaceutically active component, which is a siRNA conjugate and / or a pharmaceutically acceptable salt thereof, wherein the siRNA conjugate comprises a siRNA group and a conjugated group covalently linked to the siRNA group, the siRNA group is formed by removing one or more atoms or functional groups from a siRNA capable of inhibiting CC3 mRNA, and the conjugated group contains one or more targeting groups having affinity for mammalian asialoglycoprotein receptor (ASGPR).

[0039] In some embodiments, in the siRNA conjugates described in the applications or methods provided by the present disclosure, the siRNA group comprises a sense strand and an antisense strand, the sense strand and the antisense strand are at least partially reverse complementary to form a double-stranded region, each nucleotide in the sense strand and the antisense strand is independently a modified or unmodified nucleotide, and the antisense strand comprises a nucleotide sequence that is completely reverse complementary to at least 15 consecutive nucleotides in CC3 mRNA. In some embodiments, the antisense strand comprises a nucleotide sequence that is completely reverse complementary to at least 17, 18 or 19 consecutive nucleotides in CC3 mRNA. In some embodiments, the antisense strand comprises a nucleotide sequence that is completely reverse complementary to at least 15 consecutive nucleotides in CC3 mRNA, and at least 19 consecutive nucleotides are substantially reverse complementary.

[0040] In some embodiments, in the siRNA group, the sense strand comprises a nucleotide sequence I, and the antisense strand comprises a nucleotide sequence II. The length of the nucleotide sequence I and the nucleotide sequence II are both 19 nucleotides, and the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region, and the nucleotide sequence II is completely reverse complementary to at least 15 consecutive nucleotides of the first nucleotide sequence, and the first nucleotide sequence is a nucleotide sequence of 19 nucleotides in length in CC3 mRNA. In some embodiments, the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary or completely reverse complementary, and the nucleotide sequence II is completely reverse complementary to 16, 17, 18 or 19 consecutive nucleotides in the first nucleotide sequence.

[0041] In some embodiments, in the siRNA group:

[0042] (1) the sense strand further comprises nucleotide sequence III, and the antisense strand nucleotide sequence further comprises nucleotide sequence IV; and / or

[0043] (2) the sense strand further comprises nucleotide sequence VI; and / or

[0044] (3) The antisense strand further contains nucleotide sequence V.

[0045] Wherein, the nucleotide sequence III and the nucleotide sequence IV are respectively composed of 1-4 nucleotides, the nucleotide sequence IV and the nucleotide sequence III are equal in length and are substantially reverse complementary or completely reverse complementary, the nucleotide sequence III is connected to the 5' end of the nucleotide sequence I, the nucleotide sequence IV is connected to the 3' end of the nucleotide sequence II, and the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to the second segment of nucleotide sequence, and the second segment of nucleotide sequence refers to a nucleotide sequence in CC3 mRNA that is adjacent to the 5' end of the first segment of nucleotide sequence and has the same length as the nucleotide sequence IV.

[0046] The nucleotide sequence V and the nucleotide sequence VI are each independently composed of 1-3 nucleotides, the nucleotide sequence V is connected to the 3' end of the nucleotide sequence II or the nucleotide sequence IV, and the nucleotide sequence VI is connected to the 3' end of the nucleotide sequence I or the nucleotide sequence III, respectively constituting the 3' overhang of the antisense strand or the sense strand.

[0047] In some embodiments, the sense strand consists of nucleotide sequence I, and the antisense strand consists of nucleotide sequence II and nucleotide sequence V.

[0048] In some embodiments, the first nucleotide sequence is the nucleotide sequence shown in SEQ ID NO: 1, and there are no more than 3 nucleotide differences between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 1; the nucleotide sequence II is completely reverse complementary to the nucleotide sequence shown in SEQ ID NO: 1 in 16-19 consecutive nucleotides:

[0049] 5'-UCAUGGUGUUCGUGACGAA-3' (SEQ ID NO: 1);

[0050] Alternatively, the first nucleotide sequence is the nucleotide sequence shown in SEQ ID NO:46, and there are no more than 3 nucleotide differences between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:46; and the nucleotide sequence II is completely reverse complementary to the nucleotide sequence shown in SEQ ID NO:46 in 16-19 consecutive nucleotides:

[0051] 5'-CCUACGUGGUCAAGGUCUU-3' (SEQ ID NO: 46);

[0052] Alternatively, the first nucleotide sequence is the nucleotide sequence shown in SEQ ID NO: 91, and there are no more than 3 nucleotide differences between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 91; and the nucleotide sequence II is completely reverse complementary to the nucleotide sequence shown in SEQ ID NO: 91 in 16-19 consecutive nucleotides:

[0053] 5'-UGGCCCUACUGCAGCUAA A -3' (SEQ ID NO:91);

[0054] Alternatively, the first nucleotide sequence is the nucleotide sequence shown in SEQ ID NO: 136, and there are no more than 3 nucleotide differences between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 136; and the nucleotide sequence II is completely reverse complementary to the nucleotide sequence shown in SEQ ID NO: 136 in a continuous 16-19 nucleotide sequence:

[0055] 5'-GGAACACCCUCAUCAUCUA-3' (SEQ ID NO: 136).

[0057] In some embodiments, in the direction from the 5' end to the 3' end, the nucleotides at positions 3-19 of the nucleotide sequence II are completely reverse complementary to the nucleotides at positions 1-17 of the first nucleotide sequence.

[0058] In some embodiments, the nucleotide sequence II is substantially reverse complementary, substantially reverse complementary, or completely reverse complementary to the first nucleotide sequence; the substantially reverse complementary means that there are no more than 3 base mismatches between the two nucleotide sequences; the substantially reverse complementary means that there are no more than 1 base mismatches between the two nucleotide sequences; the completely reverse complementary means that there are no mismatches between the two nucleotide sequences. In some embodiments, in the direction from the 5' end to the 3' end, the nucleotides 3-19 of the nucleotide sequence II are completely reverse complementary to the nucleotides 1-17 of the first nucleotide sequence.

[0059] In some embodiments, the nucleotide sequence II is completely reverse complementary to the nucleotide sequence I; or, in the direction from the 5' end to the 3' end, the nucleotides at positions 2-19 of the nucleotide sequence II are completely reverse complementary to the nucleotides at positions 1-18 of the first nucleotide sequence; or, in the direction from the 5' end to the 3' end, the nucleotides at positions 1 and 3-19 of the nucleotide sequence II are completely reverse complementary to the nucleotides at positions 1-17 and 19 of the first nucleotide sequence, that is, in the direction from the 5' end to the 3' end, there is a base mismatch between the second nucleotide in the nucleotide sequence II and the second nucleotide in the nucleotide sequence I in the direction from the 3' end to the 5' end.

[0060] In some embodiments, the nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6, and the nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO:7:

[0061] 5'-UCAUGGUGUUCGUGACGGZ3-3' (SEQ ID NO: 3);

[0062] 5'-UCAUGGUGUUCGUGACGAZ3-3' (SEQ ID NO:4);

[0063] 5'-UCAUGGUGUUCGUGACGUZ3-3' (SEQ ID NO:5);

[0064] 5'-UCAUGGUGUUCGUGACGCZ3-3' (SEQ ID NO: 6);

[0065] 5'-Z4UCGUCACGAACACCAUGA-3'(SEQ ID NO:7),

[0066] wherein Z3 is selected from A, U, G or C, and Z4 is a nucleotide complementary to Z3;

[0067] Alternatively, the nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50 or SEQ ID NO:51, and the nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO:52:

[0068] 5'-CCUACGUGGUCAAGGUCUZ7-3' (SEQ ID NO: 48);

[0069] 5'-CCUACGUGGUCAAGGUCAZ7-3' (SEQ ID NO: 49);

[0070] 5'-CCUACGUGGUCAAGGUCGZ7-3' (SEQ ID NO: 50);

[0071] 5'-CCUACGUGGUCAAGGUCCZ7-3' (SEQ ID NO: 51);

[0072] 5'-Z8AGACCUUGACCACGUAGG-3'(SEQ ID NO:52),

[0073] wherein Z7 is selected from A, U, G or C, and Z8 is a nucleotide complementary to Z7;

[0074] Alternatively, the nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95 or SEQ ID NO:96, and the nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO:97:

[0075] 5'-UGGCCCUACUGCAGCUAAZ 11 -3' (SEQ ID NO:93);

[0076] 5'-UGGCCCUACUGCAGCUAUZ 11 -3' (SEQ ID NO:94);

[0077] 5'-UGGCCCUACUGCAGCUAGZ 11 -3' (SEQ ID NO:95);

[0078] 5'-UGGCCCUACUGCAGCUACZ 11 -3' (SEQ ID NO:96);

[0079] 5'-Z 12 UUAGCUGCAGUAGGGCCA-3'(SEQ ID NO:97),

[0080] Among them, Z 11 Selected from A, U, G or C, Z 12 Yes and Z 11 complementary nucleotides;

[0081] Alternatively, the nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140 or SEQ ID NO: 141, and the nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO: 142:

[0082] 5'-GGAACACCCUCAUCAUCUZ 15 -3' (SEQ ID NO: 138);

[0083] 5'-GGAACACCCUCAUCAUCAZ 15 -3' (SEQ ID NO: 139);

[0084] 5'-GGAACACCCUCAUCAUCGZ 15 -3' (SEQ ID NO: 140);

[0085] 5'-GGAACACCCUCAUCAUCCZ 15 -3' (SEQ ID NO: 141);

[0086] 5'-Z 16 AGAUGAUGAGGGUGUUCC-3'(SEQ ID NO:142),

[0087] Among them, Z 15 Selected from A, U, G or C, Z 16 Yes and Z 15 Complementary nucleotides.

[0088] In some embodiments, Z3 is A and Z4 is U; or Z7 is U and Z8 is A; or Z 11 A, Z 12 U; or Z 15 A, Z 16 For U.

[0089] In some embodiments, the sense strand and antisense strand are the same or different in length, the sense strand is 19-23 nucleotides long, and the antisense strand is 19-26 nucleotides long.

[0090] In the siRNA group of the siRNA conjugate described in the application or method provided by the present disclosure, the nucleotide sequence III and the nucleotide sequence IV are respectively composed of 1-4 nucleotides, the nucleotide sequence IV and the nucleotide sequence III are equal in length, and the nucleotide sequence IV and the nucleotide sequence III are substantially reverse complementary or completely reverse complementary, the nucleotide sequence III is connected to the 5' end of the nucleotide sequence I, the nucleotide sequence IV is connected to the 3' end of the nucleotide sequence II, and the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to the second segment of nucleotide sequence, and the second segment of nucleotide sequence refers to a nucleotide sequence in CC3 mRNA that is adjacent to the 5' end of the first segment of nucleotide sequence and has the same length as the nucleotide sequence IV. Those skilled in the art know that based on the action principle of RNAi, relative to nucleotide sequences I and II, the presence of nucleotide sequences III and IV will not significantly affect the activity and stability of siRNA, and the selection and determination of nucleotide sequences III and IV are also achievable by those skilled in the art.

[0091] In some embodiments, the first nucleotide sequence is the nucleotide sequence shown in SEQ ID NO: 1, and there are no more than 3 nucleotide differences between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 1; the nucleotide sequence II is identical to the nucleotide sequence shown in SEQ ID NO: 2 and 16 to 19 consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO: 2:

[0092] 5'-UCAUGGUGUUCGUGACGAA-3' (SEQ ID NO: 1);

[0093] 5'-UUCGUCACGAACACCAUGA-3'(SEQ ID NO:2),

[0094] The length of the nucleotide sequences III and IV is 1 nucleotide each, the base of the nucleotide sequence III is C, and the base of the nucleotide sequence IV is G; or, the length of the nucleotide sequences III and IV is 2 nucleotides each, the base composition of the nucleotide sequence III is CC, and the base composition of the nucleotide sequence IV is GG; or, the length of the nucleotide sequences III and IV is 3 nucleotides each, the base composition of the nucleotide sequence III is ACC, and the base composition of the nucleotide sequence IV is GGU; or, the length of the nucleotide sequences III and IV is 4 nucleotides each, the base composition of the nucleotide sequence III is GACC, and the base composition of the nucleotide sequence IV is GGUC; or

[0095] The first nucleotide sequence is the nucleotide sequence shown in SEQ ID NO:46, and there are no more than 3 nucleotide differences between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:46; the nucleotide sequence II is completely reverse complementary to the nucleotide sequence shown in SEQ ID NO:47 for 16 to 19 consecutive nucleotides:

[0096] 5'-CCUACGUGGUCAAGGUCUU-3' (SEQ ID NO: 46);

[0097] 5'-AAGACCUUGACCACGUAGG-3'(SEQ ID NO:47),

[0098] The length of the nucleotide sequences III and IV is 1 nucleotide each, the base of the nucleotide sequence III is G, and the base of the nucleotide sequence IV is C; or, the length of the nucleotide sequences III and IV is 2 nucleotides each, the base composition of the nucleotide sequence III is CG, and the base composition of the nucleotide sequence IV is CG; or, the length of the nucleotide sequences III and IV is 3 nucleotides each, the base composition of the nucleotide sequence III is CCG, and the base composition of the nucleotide sequence IV is CGG; or, the length of the nucleotide sequences III and IV is 4 nucleotides each, the base composition of the nucleotide sequence III is ACCG, and the base composition of the nucleotide sequence IV is CGGU; or

[0099] The first nucleotide sequence is the nucleotide sequence shown in SEQ ID NO:91, and there are no more than 3 nucleotide differences between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:91; the nucleotide sequence II is completely reverse complementary to the nucleotide sequence shown in SEQ ID NO:92 for 16 to 19 consecutive nucleotides:

[0100] 5'-UGGCCCUACUGCAGCUAAA-3' (SEQ ID NO:91);

[0101] 5'-UUUAGCUGCAGUAGGGCCA-3'(SEQ ID NO:92),

[0102] The length of the nucleotide sequences III and IV is 1 nucleotide each, the base of the nucleotide sequence III is U, and the base of the nucleotide sequence IV is A; or, the length of the nucleotide sequences III and IV is 2 nucleotides each, the base composition of the nucleotide sequence III is CU, and the base composition of the nucleotide sequence IV is AG; or, the length of the nucleotide sequences III and IV is 3 nucleotides each, the base composition of the nucleotide sequence III is UCU, and the base composition of the nucleotide sequence IV is AGA; or, the length of the nucleotide sequences III and IV is 4 nucleotides each, the base composition of the nucleotide sequence III is CUCU, and the base composition of the nucleotide sequence IV is AGAG; or

[0103] The first nucleotide sequence is the nucleotide sequence shown in SEQ ID NO: 136, and there are no more than 3 nucleotide differences between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 136; the nucleotide sequence II is completely reverse complementary to the nucleotide sequence shown in SEQ ID NO: 137 in 16-19 consecutive nucleotides:

[0104] 5'-GGAACACCCUCAUCAUCUA-3' (SEQ ID NO: 136);

[0105] 5'-UAGAUGAUGAGGGUGUUCC-3'(SEQ ID NO:137),

[0106] The length of the nucleotide sequences III and IV is 1 nucleotide each, the base of the nucleotide sequence III is G, and the base of the nucleotide sequence IV is C; or, the length of the nucleotide sequences III and IV is 2 nucleotides each, the base composition of the nucleotide sequence III is UG, and the base composition of the nucleotide sequence IV is CA; or, the length of the nucleotide sequences III and IV is 3 nucleotides each, the base composition of the nucleotide sequence III is CUG, and the base composition of the nucleotide sequence IV is CAG; or, the length of the nucleotide sequences III and IV is 4 nucleotides each, the base composition of the nucleotide sequence III is ACUG, and the base composition of the nucleotide sequence IV is CAGU.

[0107] In the siRNA group of the siRNA conjugate described in the application or method provided by the present disclosure, the nucleotide sequence V and the nucleotide sequence VI are each independently composed of 1-3 nucleotides, and are independently connected to the 3' end of the antisense strand or the sense strand, thereby forming the 3' overhang of the antisense strand or the sense strand. Those skilled in the art know that the overhang generally may play a role in improving the stability of the siRNA, thereby maintaining the activity of the siRNA, and the length and selection of the overhang are also achievable by those skilled in the art.

[0108] In some embodiments, the nucleotide sequence V consists of 2 nucleotides, and in the direction from the 5' end to the 3' end, the nucleotide sequence V is 2 consecutive thymine deoxyribonucleotides, 2 consecutive uracil ribonucleotides, or is completely reverse complementary to the third nucleotide sequence, and the third nucleotide sequence refers to a nucleotide sequence in CC3 mRNA that is adjacent to the 5' end of the first nucleotide sequence or the 5' end of the second nucleotide sequence and has a length of 2 nucleotides. In some embodiments, the nucleotide sequence V consists of 2 nucleotides, and in the direction from the 5' end to the 3' end, the nucleotide sequence V is 2 consecutive thymine deoxyribonucleotides, 2 consecutive uracil ribonucleotides, or is the same as the fourth nucleotide sequence, and the fourth nucleotide sequence refers to a nucleotide sequence in CC3 mRNA that is adjacent to the 3' end of the first nucleotide sequence and has a length of 2 nucleotides.

[0109] In some embodiments, in the siRNA group of the siRNA conjugate described in the applications or methods provided by the present disclosure, the sense strand contains the nucleotide sequence shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO: 8:

[0110] 5'-UCAUGGUGUUCGUGACGGZ3-3' (SEQ ID NO: 3);

[0111] 5'-UCAUGGUGUUCGUGACGAZ3-3' (SEQ ID NO:4);

[0112] 5'-UCAUGGUGUUCGUGACGUZ3-3' (SEQ ID NO:5);

[0113] 5'-UCAUGGUGUUCGUGACGCZ3-3' (SEQ ID NO: 6);

[0114] 5'-Z4UCGUCACGAACACCAUGAGG-3'(SEQ ID NO:8),

[0115] wherein Z3 is selected from A, U, G or C, and Z4 is a nucleotide complementary to Z3;

[0116] Alternatively, the sense strand contains the nucleotide sequence shown in SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO:13:

[0117] 5'-CCUCAUGGUGUUCGUGACGGZ3-3' (SEQ ID NO:9);

[0118] 5'-CCUCAUGGUGUUCGUGACGAZ3-3' (SEQ ID NO: 10);

[0119] 5'-CCUCAUGGUGUUCGUGACGUZ3-3' (SEQ ID NO: 11);

[0120] 5'-CCUCAUGGUGUUCGUGACGCZ3-3' (SEQ ID NO: 12);

[0121] 5'-Z4UCGUCACGAACACCAUGAGGUC-3'(SEQ ID NO:13),

[0122] wherein Z3 is selected from A, U, G or C, and Z4 is a nucleotide complementary to Z3;

[0123] Alternatively, the sense strand contains the nucleotide sequence shown in SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50 or SEQ ID NO:51, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO:53:

[0124] 5'-CCUACGUGGUCAAGGUCUZ7-3' (SEQ ID NO: 48);

[0125] 5'-CCUACGUGGUCAAGGUCAZ7-3' (SEQ ID NO: 49);

[0126] 5'-CCUACGUGGUCAAGGUCGZ7-3' (SEQ ID NO: 50);

[0127] 5'-CCUACGUGGUCAAGGUCCZ7-3' (SEQ ID NO: 51);

[0128] 5'-Z8AGACCUUGACCACGUAGGCG-3'(SEQ ID NO:53),

[0129] wherein Z7 is selected from A, U, G or C, and Z8 is a nucleotide complementary to Z7;

[0130] Alternatively, the sense strand contains the nucleotide sequence shown in SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56 or SEQ ID NO:57, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO:58:

[0131] 5'-CGCCUACGUGGUCAAGGUCUZ7-3' (SEQ ID NO:54);

[0132] 5'-CGCCUACGUGGUCAAGGUCAZ7-3' (SEQ ID NO:55);

[0133] 5'-CGCCUACGUGGUCAAGGUCGZ7-3' (SEQ ID NO: 56);

[0134] 5'-CGCCUACGUGGUCAAGGUCCZ7-3' (SEQ ID NO: 57);

[0135] 5'-Z8AGACCUUGACCACGUAGGCGGU-3'(SEQ ID NO:58),

[0136] wherein Z7 is selected from A, U, G or C, and Z8 is a nucleotide complementary to Z7;

[0137] Alternatively, the sense strand contains the nucleotide sequence shown in SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95 or SEQ ID NO:96, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO:98:

[0138] 5'-UGGCCCUACUGCAGCUAAZ 11 -3' (SEQ ID NO:93);

[0139] 5'-UGGCCCUACUGCAGCUAUZ 11 -3' (SEQ ID NO:94);

[0140] 5'-UGGCCCUACUGCAGCUAGZ 11 -3' (SEQ ID NO:95);

[0141] 5'-UGGCCCUACUGCAGCUACZ 11 -3' (SEQ ID NO:96);

[0142] 5'-Z 12 UUAGCUGCAGUAGGGCCAAG-3'(SEQ ID NO:98),

[0143] Among them, Z 11 is selected from A, U, G or C, and Z 12 Yes and Z 11 complementary nucleotides;

[0144] Alternatively, the sense strand contains the nucleotide sequence shown in SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101 or SEQ ID NO:102, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO:103:

[0145] 5'-CUUGGCCCUACUGCAGCUAAZ 11 -3' (SEQ ID NO:99);

[0146] 5'-CUUGGCCCUACUGCAGCUAUZ 11 -3' (SEQ ID NO: 100);

[0147] 5'-CUUGGCCCUACUGCAGCUAGZ 11 -3' (SEQ ID NO: 101);

[0148] 5'-CUUGGCCCUACUGCAGCUACZ 11 -3' (SEQ ID NO: 102);

[0149] 5'-Z 12 UUAGCUGCAGUAGGGCCAAGAG-3'(SEQ ID NO:103),

[0150] Among them, Z 11 is selected from A, U, G or C, and Z 12 Yes and Z 11 complementary nucleotides;

[0151] Alternatively, the sense strand contains the nucleotide sequence shown in SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140 or SEQ ID NO: 141, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO: 143:

[0152] 5'-GGAACACCCUCAUCAUCUZ 15 -3' (SEQ ID NO: 138);

[0153] 5'-GGAACACCCUCAUCAUCAZ 15 -3' (SEQ ID NO: 139);

[0154] 5'-GGAACACCCUCAUCAUCGZ 15 -3' (SEQ ID NO: 140);

[0155] 5'-GGAACACCCUCAUCAUCCZ 15 -3' (SEQ ID NO: 141);

[0156] 5'-Z 16 AGAUGAUGAGGGUGUUCCUA-3'(SEQ ID NO:143),

[0157] Among them, Z 15 is selected from A, U, G or C, and Z 16 Yes and Z 15 complementary nucleotides;

[0158] Alternatively, the sense strand contains the nucleotide sequence shown in SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146 or SEQ ID NO: 147, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO: 148:

[0159] 5'-UAGGAACACCCUCAUCAUCUZ 15 -3' (SEQ ID NO: 144);

[0160] 5'-UAGGAACACCCUCAUCAUCAZ 15 -3' (SEQ ID NO: 145);

[0161] 5'-UAGGAACACCCUCAUCAUCGZ 15 -3' (SEQ ID NO: 146);

[0162] 5'-UAGGAACACCCUCAUCAUCCZ 15 -3' (SEQ ID NO: 147);

[0163] 5'-Z 16 AGAUGAUGAGGGUGUUCCUAUC-3'(SEQ ID NO:148),

[0164] Among them, Z 15 is selected from A, U, G or C, and Z 16 Yes and Z 15 Complementary nucleotides.

[0165] As mentioned above, in the siRNA group of the siRNA conjugate described in the application or method provided by the present disclosure, each nucleotide group is independently a modified or unmodified nucleotide group. Common modification methods of nucleotide groups are known to those skilled in the art, including but not limited to: ribose ring modification, phosphate group modification, base modification, etc. In the context of the present disclosure, nucleotide analog groups are also treated as modified nucleotide groups. In some embodiments, some or all of the nucleotide groups in the siRNA of the present disclosure are modified nucleotide groups. In some embodiments, at most 2 nucleotide groups in the siRNA group are unmodified nucleotide groups, and the remaining nucleotide groups are all modified nucleotide groups. These modifications on the nucleotide groups will not cause the siRNA conjugate described in the application or method provided by the present disclosure to inhibit CC3mRNA. Function is significantly weakened or lost. For example, the modified nucleotide groups disclosed in JKWatts, GF Deleavey, and MJ Damha, Chemically modified siRNA: tools and applications. Drug Discov Today, 2008, 13 (19-20): 842-55 can be selected.

[0166] Nucleotides in which the hydroxyl group at the 2' position of the ribose group is replaced by a non-fluorinated group are well known to those skilled in the art, and these nucleotides can be selected from 2'-alkoxy-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-substituted alkyl-modified nucleotides, 2'-amino-modified nucleotides, 2'-substituted amino-modified nucleotides, and 2'-deoxynucleotides.

[0167] In some embodiments, the 2'-alkoxy modified nucleotide is a ribose 2'-methoxy modified nucleotide (2'-OMe), as shown in formula (8). In some embodiments, the 2'-amino modified nucleotide (2'-NH2) is as shown in formula (9). In some embodiments, the 2'-deoxynucleotide (DNA) is as shown in formula (10):

[0168]

[0169] Nucleotide analogs refer to groups that can replace nucleotides in nucleic acids but have structures different from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides or thymine deoxyribonucleotides. In some embodiments, nucleotide analogs can be isonucleotides, bridged nucleic acids (BNAs) or acyclic nucleotides.

[0170] BNA refers to a constrained or inaccessible nucleotide. BNA may contain a five-membered ring, a six-membered ring, or a seven-membered ring with a "fixed" C3'-endosugar condensed bridge structure. The bridge is usually incorporated into the 2'-, 4'-position of the ribose to provide a 2', 4'-BNA nucleotide. In some embodiments, BNA may be LNA, ENA, cET BNA, etc., wherein LNA is shown in formula (12), ENA is shown in formula (13), and cET BNA is shown in formula (14):

[0171]

[0172] Acyclic nucleotides are a type of nucleotides formed by opening the sugar ring of a nucleotide. In some embodiments, the acyclic nucleotide can be an unlocked nucleic acid (UNA) or a glycerol nucleic acid (GNA), wherein UNA is shown in formula (15) and GNA is shown in formula (16):

[0173]

[0174] In the above formulae (15) and (16), R is selected from H, OH or alkoxy (O-alkyl).

[0175] Isonucleotides are compounds formed by a change in the position of a base on the ribose ring of a nucleotide. In some embodiments, an isonucleotide may be a compound formed by a base moving from the 1'-position to the 2'-position or the 3'-position of the ribose ring, as shown in formula (17) or (18).

[0176]

[0177] In the compounds of formula (17)-(18) above, Base represents a nucleic acid base, such as A, U, G, C or T; and R is selected from H, OH, F or a non-fluorine group as described above.

[0178] In some embodiments, the nucleotide analog is selected from one of isonucleotides, LNA, ENA, cET, UNA and GNA. In some embodiments, each non-fluorinated modified nucleotide is a ribose 2'-methoxy modified nucleotide, and in the above and below, the ribose 2'-methoxy modified nucleotide refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribose moiety with a methoxy group.

[0179] In some embodiments, each nucleotide group in the siRNA group is a modified nucleotide group, and in the direction from the 5' end to the 3' end, in the sense strand, the nucleotide groups at positions 7, 8, 9 or positions 5, 7, 8, 9 of the nucleotide sequence I are fluorinated modified nucleotide groups, and the nucleotide groups at the remaining positions in the sense strand are each non-fluorinated modified nucleotide groups; in the antisense strand, the nucleotide groups at positions 2, 6, 14, 16 or positions 2, 6, 8, 9, 14, 16 of the nucleotide sequence II are fluorinated modified nucleotide groups, and the nucleotide groups at the remaining positions in the antisense strand are non-fluorinated modified nucleotide groups;

[0180] Wherein, each of the non-fluorinated modified nucleotide groups is independently selected from one of the nucleotide groups or nucleotide analog groups formed by replacing the hydroxyl group at the 2' position of the ribose group of the nucleotide group with a non-fluorinated group. In some embodiments, at least one or each of the non-fluorinated modified nucleotides is a methoxy-modified nucleotide. In some embodiments, the nucleotide groups at positions 7, 8, and 9 of the nucleotide sequence I are fluorinated modified nucleotide groups, and the nucleotide groups at the remaining positions in the sense strand are each ribose 2'-O-methoxy-modified nucleotide groups; in the antisense strand, the nucleotide groups at positions 2, 6, 14, and 16 of the nucleotide sequence II are fluorinated modified nucleotide groups, and the nucleotide groups at the remaining positions in the antisense strand are each ribose 2'-methoxy-modified nucleotide groups.

[0181] In some embodiments, in the direction from the 5' end to the 3' end, at least one of the 3rd to 5th nucleotide groups of the nucleotide sequence II is a stabilizing modified nucleotide group, and the stabilizing modified nucleotide group refers to a nucleotide group formed by replacing the 2'-hydroxyl group of the ribose of the nucleotide group with a stabilizing modified group, and, compared with the siRNA in which the nucleotide group at the corresponding position is an unmodified nucleotide, the thermal stability of the siRNA containing the stabilizing modified nucleotide group is increased, and the steric hindrance of the stabilizing modified group is greater than that of the methoxy group.

[0182] In some embodiments, except for the stabilizing modified nucleotides, no more than 5 of the non-fluorinated modified nucleotides are ribose 2'-deoxynucleotides, and each of the remaining non-fluorinated modified nucleotides is a methoxy-modified nucleotide; or, except for the stabilizing modified nucleotides, no more than 3 of the non-fluorinated modified nucleotides are ribose 2'-deoxynucleotides, and each of the remaining non-fluorinated modified nucleotides is a methoxy-modified nucleotide; or, each of the non-fluorinated modified nucleotides is a methoxy-modified nucleotide. In some embodiments, from the 5' end to the 3' end, no more than 2 nucleotides from the 3rd to 9th nucleotides in the nucleotide sequence II are the stabilizing modified nucleotides. In some embodiments, from the 5' end to the 3' end, the 3rd nucleotide or the 5th nucleotide in the nucleotide sequence II is the stabilizing modified nucleotide.

[0183] In some embodiments, the increased thermal stability of siRNA refers to an increase in the Tm of the siRNA, where Tm is the double-stranded thermal dissociation temperature of the siRNA. In some embodiments, the increased thermal stability of the siRNA refers to an increase in the Tm of the siRNA of at least 0.05°C. In some embodiments, the increased thermal stability of the siRNA refers to an increase in the Tm of the siRNA of 0.1-6°C. In some embodiments, the increased thermal stability of the siRNA refers to an increase in the Tm of the siRNA of 0.5-4°C.

[0184] In some embodiments, each of the stabilizing modifying groups independently has a structure represented by -XR, wherein X is O, NR', S, SiR'2 or absent; R is one of C2-C6 alkyl, substituted C2-C6 alkyl, C6-C8 aryl, and substituted C6-C8 aryl, and each R' is independently one of H, C1-C6 alkyl, substituted C1-C6 alkyl, C6-C8 aryl, and substituted C6-C8 aryl, and the substituted C2-C6 alkyl, substituted C6-C8 aryl, or substituted C1-C6 alkyl refers to a group formed by replacing one or more hydrogen atoms in the C2-C6 alkyl, C6-C8 aryl, or C1-C6 alkyl with a substituent, and the substituent is selected from one or more of the following substituents: C1-C3 alkyl, C6-C8 aryl, C1-C3 alkoxy, halogen, oxysubyl and thiosubyl. In some embodiments, each of the stabilizing modifying groups is independently selected from one of 2'-O-methoxyethyl, 2'-O-allyl, 2'-allyl, 2'-O-2-N-methylamino-2-oxyylideneethyl, 2'-O-2-N,N-dimethylaminoethyl, 2'-O-3-aminopropyl and 2'-O-2,4-dinitrophenyl. In some embodiments, each of the stabilizing modifying groups is 2'-O-methoxyethyl.

[0185] In some embodiments, the siRNA group is a group formed by removing one atom or functional group from one of the following siRNAs in Tables 1a-1d: siCC3a1-M1, siCC3a1-M2, siCC3a2-M1, siCC3a2-M2, siCC3a3-M1, siCC3a3-M2, siCC3a4-M1, siCC3a4-M2, siCC3a5-M1, siCC3a5-M2, siCC3a6-M1, siCC3a6-M2, siCC 3a7-M1, siCC3a7-M2, siCC3a8-M1, siCC3a8-M2, siCC3b1-M1, siCC3b1-M2, siCC3b2-M1, siCC3b2-M2, siCC3b3-M1, siCC3b3-M2, siCC3b4-M1, siCC3b4-M2, siCC3b5-M1, siCC3b5-M2, siCC3b6-M1, siCC3b6-M2, siCC3b7-M1, siCC3b7 -M2, siCC3b8-M1, siCC3b8-M2, siCC3c1-M1, siCC3c1-M2, siCC3c2-M1, siCC3c2-M2, siCC3CC3-M1, siCC3CC3-M2, s iCC3c4-M1, siCC3c4-M2, siCC3c5-M1, siCC3c5-M2, siCC3c6-M1, siCC3c6-M2, siCC3c7-M1, siCC3c7-M2, siCC3c8- M1, siCC3c8-M2, siCC3d1-M1, siCC3d1-M2, siCC3d2-M1, siCC3d2-M2, siCC3d3-M1, siCC3d3-M2, siCC3d4-M1, siCC 3d4-M2, siCC3d5-M1, siCC3d5-M2, siCC3d6-M1, siCC3d6-M2, siCC3d7-M1, siCC3d7-M2, siCC3d8-M1 or siCC3d8-M2.

[0186] In some embodiments, at least a portion of the phosphate groups in the phosphate-sugar backbone of at least one single strand in the sense strand and the antisense strand of the siRNA is a phosphate group with a modified group. In some embodiments, the phosphate group with a modified group is a thiophosphate group formed by replacing at least one oxygen atom in the phosphodiester bond in the phosphate group with a sulfur atom; in some embodiments, the phosphate group with a modified group is a thiophosphate subunit having a structure as shown in formula (1):

[0187]

[0188] This modification can stabilize the double-stranded structure of siRNA and maintain high specificity and high affinity of base pairing.

[0189] In some embodiments, the phosphate group having a modifying group is present in at least one of the group consisting of:

[0190] Between the first nucleotide and the second nucleotide at the 5' end of the sense strand;

[0191] Between the second nucleotide and the third nucleotide at the 5' end of the sense strand;

[0192] Between the first nucleotide and the second nucleotide at the 3' end of the sense strand;

[0193] Between the second nucleotide and the third nucleotide at the 3' end of the sense strand;

[0194] Between the first nucleotide and the second nucleotide at the 5' end of the antisense strand;

[0195] Between the second nucleotide and the third nucleotide at the 5' end of the antisense strand;

[0196] between the first nucleotide and the second nucleotide at the 3' end of the antisense strand; and

[0197] The 3' end of the antisense strand is between the second nucleotide and the third nucleotide.

[0198] In some embodiments, the siRNA group is a group formed by removing one atom or functional group from one of the following siRNAs in Tables 1a-1d: siCC3a1-M1S, siCC3a1-M2S, siCC3a2-M1S, siCC3a2-M2S, siCC3a3-M1S, siCC3a3-M2S, siCC3a4-M1S, siCC3a4-M2S, siCC3a5-M1S, siCC3a5-M2S, siCC3a6-M1S, siCC3a6-M2S, siCC3a7- M1S, siCC3a7-M2S, siCC3a8-M1S, siCC3a8-M2S, siCC3b1-M1S, siCC3b1-M2S, siCC3b2-M1S, siCC3b2-M2S, siCC3b3-M1S, siC C3b3-M2S, siCC3b4-M1S, siCC3b4-M2S, siCC3b5-M1S, siCC3b5-M2S, siCC3b6-M1S, siCC3b6-M2S, siCC3b7-M1S, siCC3b7-M2 S, siCC3b8-M1S, siCC3b8-M2S, siCC3c1-M1S, siCC3c1-M2S, siCC3c2-M1S, siCC3c2-M2S, siCC3CC3-M1S, siCC3CC3-M2S, siC C3c4-M1S, siCC3c4-M2S, siCC3c5-M1S, siCC3c5-M2S, siCC3c6-M1S, siCC3c6-M2S, siCC3c7-M1S, siCC3c7-M2S, siCC3c8-M1 S, siCC3c8-M2S, siCC3d1-M1S, siCC3d1-M2S, siCC3d2-M1S, siCC3d2-M2S, siCC3d3-M1S, siCC3d3-M2S, siCC3d4-M1S, siCC3 d4-M2S, siCC3d5-M1S, siCC3d5-M2S, siCC3d6-M1S, siCC3d6-M2S, siCC3d7-M1S, siCC3d7-M2S, siCC3d8-M1S or siCC3d8-M2S.

[0199] In some embodiments, the 5' terminal nucleotide of the antisense strand in the siRNA group is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide.

[0200] The commonly used 5'-phosphate nucleotides or 5'-phosphate analogue modified nucleotides are well known to those skilled in the art, such as 5'-phosphate nucleotides may have the following structure:

[0201]

[0202] For example, Anastasia Khvorova and Jonathan K. Watts, The chemical evolution of oligonucleotide therapies of clinical utility. Nature Biotechnology, 2017, 35(3): 238-48 disclose the following four 5'-phosphate analog modified nucleotides:

[0203]

[0204] Wherein, R is selected from H, OH, methoxy, and fluorine; Base represents a nucleic acid base, selected from A, U, C, G, or T.

[0205] In some embodiments, the 5'-phosphate nucleotide is a nucleotide containing a 5'-phosphate modification as shown in formula (2), and the 5'-phosphate analog modified nucleotide is a nucleotide containing a vinyl phosphate (5'-(E)-vinylphosphonate, E-VP) modification as shown in formula (3), or a nucleotide modified with a thiophosphate as shown in formula (5).

[0206] In some embodiments, the siRNA group is a group formed by removing one atom or functional group from one of the following siRNAs in Tables 1a-1d: siCC3a1-M1P1, siCC3a1-M2P1, siCC3a2-M1P1, siCC3a2-M2P1, siCC3a3-M1P1, siCC3a3-M2P1, siCC3a4-M1P1, siCC3a4-M2P1, siCC3a5-M1P1, siCC3a5-M2P1, siCC3a6-M1P1, siCC3a6-M2P1, siCC3a7-M1P1, siCC3a7-M2P1, siCC3a8-M1P1, siCC3a8-M2P1, siCC3a9-M1P1, siCC3a10-M1P1, siCC3a11-M2P1, siCC3a12-M1P1, siCC3a13-M2P1, siCC3a14-M1P1, siCC3a14-M2P1, siCC3a15-M1P1, siCC3a15-M2P1, siCC3a16-M1P1, siCC3a16-M2P1, siCC3a17-M1P1, siCC3a17-M2P1, siCC3a18-M1P1, siCC3a19-M1P1, siCC3a2 iCC3a8-M2P1, siCC3a1-M1SP1, siCC3a1-M2SP1, siCC3a2-M1SP1, siCC3a2-M2SP1, siCC3a3-M1SP1, siCC3a3-M2SP1, siCC3a4-M1SP1, siCC3a4-M2SP1, siCC3a5-M1SP1, siCC3a5-M2SP1, siCC3a6-M1SP1, siCC3a6-M2SP1, siCC3a7-M1SP1, siCC3a7-M2SP1, siCC3a8-M1SP1, siCC3a8-M2SP1, siCC3b1-M1P1, siCC3b1-M2P1, siCC3b2-M1P1, siCC3b2-M2P1, siCC3b3-M1P1, siCC3b3-M2P1, siCC3b4-M1P1, siCC3b4-M2P1, siCC3b5-M1P1, siCC3b5-M2P1, siCC3b6 -M1P1, siCC3b6-M2P1, siCC3b7-M1P1, siCC3b7-M2P1, siCC3b8-M1P1, siCC3b8-M2P1, siCC3b1-M1SP1, siCC3b1-M2SP1, siCC3b2-M1SP1, siCC3b2-M2SP 1. siCC3b3-M1SP1, siCC3b3-M2SP1, siCC3b4-M1SP1, siCC3b4-M2SP1, siCC3b5-M1SP1, siCC3b5-M2SP1, siCC3b6-M1SP1, siCC3b6-M2SP1, siCC3b7-M1S P1, siCC3b7-M2SP1, siCC3b8-M1SP1, siCC3b8-M2SP1, siCC3c1-M1P1, siCC3c1-M2P1, siCC3c2-M1P1, siCC3c2-M2P1, siCC3CC3-M1P1, siCC3CC3-M2P1,siCC3c4-M1P1, siCC3c4-M2P1, siCC3c5-M1P1, siCC3c5-M2P1, siCC3c6-M1P1, siCC3c6-M2P1, siCC3c7-M1P1, siCC3c7 -M2P1, siCC3c8-M1P1, siCC3c8-M2P1, siCC3c1-M1SP1, siCC3c1-M2SP1, siCC3c2-M1SP1, siCC3c2-M2SP1, siCC3CC3-M1 SP1, siCC3CC3-M2SP1, siCC3c4-M1SP1, siCC3c4-M2SP1, siCC3c5-M1SP1, siCC3c5-M2SP1, siCC3c6-M1SP1, siCC3c6-M 2SP1, siCC3c7-M1SP1, siCC3c7-M2SP1, siCC3c8-M1SP1, siCC3c8-M2SP1, siCC3d1-M1P1, siCC3d1-M2P1, siCC3d2-M1P1 , siCC3d2-M2P1, siCC3d3-M1P1, siCC3d3-M2P1, siCC3d4-M1P1, siCC3d4-M2P1, siCC3d5-M1P1, siCC3d5-M2P1, siCC3d 6-M1P1, siCC3d6-M2P1, siCC3d7-M1P1, siCC3d7-M2P1, siCC3d8-M1P1, siCC3d8-M2P1, siCC3d1-M1SP1, siCC3d1-M2SP1 , siCC3d2-M1SP1, siCC3d2-M2SP1, siCC3d3-M1SP1, siCC3d3-M2SP1, siCC3d4-M1SP1, siCC3d4-M2SP1, siCC3d5-M1SP1, siCC3d5-M2SP1, siCC3d6-M1SP1, siCC3d6-M2SP1, siCC3d7-M1SP1, siCC3d7-M2SP1, siCC3d8-M1SP1 or siCC3d8-M2SP1. ,

[0207] The inventors of the present disclosure have found that the siRNA groups formed by these siRNAs have high CC3 mRNA inhibitory activity, and also have low off-target effects and low hepatotoxic reactions, and are therefore suitable for use in the preparation of drugs for treating and / or preventing periodontitis.

[0208] The above-mentioned siRNA groups can be obtained by conventional siRNA 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. The modified nucleotide groups can be introduced into the siRNA conjugates described in the present disclosure by using nucleoside monomers with corresponding modifications. The methods for preparing nucleoside monomers with corresponding modifications and the methods for introducing modified nucleotide groups into siRNA are also well known to those skilled in the art.

[0209] As mentioned above, the siRNA conjugate described in the application or method provided by the present disclosure contains the above-mentioned siRNA group capable of inhibiting CC3mRNA, and a conjugated group covalently linked to the siRNA group, wherein the conjugated group contains one or more targeting groups having affinity for mammalian asialoglycoprotein receptor (ASGPR). In some embodiments, the conjugated group further comprises a linker, and the siRNA group, the linker and the targeting group are sequentially covalently or non-covalently linked.

[0210] Each of the targeting groups is independently a ligand with affinity to the asialoglycoprotein receptor on the surface of mammalian hepatocytes. In some embodiments, each of the targeting groups is independently an asialoglycoprotein or a sugar. In some embodiments, each of the targeting groups is independently selected from one of D-mannopyranose, L-mannopyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-galactose, and L-galactose. In some embodiments, each targeting group is independently selected from α-D-mannofuranose, β-D-mannofuranose, α-D-mannopyranose, β-D-mannopyranose, α-D-glucopyranose, β-D-glucopyranose, α-D-glucopyranose, β-D-glucopyranose, α-D-glucofuranose, β-D-glucofuranose, α-D-fructofuranose, α-D-fructopyranose, α-D-galactopyranose, β-D-galactopyranose, α-D-galactofuranose, β-D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-butyrylgalactosamine, N-isobutyrylgalactosamine, 2-amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-β-D-pyranose. one of glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-formamido-2,3-di-O-methyl-D-pyranose mannoside, 2-deoxy-2-sulfoamino-D-pyranose glucopyranose, N-glycolyl-α-neuraminic acid, 5-thio-β-D-pyranose glucopyranose, 2,3,4-tri-O-acetyl-1-thio-6-O-trityl-α-D-pyranose glucopyranoside methyl ester, 4-thio-β-D-galactopyranose, 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-pyranose glucopyranose heptanoside ethyl ester, 2,5-anhydro-D-allose nitrile, D-ribose, D-4-thioribose, L-ribose, and L-4-thioribose. In some embodiments, each of the targeting groups having affinity for ASGPR is independently selected from one of asialoglycoprotein, lactose, galactose or N-acetylgalactosamine. In some embodiments, at least one or each of the targeting groups is galactose or N-acetylgalactosamine.

[0211] In the context of the present disclosure, unless otherwise specified, "conjugation" refers to the connection between two or more chemical moieties, each having a specific function, in a covalently linked manner; accordingly, "conjugate" refers to a compound formed by covalently linking the chemical moieties. Further, "siRNA conjugate" means a compound formed by covalently linking one or more chemical moieties having a specific function to a siRNA group. Depending on the context, siRNA conjugates should be understood as a general term for multiple siRNA conjugates or a siRNA conjugate represented by a certain chemical formula. In the context of the present disclosure, "conjugated molecule" should be understood as a specific compound that can be conjugated to a siRNA group through a reaction to ultimately form the siRNA conjugate provided by the present disclosure.

[0212] In some embodiments, the targeting group is 1-6. In some embodiments, the targeting group is 2-4. The conjugation site of the siRNA group and the conjugation group can be at the 3' end or 5' end of the siRNA sense strand, or at the 5' end of the antisense strand, or in the internal sequence of the siRNA. In some embodiments, the conjugation site of the siRNA and the conjugation group is at the 3' end of the siRNA sense strand.

[0213] In some embodiments, the conjugate group can be connected to the phosphate group, 2'-hydroxyl group or base of the nucleotide. In some embodiments, the conjugate group can also be connected to the 3'-hydroxyl group, in which case the nucleotides are connected by a 2'-5' phosphodiester bond. When the conjugate group is connected to the end of the siRNA chain, the conjugate group is usually connected to the phosphate group of the nucleotide; when the conjugate group is connected to the internal sequence of the siRNA, the conjugate group is usually connected to the ribose sugar ring or the base. Various connection methods can be referred to in the literature: Muthiah Manoharan et.al.siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo inhepatocytes.ACS Chemical biology, 2015, 10(5): 1181-7.

[0214] The targeting group with affinity for ASGPR can be connected to the siRNA group via a suitable linker, and a person skilled in the art can select a suitable linker as needed. In some embodiments, the siRNA group and the conjugated group can be connected via an acid-labile or reducible chemical bond, and these chemical bonds can be degraded in the acidic environment of the cell endosome, thereby making the siRNA free. For non-degradable conjugation methods, the conjugated group can be connected to the sense strand of the siRNA, thereby minimizing the effect of the conjugation on the activity of the siRNA.

[0215] In some embodiments, the linker in the siRNA conjugate described in the application or method provided by the present disclosure has a structure as shown in formula (301):

[0216]

[0217] Wherein, k is an integer from 1 to 3;

[0218] L A has a structure including an amide bond as shown in formula (302), L B It has a structure containing N-acylpyrrolidine as shown in formula (303), containing a carbonyl group and an oxygen atom, L C is a linking group based on hydroxymethylaminomethane, dihydroxymethylaminomethane or trishydroxymethylaminomethane;

[0219]

[0220]

[0221] Among them, n 302 ,q 302 and p 302 Each independently represents an integer from 2 to 6, optionally, n 302 ,q 302 and p 302 Each independently is 2 or 3; n 303 An integer between 4 and 16. Optionally, n 303 is an integer between 8 and 12, Indicates the site to which a group is covalently attached.

[0222] In the connector, each L A Each of the targeting groups is connected via an ether bond and L C The oxygen atom of the hydroxyl group in the part is C Partially form an ether bond and connect; L B Through the carbonyl group in formula (303) and L CThe nitrogen atom of the amino group in the moiety forms an amide bond and is connected to the siRNA group through the oxygen atom in formula (303) to form a phosphate bond or a phosphorothioate bond.

[0223] In some embodiments, the siRNA conjugate described in the application or method provided by the present disclosure has a structure as shown in formula (305):

[0224]

[0225] Wherein, Nu represents the siRNA group.

[0226] In some embodiments, the linker in the siRNA conjugate described in the application or method provided by the present disclosure has a structure shown in formula (306):

[0227]

[0228] Among them, n 306 is an integer from 0 to 3, each p 306 are independently an integer from 1 to 6, Indicates the site where the group is covalently attached; the linking group is connected to the targeting group by forming an ether bond through the oxygen atom marked by *; the linking group is connected to the siRNA by forming a phosphate bond or a phosphorothioate bond through at least one of the oxygen atoms marked by #, and the remaining oxygen atoms marked by # are connected to hydrogen atoms to form hydroxyl groups, or are connected to C1-C3 alkyl groups to form C1-C3 alkoxy groups;

[0229] In some embodiments, the siRNA conjugate described in the application or method provided by the present disclosure has a structure as shown in formula (307):

[0230]

[0231] Wherein, Nu represents the siRNA group.

[0232] In some embodiments, the siRNA conjugate described in the application or method provided by the present disclosure has a structure shown in formula (308):

[0233]

[0234]

[0235] in,

[0236] n1 is an integer selected from 1-3, n3 is an integer selected from 0-4;

[0237] Each m1, m2 or m3 is independently an integer selected from 2-10;

[0238] R 10 , R 11 , R 12 , R 13 , R 14 or R 15 Each is independently H, or is selected from the group consisting of: C1-C 10 Alkyl, C1-C 10 Haloalkyl and C1-C 10 Alkoxy;

[0239] R3 has the structure shown in formula A59:

[0240]

[0241] Wherein, E1 is OH, SH or BH2, and Nu represents the above-mentioned siRNA group;

[0242] R2 is a straight chain alkylene group having a length of 1 to 20 carbon atoms, wherein one or more carbon atoms are optionally replaced by any one or more selected from the group consisting of: C(O), NH, O, S, CH=N, S(O)2, C2-C 10 Alkenylene, C2-C 10 Alkynylidene, C6-C 10 Arylene, C3-C 18 Heterocyclylene and C5-C 10 wherein R2 may optionally have any one or more substituents selected from the group consisting of: C1-C 10 Alkyl, C6-C 10 Aryl, C5-C 10 Heteroaryl, C1-C 10 Haloalkyl, -OC1-C 10 Alkyl, -OC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-OH, -OC1-C 10 Halogenated alkyl, -SC1-C 10 Alkyl, -SC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-SH, -SC1-C 10 Haloalkyl, halogen substituent, -OH, -SH, -NH2, -C1-C 10 Alkyl-NH2, -N(C1-C 10 Alkyl)(C1-C 10 Alkyl), -NH(C1-C 10 Alkyl), -N(C1-C 10 Alkyl)(C1-C 10Alkylphenyl), -NH(C1-C 10 alkylphenyl), cyano, nitro, -CO2H, -C(O)O(C1-C 10 Alkyl), -CON(C1-C 10 Alkyl)(C1-C 10 Alkyl), -CONH(C1-C 10 Alkyl), -CON(C1-C 10 Alkyl)(C1-C 10 Alkylphenyl), -CONH(C1-C 10 alkylphenyl), -CONH2, -NHC(O)(C1-C 10 alkyl), -NHC(O)(phenyl), -N(C1-C 10 Alkyl)C(O)(C1-C 10 Alkyl), -N(C1-C 10 alkyl)C(O)(phenyl), -C(O)C1-C 10 Alkyl, -C(O)C1-C 10 Alkylphenyl, -C(O)C1-C 10 Haloalkyl, -OC(O)C1-C 10 Alkyl, -SO2(C1-C 10 Alkyl), -SO2(phenyl), -SO2(C1-C 10 haloalkyl), -SO2NH2, -SO2NH(C1-C 10 Alkyl), -SO2NH(phenyl), -NHSO2(C1-C 10 alkyl), -NHSO2(phenyl) and -NHSO2(C1-C 10 haloalkyl);

[0243] Each L1 is independently a straight chain alkylene group having a length of 1 to 70 carbon atoms, wherein one or more carbon atoms may be optionally replaced by any one or more selected from the group consisting of: C(O), NH, O, S, CH=N, S(O)2, C2-C 10 Alkenylene, C2-C 10 Alkynylidene, C6-C 10 Arylene, C3-C 18 Heterocyclylene and C5-C 10 wherein L1 optionally has any one or more substituents selected from the group consisting of: C1-C 10 Alkyl, C6-C 10 Aryl, C5-C 10 Heteroaryl, C1-C 10 Haloalkyl, -OC1-C 10Alkyl, -OC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-OH, -OC1-C 10 Halogenated alkyl, -SC1-C 10 Alkyl, -SC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-SH, -SC1-C 10 Haloalkyl, halogen substituent, -OH, -SH, -NH2, -C1-C 10 Alkyl-NH2, -N(C1-C 10 Alkyl)(C1-C 10 Alkyl), -NH(C1-C 10 Alkyl), -N(C1-C 10 Alkyl)(C1-C 10 Alkylphenyl), -NH(C1-C 10 alkylphenyl), cyano, nitro, -CO2H, -C(O)O(C1-C 10 Alkyl), -CON(C1-C 10 Alkyl)(C1-C 10 Alkyl), -CONH(C1-C 10 alkyl), -CONH2, -NHC(O)(C1-C 10 alkyl), -NHC(O)(phenyl), -N(C1-C 10 Alkyl)C(O)(C1-C 10 Alkyl), -N(C1-C 10 alkyl)C(O)(phenyl), -C(O)C1-C 10 Alkyl, -C(O)C1-C 10 Alkylphenyl, -C(O)C1-C 10 Haloalkyl, -OC(O)C1-C 10 Alkyl, -SO2(C1-C 10 Alkyl), -SO2(phenyl), -SO2(C1-C 10 haloalkyl), -SO2NH2, -SO2NH(C1-C 10 Alkyl), -SO2NH(phenyl), -NHSO2(C1-C 10 alkyl), -NHSO2(phenyl) and -NHSO2(C1-C 10 haloalkyl);

[0244] represents the site of covalent attachment of a group;

[0245] Each M1 is independently selected from one of the targeting groups having affinity for ASGPR.

[0246] The skilled artisan will appreciate that although L1 is defined as a linear alkylene for convenience, it may not be a linear group or be named differently, such as an amine or alkenyl group resulting from the above substitutions and / or replacements. For the purposes of this disclosure, the length of L1 is the number of atoms in the chain connecting the two points of attachment. For this purpose, a ring resulting from the replacement of a carbon atom of the linear alkylene group (such as a heterocyclylene or heteroarylene) is counted as one atom.

[0247] In some embodiments, n1 can be an integer of 1-3, and n3 can be an integer of 0-4, ensuring that the number of M1 ligands in the conjugate is at least 2; in some embodiments, n1+n3≥2, so that the number of M1 ligands can be at least 3, making it easier for M1 ligands to bind to liver surface asialoglycoprotein receptors, thereby promoting the conjugate to enter cells through endocytosis. Experiments show that when the number of M1 ligands is greater than 3, the ease of binding of M1 ligands to liver surface asialoglycoprotein receptors is not significantly increased. Therefore, considering the ease of synthesis, structure / process cost and delivery efficiency, in some embodiments, n1 is an integer of 1-2, n3 is an integer of 0-1, and n1+n3=2-3.

[0248] In some embodiments, when m1, m2 and m3 are independently selected from integers of 2-10, the spatial positions between multiple M1 ligands can be suitable for the binding of M1 ligands to the asialoglycoprotein receptor on the liver surface. In order to make the above-mentioned siRNA conjugate simpler, easier to synthesize and / or reduce costs, in some embodiments, m1, m2 and m3 are each independently an integer of 2-5. In some embodiments, m1=m2=m3.

[0249] Those skilled in the art will appreciate that when R 10 , R 11 , R 12 , R 13 , R 14 and R 15 Each independently selected from H, C1-C 10 Alkyl, C1-C 10 Haloalkyl, and C1-C 10 When one of the alkoxy groups is present, the properties of the above siRNA conjugates will not be changed, and the purpose of the present disclosure can be achieved. 10 , R 11 , R 12 , R 13 , R 14 and R 15 Each is independently selected from H, methyl and ethyl. 10 , R 11, R 12 , R 13 , R 14 and R 15 Both are H.

[0250] In the siRNA conjugate described in the application or method provided by the present disclosure, R3 is a group with a structure shown in formula A59, wherein E1 is OH, SH or BH2. Based on the consideration of the availability of raw materials for preparation, in some embodiments, E1 is OH or SH.

[0251] In some embodiments, R2 is selected to achieve the connection with the nitrogen atom on the nitrogen-containing skeleton and A59. In the context of the present disclosure, "nitrogen-containing skeleton" refers to a skeleton having R 10 , R 11 , R 12 , R 13 , R 14 and R 15 The carbon atoms and N atoms are connected to each other in a chain structure. Therefore, R2 can be any connecting group that can connect the A59 group to the N atom on the nitrogen-containing skeleton in an appropriate manner. In some embodiments, when the siRNA conjugate described in the application or method provided by the present disclosure is prepared by a solid phase synthesis process, the R2 group needs to contain both a connection site connected to the N atom on the nitrogen-containing skeleton and a connection site connected to the P atom in R3. In some embodiments, the site in R2 that is connected to the N atom on the nitrogen-containing skeleton forms an amide bond with the N atom, and the site that is connected to the P atom on R3 forms a phosphate bond with the P atom. In some embodiments, the length of R2 is 2-20 atoms, or 4-15 atoms. In some embodiments, R2 is B5, B6, B5' or B6':

[0252]

[0253]

[0254] in, Indicates the site of covalent attachment of a group.

[0255] The value range of q2 may be an integer from 1 to 10. In some implementations, q2 is an integer from 1 to 5.

[0256] The role of L1 is to connect the M1 ligand to the N on the nitrogen-containing skeleton, providing a targeting function for the siRNA conjugate described in the application or method provided by the present disclosure. In some embodiments, L1 is selected from one or more connection combinations of the formula A1-A26 groups. In some embodiments, L1 is selected from one or more connection combinations of A1, A4, A5, A6, A8, A10, A11 and A13; in some embodiments, L1 is selected from a connection combination of at least 2 of A1, A4, A8, A10 and A11; in some embodiments, L1 is selected from a connection combination of at least 2 of A1, A8, A10.

[0257]

[0258]

[0259] In some embodiments, L1 can be 3-25 atoms, 3-20 atoms, 4-15 atoms, or 5-12 atoms in length. In some embodiments, L1 is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60 atoms in length.

[0260] In some embodiments, j1 is an integer of 2-10, and in some embodiments, j1 is an integer of 3-5. In some embodiments, j2 is an integer of 2-10, and in some embodiments, j2 is an integer of 3-5. R' is a C1-C4 alkyl group, and in some embodiments, R' is one of a methyl group, an ethyl group, and an isopropyl group. Ra is one of A27, A28, A29, A30, and A31, and in some embodiments, Ra is A27 or A28. Rb is a C1-C5 alkyl group, and in some embodiments, Rb is one of a methyl group, an ethyl group, an isopropyl group, and a butyl group. In some embodiments, j1, j2, R', Ra, and Rb are each selected in formulas A1-A26 to achieve the connection of the M1 ligand to the N on the nitrogen-containing skeleton, and to make the spatial position between the M1 ligands more suitable for the M1 ligand to bind to the liver surface asialoglycoprotein receptor.

[0261]

[0262] In some embodiments, the siRNA conjugates described in the uses or methods provided herein have a structure as shown in formula (403), (404), (405), (406), (407), (408), (409), (410), (411), (412), (413), (414), (415), (416), (417), (418), (419), (420), (421) or (422):

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

[0270] Wherein, Nu represents a siRNA group. In addition, as described above, when the pharmaceutically active component in the RNAi agent is at least partially a pharmaceutically acceptable salt of the conjugate, the hydrogen in the hydroxyl group connected to the P atom in the above formulas (403)-(422) is replaced by a cation, such as an alkali metal ion or an ammonium ion. In some embodiments, the pharmaceutically active component is an alkali metal salt or a partial alkali metal salt of the siRNA conjugate having the structure shown in formula (403).

[0271] In some embodiments, the P atom in formula A59 can be connected to any possible position in the siRNA sequence, for example, the P atom in formula A59 can be connected to any nucleotide of the sense strand or antisense strand of the siRNA; in some embodiments, the P atom in formula A59 is connected to any nucleotide of the sense strand of the siRNA. In some embodiments, the P atom in formula A59 is connected to the end of the sense strand or antisense strand of the siRNA; in some embodiments, the P atom in formula A59 is connected to the end of the sense strand of the siRNA. The end refers to the first 4 nucleotides of the sense strand or the antisense strand from one end thereof. In some embodiments, the P atom in formula A59 is connected to the end of the sense strand or antisense strand of the siRNA; in some embodiments, the P atom in formula A59 is connected to the 3' end of the sense strand of the siRNA. In the case of being connected to the above-mentioned position of the sense strand of the siRNA, after the above-mentioned siRNA conjugate enters the cell, when unwinding, a separate siRNA antisense strand can be released to inhibit the expression of the target gene through the RNAi mechanism.

[0272] The P atom in formula A59 can be connected to any possible position on the nucleotide in the siRNA, for example, the 5' position of the nucleotide, the 2' position of the nucleotide, the 3' position of the nucleotide or the base of the nucleotide. In some embodiments, the P atom in formula A59 can be connected to the 2' position, 3' position or 5' position of the nucleotide in the siRNA by forming a phosphodiester bond. In some embodiments, the P atom in formula A59 is connected to the oxygen atom formed after dehydrogenation of the 3' hydroxyl of the 3' terminal nucleotide of the siRNA sense strand, or the P atom in formula A59 is connected to the nucleotide by replacing the hydrogen in the 2'-hydroxyl of a nucleotide in the siRNA sense strand, or the P atom in formula A59 is connected to the nucleotide by replacing the hydrogen in the 5' hydroxyl of the 5' terminal nucleotide of the siRNA sense strand.

[0273] The inventors of the present disclosure have found that, under the premise that the siRNA conjugates described in the application or method provided by the present disclosure show higher CC3 mRNA silencing activity, lower off-target effects and lower hepatotoxic reactions, it is more conducive to the application or method provided by the present disclosure to obtain better treatment and prognosis of periodontitis and related diseases. Therefore, in some embodiments, in the application or method provided by the present disclosure, the siRNA group in the siRNA conjugate in the RNAi agent used can be a group formed by removing one atom or functional group from one of the siRNAs shown in Tables 1a, 1b, 1c and 1d.

[0274] Table 1a The first siRNA sequence

[0275]

[0276]

[0277]

[0278]

[0279]

[0280] Table 1b Second siRNA sequence

[0281]

[0282]

[0283]

[0284]

[0285]

[0286] Table 1c The third siRNA sequence

[0287]

[0288]

[0289]

[0290]

[0291] Table 1d The fourth siRNA sequence

[0292]

[0293]

[0294]

[0295]

[0296] Among them, the capital letters C, G, U, and A represent the base composition of the nucleotide; the lowercase letter m represents that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide; the lowercase letter f represents that the nucleotide adjacent to the left of the letter f is a fluorinated-modified nucleotide; the lowercase letter d represents that the nucleotide adjacent to the right of the letter d is a 2'-deoxynucleotide; the capital letters marked with underlines S Indicates the letter SThe adjacent nucleotide on the left is a stabilized modified nucleotide; the lowercase letter s indicates that the two nucleotides on the left and right of the letter s are connected by phosphorothioate subunits; P1 indicates that the adjacent nucleotide on the right of P1 is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide. In some embodiments, S is a specific stabilizing modification such as moe , where the underlined letter combinations moe Indicates that in this letter combination moe The adjacent nucleotide on the left is a nucleotide with a 2'-O-methoxyethyl modification. In some embodiments, P1 is VP, Ps or P representing a specific modification, wherein the letter combination VP indicates that the adjacent nucleotide on the right side of the letter combination VP is a nucleotide modified with vinyl phosphate (5'-(E)-vinylphosphonate, E-VP), the letter combination Ps indicates that the adjacent nucleotide on the right side of the letter combination Ps is a nucleotide modified with phosphorothioate, and the capital letter P indicates that the adjacent nucleotide on the right side of the letter P is a 5'-phosphate nucleotide. In addition, each U or T in the sequence listed in Tables 1a-1d above can be arbitrarily replaced with each other without significantly affecting the activity or off-target effect of the siRNA.

[0297] Preparation of siRNA conjugates

[0298] The above-mentioned siRNA conjugates can be synthesized by methods that have been described in detail in the prior art. For example, WO2015006740A2 describes in detail the preparation methods of various siRNA conjugates. The siRNA conjugates described in the application or method provided by the present disclosure can also be obtained by methods familiar to those skilled in the art. For example, WO2014025805A1 describes the preparation method of the structure shown in formula (305), and Rajeev et al. describe the preparation method of the structure shown in formula (307) in ChemBioChem 2015, 16, 903-908. Chinese patent application CN110959011A also discloses in detail the method for preparing the siRNA conjugate shown in formula (308). The contents of the above-mentioned documents are incorporated herein by reference in their entirety.

[0299] Pharmaceutically acceptable excipients

[0300] As mentioned above, the RNAi agent used in the application of the present disclosure contains a pharmaceutically active component. In some embodiments, the RNAi agent used in the application of the present disclosure further contains a pharmaceutically acceptable excipient.

[0301] The pharmaceutically acceptable excipients are one or more of various ingredients conventionally used in the art, such as one or more of solvents, protective agents, osmotic pressure regulators, and other pharmaceutically acceptable carriers.

[0302] For example, when the RNAi agent is an injection, the pharmaceutically acceptable excipient is a solvent, such as deionized water, water for injection, ethanol or a pH buffer. The pH buffer may be a tris(hydroxymethyl)aminomethane hydrochloride buffer with a pH value of 7.5-8.5 and / or a phosphate buffer with a pH value of 5.5-8.5, for example, a phosphate buffer with a pH value of 5.5-8.5.

[0303] The amount of the solvent is adjusted according to the required solution concentration. Based on the siRNA groups in the siRNA conjugate, the concentration of the siRNA conjugate in the injection can be 0.01 mg / mL-5 mg / mL siRNA, 0.1 mg / mL-5 mg / mL, or 0.5 mg / mL-3 mg / mL.

[0304] The protective agent may be at least one of inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose and glucose. Based on the total weight of the pharmaceutical composition, the content of the protective agent may be 0.01-30% by weight.

[0305] The osmotic pressure regulator can be sodium chloride and / or potassium chloride. The content of the osmotic pressure regulator makes the osmotic pressure of the pharmaceutical composition 200-700 milliosmole / kilogram (mOsm / kg). According to the required osmotic pressure, those skilled in the art can easily determine the content of the osmotic pressure regulator. In some embodiments, the dosage of the preparation made of the pharmaceutical composition during administration can be adjusted due to different administration methods.

[0306] In some embodiments, the pharmaceutical composition may be a liquid preparation, such as an injection; or a lyophilized powder injection, which is mixed with a liquid excipient during administration to prepare a liquid preparation. The liquid preparation may be, but is not limited to, for subcutaneous, intramuscular or intravenous administration, and may be, but is not limited to, administered by spray to the lungs, or administered by spray through the lungs to other organs and tissues (such as the liver), or delivered orally. In some embodiments, the pharmaceutical composition is administered by subcutaneous injection.

[0307] The other pharmaceutically acceptable carriers may be carriers conventionally used in the field of siRNA administration, such as but not limited to magnetic nanoparticles (e.g., nanoparticles based on Fe3O4 or Fe2O3), carbon nanotubes, mesoporous silicon, calcium phosphate nanoparticles, polyethyleneimine (PEI), polyamidoamine (PAMAM) dendrimer, poly(L-lysine), chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), poly(D&L-lactic / glycolic acid) copolymer (PLGA), poly(2-aminoethyl ethylene phosphate), poly(L-lysine ... Phosphate), PPEEA) and poly(methacrylate-N,N-dimethylaminoethyl ester) (poly(2-dimethylaminoethylmethacrylate), PDMAEMA) and one or more of their derivatives.

[0308] In some embodiments, there are no special requirements for the content of siRNA conjugates and other pharmaceutically acceptable carriers in the pharmaceutical composition. In some embodiments, the weight ratio of siRNA groups to pharmaceutically acceptable carriers in the siRNA conjugates can be 1:(1-500). In some embodiments, the above weight ratio is 1:(1-50).

[0309] In some embodiments, the RNAi agent can be in the form of a liposome preparation. At this time, the pharmaceutically acceptable excipient is a pharmaceutically acceptable carrier used in the liposome preparation. In some embodiments, the pharmaceutically acceptable carrier used in the liposome preparation comprises an amine-containing transfection compound (hereinafter also referred to as an organic amine), an auxiliary lipid and / or a pegylated lipid. Among them, the organic amine, auxiliary lipid and pegylated lipid can be selected from one or more of the amine-containing transfection compounds or pharmaceutically acceptable salts or derivatives thereof, auxiliary lipids and pegylated lipids described in Chinese patent application CN103380113A (incorporated herein in their entirety by reference).

[0310] In some embodiments, the organic amine may be a compound described in Chinese patent application CN103380113A as shown in formula (201) or a pharmaceutically acceptable salt thereof:

[0311]

[0312] in:

[0313] Each X 101 or X 102 are each independently O, S, NA or CA, wherein A is hydrogen or C1-C 20 hydrocarbon chain;

[0314] Each Y 101 or Z 101 Each independently is C=O, C=S, S=O, CH-OH or SO2;

[0315] Each R 101 , R 102 , R 103 , R 104 , R 105 , R 106 or R 107 each independently represents hydrogen, a cyclic or acyclic, substituted or unsubstituted, branched or straight-chain aliphatic group, a cyclic or acyclic, substituted or unsubstituted, branched or straight-chain heteroaliphatic group, a substituted or unsubstituted, branched or straight-chain acyl group, a substituted or unsubstituted, branched or straight-chain aryl group, a substituted or unsubstituted, branched or straight-chain heteroaryl group;

[0316] x is an integer from 1 to 10;

[0317] n is an integer of 1-3, m is an integer of 0-20, and p is 0 or 1; wherein, if m=p=0, then R 102 It is hydrogen;

[0318] And, if at least one of n or m is 2, then R 103 and the nitrogen in formula (201) to form a structure as shown in formula (202) or formula (203):

[0319]

[0320] wherein g, e and f are each independently an integer of 1 to 6, "HCC" represents a hydrocarbon chain, and each *N represents a nitrogen atom in formula (201).

[0321] In some embodiments, R 103 is a polyamine. In other embodiments, R 103 In some embodiments, R in formula (201)101 and R 102 Each of is independently an optionally substituted or unsubstituted, branched or straight chain alkyl or alkenyl group having 3 to about 20 carbon atoms, such as 8 to about 18 carbon atoms, and 0 to 4 double bonds, such as 0 to 2 double bonds.

[0322] In some embodiments, if each of n and m independently has a value of 1 or 3, then R 103 It can be any one of the following formulas (204) to (213):

[0323]

[0324]

[0325] Wherein, in formula (204) to formula (213), g, e and f are each independently an integer of 1 to 6, each "HCC" represents a hydrocarbon chain, and each * indicates R 103 Possible points of attachment to the nitrogen atom in formula (201), wherein each H at any * position can be replaced to achieve attachment to the nitrogen atom in formula (201).

[0326] Among them, the compound represented by formula (201) can be prepared according to the description in Chinese patent application CN103380113A.

[0327] In some embodiments, the organic amine is an organic amine as shown in formula (214) and / or an organic amine as shown in formula (215):

[0328]

[0329]

[0330] The auxiliary lipid is cholesterol, a cholesterol analog and / or a cholesterol derivative;

[0331] The PEGylated lipid is 1,2-dipalmitamide-sn-glycerol-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)]-2000.

[0332] In some embodiments, in the pharmaceutical composition, the molar ratio of the organic amine, the auxiliary lipid and the pegylated lipid is (19.7-80):(19.7-80):(0.3-50), for example, it can be (50-70):(20-40):(3-20).

[0333] In some embodiments, the pharmaceutical composition particles formed by the siRNA conjugate described in the uses or methods provided by the present disclosure and the above-mentioned amine-containing transfection reagent have an average diameter of about 30 nm to about 200 nm, typically about 40 nm to about 135 nm, more typically, the average diameter of the liposome particles is about 50 nm to about 120 nm, about 50 nm to about 100 nm, about 60 nm to about 90 nm or about 70 nm to about 90 nm, for example, the average diameter of the liposome particles is about 30, 40, 50, 60, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, 150 or 160 nm.

[0334] In some embodiments, in the RNAi agent formed by the siRNA conjugate described in the uses or methods provided by the present disclosure and the above-mentioned amine-containing transfection reagent, the weight ratio (weight / weight ratio) of the siRNA group to the total lipids (e.g., organic amines, helper lipids and / or pegylated lipids) is from about 1:1 to about 1:50, from about 1:1 to about 1:30, from about 1:3 to about 1:20, from about 1:4 to about 1:18, from about 1:5 to about 1:17, from about 1:5 to about 1:15, from about 1:5 to about 1:12, from about 1:6 to about 1:12, or from about 1:6 to about 1:10, for example, the weight ratio of the siRNA group to the total lipids is about 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17 or 1:18.

[0335] In some embodiments, the components of the RNAi agent can exist independently when sold, and can exist in the form of a liquid preparation when used. In some embodiments, the RNAi agent described in the application or method provided by the present disclosure can be prepared according to various known methods, except that the siRNA conjugate described in the application or method provided by the present disclosure is substituted for the siRNA in the pharmaceutical composition known in the art; in some embodiments, it can be prepared according to the following method:

[0336] The organic amine, the auxiliary lipid and the PEGylated lipid are suspended in alcohol according to the above molar ratio and mixed to obtain a lipid solution; the amount of alcohol used is such that the total mass concentration of the obtained lipid solution is 2-25 mg / mL, for example, 8-18 mg / mL. The alcohol is selected from pharmaceutically acceptable alcohols, such as alcohols that are liquid at around room temperature, for example, ethanol, propylene glycol, benzyl alcohol, glycerol, polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400, one or more, for example, ethanol.

[0337] The siRNA conjugate described in the application or method provided by the present disclosure is dissolved in a buffered saline solution to obtain an siRNA conjugate aqueous solution. The concentration of the buffered saline solution is 0.05-0.5M, for example, it can be 0.1-0.2M, and the pH of the buffered saline solution is adjusted to 4.0-5.5, for example, it can be 5.0-5.2, and the amount of the buffered saline solution is such that the concentration of the siRNA group does not exceed 0.6mg / mL, for example, it can be 0.2-0.4mg / mL. The buffer salt is selected from one or more of soluble acetate and soluble citrate, for example, it can be sodium acetate and / or potassium acetate.

[0338] The lipid solution and the siRNA conjugate aqueous solution are mixed, and the mixed product is incubated at 40-60° C. for at least 2 minutes, for example, 5-30 minutes, to obtain an incubated liposome preparation. The volume ratio of the lipid solution to the siRNA conjugate aqueous solution is 1:(2-5), for example, 1:4.

[0339] The incubated liposome preparation is concentrated or diluted, impurities are removed, and sterilization is performed to obtain the pharmaceutical composition provided by the present disclosure, wherein the physicochemical parameters are a pH value of 6.5-8, an encapsulation rate of not less than 80%, a particle size of 40-200 nm, a polydispersity index of not more than 0.30, and an osmotic pressure of 250-400 mOsm / kg; for example, the physicochemical parameters may be a pH value of 7.2-7.6, an encapsulation rate of not less than 90%, a particle size of 60-100 nm, a polydispersity index of not more than 0.20, and an osmotic pressure of 300-400 mOsm / kg.

[0340] The concentration or dilution can be performed before, after or simultaneously with the removal of impurities. The impurity removal method can adopt various existing methods, such as using a tangential flow system, a hollow fiber column, ultrafiltration under 100K Da conditions, and the ultrafiltration exchange solution is a phosphate buffered saline (PBS) with a pH of 7.4. The sterilization method can adopt various existing methods, such as filtration sterilization on a 0.22 μm filter.

[0341] As described above, in one aspect, the present disclosure provides the use of the above-mentioned RNAi agent in the preparation of a drug for treating and / or preventing periodontitis and periodontitis-related diseases. On the other hand, the present disclosure also provides a method for treating and / or preventing a disease or symptom associated with periodontitis or periodontitis, the method comprising administering an effective amount of the above-mentioned RNAi agent to a subject in need thereof. In some embodiments, the periodontitis-related disease is one or more of diabetes, stroke, myocardial infarction, atherosclerosis, hypertension, osteoporosis, and premature birth.

[0342] By administering the RNAi agent, such as siRNA conjugate and / or pharmaceutical composition, described in the application or method provided by the present disclosure to a subject in need, the level of CC3 protein expressed by hepatocytes can be reduced by regulating gene expression, thereby preventing and / or treating pathological conditions or diseases associated with periodontitis. In some embodiments, the amount of the RNAi agent in the drug or the amount used in the method is such that the maximum inhibition rate of CC3 mRNA in the liver of the subject is greater than 50%.

[0343] Therefore, the RNAi agent described in the use or method provided by the present disclosure can be used to prevent and / or treat the pathological conditions or diseases, or to prepare a medicament for preventing and / or treating the pathological conditions or diseases described herein.

[0344] The term "administration / administration" as used herein refers to placing an siRNA conjugate and / or a pharmaceutical composition into a subject's body by a method or approach that at least partially localizes the siRNA conjugate and / or the pharmaceutical composition to a desired site to produce a desired effect. Routes of administration suitable for the methods of the present disclosure include local administration and systemic administration. In general, local administration results in more siRNA conjugates and / or pharmaceutical compositions being delivered to a specific site compared to the entire body of the subject; whereas systemic administration results in the siRNA conjugate and / or pharmaceutical composition being delivered to essentially the entire body of the subject. Considering that the present disclosure is intended to provide a means for preventing and / or treating CC3-related diseases or symptoms, in some embodiments, an administration method that is capable of delivering the drug to the liver is employed.

[0345] The subject can be administered by any suitable route known in the art, including but not limited to oral or parenteral routes, such as intravenous administration, intramuscular administration, subcutaneous administration, transdermal administration, airway administration (aerosol), pulmonary administration, nasal administration, rectal administration and topical administration (including oral administration and sublingual administration). The frequency of administration can be once or more every day, every week, every two weeks, every three weeks, every month, every 2 months, every 3 months, every six months, or every year.

[0346] The dosage provided and / or used in the pharmaceutical composition may be a dosage conventional in the art, which may be determined based on various parameters, particularly the age, weight and sex of the subject. Toxicity and efficacy may be determined by standard pharmaceutical procedures in cell culture or experimental animals, such as determination of LD50 (the lethal dose that causes 50% of the population to die) and ED50 (the dose that causes 50% of the maximum response intensity in quantitative reactions, and the dose that causes 50% of the experimental subjects to have a positive reaction in qualitative reactions). The range of human dosages may be derived based on data obtained from cell culture analysis and animal studies.

[0347] When administering the siRNA conjugate and / or pharmaceutical composition described in the application or method provided by the present disclosure, for example, for male or female C57BL / 6J or C3H / HeNCrlVr mice of 6-12 weeks of age and weighing 18-25 g, the amount of siRNA in the siRNA conjugate and / or pharmaceutical composition is calculated as follows: for the siRNA conjugate formed by siRNA and a pharmaceutically acceptable conjugated molecule, the amount of siRNA can be 0.001-100 mg / kg body weight, in some embodiments, 0.01-50 mg / kg body weight, in further embodiments, 0.05-20 mg / kg body weight, in further embodiments, 0.1-15 mg / kg body weight, and in yet further embodiments, 0.1-10 mg / kg body weight. When administering the siRNA conjugate described in the application or method provided by the present disclosure, the above dosage may be preferred.

[0348] The present disclosure will be further described below by way of examples, but the present disclosure is not limited thereby.

[0349] Example

[0350] Unless otherwise specified, the reagents and culture media used in the following examples are commercially available products, and the nucleic acid electrophoresis, real-time PCR and other operations used are all performed according to the methods described in Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)).

[0351] Preparation Example 1-6 Synthesis of siRNA Conjugate 1-6

[0352] According to the preparation method described in Preparation Example 14 of CN110959011A, conjugates 1-6 in Table 2 below were prepared, with the only difference being that the sense strand and antisense strand of siRNA contained in each siRNA conjugate were as shown in Table 2; the sense strand and antisense strand of siRNA were synthesized according to the nucleic acid sequences of siRNA numbered as conjugates 1-conjugates 6 in Table 2 below. Each siRNA conjugate was diluted to a concentration of 0.2 mg / mL (in terms of siRNA) using ultrapure water (Milli-Q ultrapure water instrument, resistivity 18.2 MΩ*cm (25°C)), and then the molecular weight was detected using a liquid chromatography-mass spectrometer (LC-MS, Liquid Chromatography-Mass SP1ectrometry, purchased from Waters, model: LCT Premier). The measured values ​​are consistent with the theoretical values, indicating that the synthesized conjugates 1-6 have the double-stranded nucleic acid sequences of the target design. Each siRNA conjugate has a structure shown in formula (403) and is a sodium salt of the siRNA conjugate, wherein Nu represents a siRNA group and has a siRNA sequence corresponding to conjugates 1-6 in Table 2, and the connection site of the siRNA group is the ribose 3' position of the 3' terminal nucleotide of the sense strand. Among them, conjugate 5 is a siRNA conjugate having a sequence composition substantially the same as conjugate 1, but not containing a stabilizing modified nucleotide; conjugate 6 is a siRNA conjugate having a sequence composition substantially the same as conjugates 2-3, but not containing a stabilizing modified nucleotide, and there is a base mismatch in the sense strand and antisense strand sequences of conjugate 6 and conjugate 4, respectively;

[0353] Comparative Preparation Example 1 Synthesis of Reference siRNA Conjugate NC

[0354] According to the preparation method described in Preparation Example 14 of CN110959011A, a reference siRNA conjugate numbered as reference conjugate NC in Table 2 was prepared, the only difference being that the sense strand and antisense strand of the siRNA contained in the reference siRNA conjugate NC were as shown in Table 2; the sense strand and antisense strand of the siRNA were synthesized according to the nucleic acid sequence of the siRNA numbered as reference conjugate NC in Table 2. Each reference siRNA conjugate was diluted to a concentration of 0.2 mg / mL (based on siRNA) using ultrapure water (Milli-Q ultrapure water instrument, resistivity 18.2 MΩ*cm (25°C)), and then the molecular weight was detected using a liquid chromatography-mass spectrometer (LC-MS, Liquid Chromatography-Mass Spectrometry, purchased from Waters, model: LCT Premier). The measured values ​​are consistent with the theoretical values, indicating that the synthesized reference conjugate NC has the structure shown in formula (403), and the siRNA group contained therein has the siRNA sequence corresponding to the reference conjugate NC in Table 2. The reference conjugate NC is a negative control siRNA conjugate whose sequence has no significant homology with any known mRNA.

[0355] Table 2 siRNA sequences in siRNA conjugates

[0356]

[0357] Among them, capital letters C, G, U, A, and T represent the base composition of nucleotides; lowercase letter m indicates that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide; lowercase letter f indicates that the nucleotide adjacent to the left of the letter f is a fluorinated modified nucleotide; the letter combination marked with an underline moe It indicates that the nucleotide adjacent to the left of the letter combination moe is a ribose 2'-O-methoxyethyl modified nucleotide; the lowercase letter s indicates that the two nucleotides on the left and right of the letter s are connected by thiophosphate subunits; the lowercase letter d indicates that the nucleotide adjacent to the right of the letter d is a 2'-deoxynucleotide.

[0358] Experimental Example 1 Inhibitory activity of siRNA conjugates against target sequences in the in vitro psi-CHECK system

[0359] According to the method described in Kumico Ui-Tei et al., Functional dissection of siRNA sequence by systematic DNA substitution: modified siRNA with a DNA seed arm is a powerful tool for mammalian gene silencing with significantly reduced off-target effect. Nucleic Acids Research, 2008. 36 (7), 2136-2151, a detection plasmid was constructed, and the detection plasmid and the conjugate to be tested were co-transfected into HEK293A cells, and the target sequence inhibition activity of siRNA was reflected by the expression level of the dual luciferase reporter gene. The conjugates to be tested in this experimental example are conjugate 1 and conjugate 5. The specific steps are as follows:

[0360] [1] Construction of detection plasmid

[0361] Using psi-CHECK TM -2(Promega TM ) plasmid to construct a detection plasmid, wherein the plasmid contains a target sequence 1, i.e., the siRNA conjugate target sequence. For the siRNA conjugate to be tested, the target sequence 1 is as follows:

[0362] 5'-CCTCATGGTGTTCGTGACGAA-3'(SEQ ID NO:189)

[0363] The target sequence 1 is homologous to a portion of CC3 mRNA and is completely complementary to the sequence of the antisense strand in the siRNA conjugate being tested. Therefore, the inhibitory effect of each siRNA conjugate on the target sequence 1 can reflect the inhibitory ability of the siRNA conjugate being tested on the expression of the target gene. TM -2 plasmid's XhoI / Not I site.

[0364] [2] Transfection

[0365] HEK293A cells (purchased from Nanjing Kebai Biotechnology Co., Ltd.) were cultured in H-DMEM complete medium (HyClone) supplemented with 10% fetal bovine serum (FBS, RMBIO) and 0.2% penicillin-streptomycin (HyClone) by volume at 37°C in an incubator containing 5% CO2 / 95% air.

[0366] HEK293A cells were cultured at 8 × 10 3 Cells / well were inoculated in a 96-well plate. After 16 hours, when the cell growth density reached 70%, the complete medium in the culture wells was aspirated, and 80 μL of opti-MEM medium (GIBCO) was added to each well and culture was continued for 1.5 hours.

[0367] The above detection plasmid was diluted with PBS to a 20 μM storage solution; each siRNA conjugate to be tested was prepared with PBS into 20 μM, 4 μM, 1 μM, 0.25 μM, 0.0625 μM, 0.015625 μM, 0.003906 μM, 0.0009765 μM, 0.0002441 μM, 0.00006104 μM and 0.00001526 μM (based on the amount of siRNA in the siRNA conjugate) 11 different concentrations of siRNA conjugate working solutions. The siRNA conjugates used were conjugate 1 and conjugate 5 prepared above.

[0368] For each siRNA conjugate, 1A1-1A11 solutions were prepared respectively. Each 1A1-1A11 solution contained 1 μL of the above 11 concentrations of siRNA working solution, 0.05 μL of detection plasmid working solution (including 10 ng of detection plasmid) and 8.95 μL of Opti-MEM culture medium.

[0369] Prepare 1B solution, each 1B solution contains 0.2μL Lipofectamine TM 2000 and 9.8 μL Opti-MEM medium.

[0370] Prepare 1C solution, each 1C solution contains 0.05 μL of detection plasmid working solution (including 10 ng of detection plasmid) and 8.95 μL of Opti-MEM culture medium.

[0371] A portion of 1B solution was mixed with a portion of 1A1-1A11 solution of each siRNA conjugate, and the mixtures were incubated at room temperature for 20 min to obtain transfection complexes 1X1-1X11 of each siRNA conjugate.

[0372] One portion of solution 1B was mixed with one portion of solution 1C, and the mixtures were incubated at room temperature for 20 min to obtain a blank transfection complex 1Xa.

[0373] In the culture wells, the transfection complexes 1X1-1X11 of each siRNA conjugate were added respectively and mixed evenly. The added amount was 20 μL / well to obtain transfection complexes with final concentrations of each siRNA conjugate of approximately 40 nM, 10 nM, 2.5 nM, 0.625 nM, 0.15625 nM, 0.03906 nM, 0.009765 nM, 0.002441 nM, 0.0006103 nM, 0.0001526 nM, and 0.00003815 nM (based on the amount of siRNA in the siRNA conjugate). The transfection complexes 1X1-1X11 of each siRNA conjugate were transfected into 3 culture wells to obtain a co-transfection mixture containing the siRNA conjugate, which was recorded as a test group.

[0374] For each siRNA conjugate, the transfection complex 1Xa was added to the other 3 culture wells at 20 μL / well to obtain a transfection mixture without siRNA conjugate, which was recorded as a blank control group.

[0375] After 4 hours of transfection in the culture wells, 100 μL of H-DMEM complete medium containing 20% ​​FBS was added to each well. The 96-well plate was placed in a CO2 incubator at 37°C and an air atmosphere containing 5 v / v% CO2 for 24 hours.

[0376] [3] Detection

[0377] Aspirate the medium in the culture wells and add 150 μL of Luciferase reagent and H-DMEM mixed solution (volume ratio 1:1), mixed thoroughly, incubated at room temperature for 10 minutes, transferred 120 μL of the mixed solution to a 96-well ELISA plate, and the chemiluminescence value (Fir) of Firefly in each culture well on the 96-well ELISA plate was read using a Synergy II multi-function ELISA reader (BioTek). Then, 60 μL of Stop& Reagents were mixed thoroughly, incubated at room temperature for 10 min, and then the chemiluminescence value (Ren) of Renilla in each culture well on the 96-well ELISA plate was read using an ELISA reader according to the arrangement of reading Fir.

[0378] Calculate the luminescence ratio of each well on the 96-well ELISA plate Ratio = Ren / Fir, and the luminescence ratio Ratio (test) or Ratio (control) of each test group or control group is the average of the Ratios of three culture wells; normalize the luminescence ratio of each test group based on the luminescence ratio of the control group to obtain the ratio Ratio (test) / Ratio (control), which represents the relative expression level of the Renilla reporter gene, that is, the residual activity. The inhibition rate of siRNA on the target sequence = (1-R) ​​× 100%.

[0379] According to the relative residual activity of Renilla in HEK293A cells after transfection with different concentrations of the tested siRNA, the nonlinear regression analysis function of Graphpad 5.0 software was used to fit the log (inhibitor) vs. response—Variable slope (four parameters) dose-effect curve.

[0380] Calculate the IC of the siRNA targeting the target sequence based on the function corresponding to the fitted dose-effect curve 50 Value, the function is as follows,

[0381]

[0382] Where:

[0383] Y is the ratio R, i.e. the relative residual activity of Renilla,

[0384] X is the logarithmic value of the transfected siRNA concentration,

[0385] Bot is the Y value at the bottom of the steady state period,

[0386] Top is the Y value at the top of the steady state period,

[0387] X' is the value of X when Y is halfway between the bottom and the top, and HillSlope is the slope of the curve at X'.

[0388] From the dose-effect curve and the corresponding function, determine the corresponding X when Y = 50% 50 The IC value of each siRNA was calculated. 50 Value = 10^X50(nM), IC 50 The values ​​are summarized in Table 3.

[0389] Table 3 IC values ​​of siRNA conjugates in the psi-CHECK system 50

[0390] Conjugate No. <![CDATA[IC 50 (GSCM)nM]]> Conjugate 1 0.004 Conjugate 5 0.011

[0391] In Table 3, the difference between conjugate 5 and conjugate 1 is that, in the direction from the 5' end to the 3' end, the base on the 5th nucleotide of the antisense strand of conjugate 1 is T, and the nucleotide has a 2'-O-methoxyethyl modification group, and correspondingly, the base on the 5th nucleotide of the antisense strand of conjugate 5 is U, and does not have a 2'-O-methoxyethyl modification. In addition, conjugate 1 and conjugate 5 have the same sequence and modification composition, and the antisense strands are both completely reverse complementary sequences to a 19-contiguous nucleotide segment in CC3 mRNA.

[0392] From the results in Table 3, it can be seen that the siRNA conjugates described in the applications or methods provided by the present disclosure have high target sequence inhibition activity in the in vitro psi-CHECK system. Furthermore, compared with conjugate 5 having the same sequence but not containing the stabilizing modified nucleotide, conjugate 1 containing the stabilizing modified nucleotide has greatly improved inhibition activity against the target sequence, and the IC value of conjugate 5 is 2.34. 50 The value is the IC of conjugate 1 50 2.75 times the value.

[0393] Experimental Example 2 Inhibitory activity of siRNA conjugates against target sequences in the in vitro psi-CHECK system

[0394] According to the method of Experimental Example 1, the inhibitory activity of conjugates 2, 3 and 6 on the target sequence in the in vitro psi-CHECK system was tested. The only difference from the method of Experimental Example 1 is that for conjugates 2-3 and conjugate 6, the target sequence 2 shown below is used instead of the target sequence 1:

[0395] Target sequence 2: 5'-TAGGAACACCCTCATCATCTA-3' (SEQ ID NO: 190).

[0396] The target sequence 2 is homologous to another part of CC3mRNA (different from the target sequence 1) and is completely complementary to the sequence of the antisense strand in the siRNA conjugate tested. Therefore, the inhibitory effect of each siRNA conjugate on the target sequence 2 can reflect the inhibitory ability of the siRNA conjugate tested on the expression of the target gene. 50 The values ​​are summarized in Table 4.

[0397] Table 4 IC values ​​of siRNA conjugates in the psi-CHECK system 50

[0398] Conjugate No. <![CDATA[IC 50 (GSCM)nM]]> Conjugate 2 0.0003 Conjugate 3 0.0003 Conjugate 6 0.0011

[0399] In Table 4, the difference between conjugate 2, conjugate 3 and conjugate 6 is that, in the direction from the 5' end to the 3' end, the third nucleotide of the antisense chain of conjugate 2 has a 2'-O-methoxyethyl modification group, the base on the fifth nucleotide of the antisense chain of conjugate 3 is T, and the nucleotide has a 2'-O-methoxyethyl modification group, and correspondingly, the base on the fifth nucleotide of the antisense chain of reference conjugate 2 is U, and does not have a 2'-O-methoxyethyl modification.

[0400] From the results in Table 4, it can be seen that the siRNA conjugates described in the application or method provided by the present disclosure have high target sequence inhibition activity in the in vitro psi-CHECK system. Furthermore, conjugates 2-3 have further improved target sequence inhibition activity compared to conjugate 6 which has the same sequence as conjugate 2-3 but does not contain stabilizing modified nucleotides. The IC 50 Values ​​are the IC of conjugate 2 and conjugate 3 50 3.7 times the value.

[0401] Experimental Example 3 Inhibitory Efficiency of siRNA Conjugates on CC3 mRNA Expression in Normal Rats

[0402] This experimental example investigates the inhibition rate of conjugate 3 and conjugate 4 on CC3 mRNA in liver tissue of normal SD rats (purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.).

[0403] Normal rats SD aged 6-8 weeks were randomly divided into groups of 5 rats per group, and different doses of conjugate 3, conjugate 4 and PBS were administered to each group of rats. The dosage of all animals was calculated according to body weight, and a single dose was administered by subcutaneous injection. The dosage of siRNA conjugate (in terms of the amount of siRNA) was 3 mg / kg (also marked as 3 mpk) and 9 mg / kg (also marked as 9 mpk) in two dosage groups, and the administration volume was 5 mL / kg. Each siRNA conjugate was provided in a PBS aqueous solution, and the drug concentration to be configured for the conjugate was calculated according to the dosage and administration volume. The rats were killed on the 7th day after administration, and the livers were collected to detect the expression of CC3 mRNA in the liver.

[0404] Real-time fluorescence quantitative PCR was used to detect the expression of CC3 mRNA in liver tissue. Specifically, a reverse transcription kit (Promega, Catalog No. A3500) was used to reverse transcribe cDNA according to the instructions. A 2×Ultra SYBR Mixture (with ROX) (Beijing Kangwei Century Biotechnology Co., Ltd., Catalog No. CW0956) kit was used to detect the expression of CC3 mRNA using cDNA as a template according to the instructions. Among them, the PCR primers used to amplify CC3 and GAPDH as an internal reference gene are shown in Table 5.

[0405] Table 5 Primer sequences

[0406]

[0407] The CC3 mRNA expression was calculated according to the following equation:

[0408] CC3 mRNA expression level=(CC3 mRNA expression level in the test group / GAPDH mRNA expression level in the test group) / (CC3 mRNA expression level in the control group / GAPDH mRNA expression level in the control group)×100%.

[0409] It was calculated that in this experimental example, the inhibition efficiency of conjugate 3 and conjugate 4 on CC3 mRNA in normal rat SD liver tissue is shown in Table 6 below.

[0410] Table 6 Inhibition efficiency of CC3 mRNA

[0411]

[0412] It can be seen from Table 6 that when the dosage is 9 mg / kg, the inhibition efficiency of conjugate 3 and conjugate 4 on CC3 mRNA can reach more than 92%, especially conjugate 4, the inhibition efficiency of CC3 mRNA reaches 97.19%, indicating that the siRNA conjugate described in the application or method provided by the present disclosure has a significant inhibition efficiency on CC3 mRNA in rat liver tissue.

[0413] Experimental Example 4 Inhibitory Efficiency of siRNA Conjugates on CC3 mRNA Expression in Mice

[0414] This experimental example investigates the inhibition rate of conjugate 3 and conjugate 4 on CC3 mRNA in liver tissue in normal mice C57 / B6 (purchased from Spefox (Beijing) Biotechnology Co., Ltd.) and CAIA model (antibody-induced arthritis model) mice (purchased from Spefox (Beijing) Biotechnology Co., Ltd.).

[0415] C57 / B6 mice aged 6-8 weeks were randomly divided into groups of 5 each, namely normal mouse group and CAIA model mouse group. Different doses of conjugate 3, conjugate 4 and PBS were administered to the normal group mice, and different doses of conjugate 3 and PBS were administered to the CAIA model mice. The drug dose of all animals was calculated according to body weight, and a single dose was administered by subcutaneous injection. The siRNA conjugate dosage (in terms of the amount of siRNA) was 3 mg / kg (also marked as 3 mpk) and 9 mg / kg (also marked as 9 mpk) in two dosage groups, and the administration volume was 5 mL / kg. Each siRNA conjugate was provided in a PBS aqueous solution, and the drug concentration to be configured for the conjugate was calculated according to the dosage and administration volume.

[0416] On the 7th day after administration, normal mice were killed, livers were collected, and the expression level of CC3 mRNA in the liver was detected. The detection and calculation process of the expression level of CC3 mRNA in the liver was performed according to the method of Experimental Example 3.

[0417] It was calculated that in this experimental example, the inhibition efficiency of conjugate 3 and conjugate 4 on CC3 mRNA in normal mouse liver tissue is shown in Table 7 below.

[0418] Table 7 Inhibition efficiency of CC3 mRNA

[0419]

[0420] On the 20th day after administration, the CAIA model mice were killed, the livers were collected, and the expression level of CC3 mRNA in the liver was detected and calculated according to the method of Experimental Example 3. When the dosage was 3 mg / kg, the inhibition rate of conjugate 3 on CC3 mRNA was 58%, and when the dosage was 9 mg / kg, the inhibition rate of conjugate 3 on CC3 mRNA was 85%.

[0421] It can be seen from Table 7 that the inhibition efficiency of conjugate 4 on CC3 mRNA can reach more than 85%, especially when the dosage is 9 mg / kg, the inhibition efficiency of CC3 mRNA reaches 93.40%, and at this dosage, the inhibition efficiency of conjugate 3 on CC3 mRNA in CAIA model mice can reach 85%, indicating that the siRNA conjugate described in the application or method provided by the present disclosure has a significant inhibition efficiency on CC3 mRNA in mouse liver tissue.

[0422] Experimental Example 5 Inhibition of CC3 mRNA by siRNA conjugates in mice in vivo

[0423] Female hC3 transgenic mice of SPF grade and 5 weeks old (purchased from Shanghai Model Organisms Science Co., Ltd.) were randomly divided into 4 groups, with 5 mice in each group, including three groups of test group mice and the remaining group of mice as blank control group mice. Each mouse in the first test group was injected subcutaneously with 1 mg / kg of conjugate 3, which was provided in the form of 1×PBS solution containing 0.2 mg / mL (in terms of siRNA) of conjugate 3, and the administration volume was 5 mL / kg; each mouse in the second test group was injected with 1 mg / kg of conjugate 4, which was provided in the form of 1×PBS solution containing 0.2 mg / mL (in terms of siRNA) of conjugate 4, and the administration volume was 5 mL / kg; each mouse in the third test group was injected with 3 mg / kg of conjugate 4, which was provided in the form of 1×PBS solution containing 0.6 mg / mL (in terms of siRNA) of conjugate 4, and the administration volume was 5 mL / kg; each blank control group mouse was given 5 mL / kg of 1×PBS solution. The injection dose is calculated based on the siRNA contained in the conjugate and the weight of the mouse.

[0424] The administration time point was calculated as the first day. The animals were killed on the 22nd day. The liver tissue of each mouse was collected and preserved with RNA later (Sigma Aldrich). 1 mL Trizol (Sigma) was added to each liver tissue, and the liver tissue was broken three times in the Tissuelyset II fully automatic tissue homogenizer for 30 seconds each time to obtain liver tissue homogenate. 0.2 mL chloroform was added to it and allowed to stand for 3 minutes. Centrifuge at 3500 rpm for 15 minutes at 4°C, and 0.4 mL supernatant was taken. 0.5 mL isopropanol was added to the supernatant and allowed to stand at room temperature for 10 minutes. Centrifuge at 3500 rpm for 15 minutes at 4°C, and the supernatant was discarded. 1 mL ethanol was added to the precipitate to wash the precipitate, and the precipitate was centrifuged at 3500 rpm for 8 minutes at 4°C, and the supernatant was discarded. 70 μL DEPC water was added to the precipitate to obtain the extracted total RNA solution.

[0425] For the total RNA of liver tissue of each mouse, 10.5 μL of total RNA aqueous solution containing 1 μg of total RNA was taken, and a reverse transcription reaction system of 20 μL was prepared according to the reverse transcription operation steps in the kit manual using a reverse transcription kit Reverse Transcription System (purchased from Promega, catalog number A3500), and the total RNA was reverse transcribed. The conditions for reverse transcription are: for each reverse transcription reaction system, the reverse transcription reaction system was incubated at 42°C for 30 min, then incubated at 95°C for 5 min, and finally incubated at 4°C for 5 min. After the reaction was completed, 80 μL of DEPC water was added to the reverse transcription reaction system to obtain a solution containing cDNA.

[0426] For each reverse transcription reaction system, 5 μL of the above solution containing cDNA was taken as a template, and 20 μL of the qPCR reaction system was prepared using the reagents provided by the SYBR select Master Mix kit (Applied biosystem), wherein the PCR primer sequences for amplifying the target gene CC3 and the internal reference gene GAPDH are shown in Table 8 below, and the final concentration of each primer is 0.25 μM. Each qPCR reaction system was placed on an ABI StepOnePlus Real-Time PCR instrument and amplified using a three-step method, with the amplification program being 95°C pre-denaturation for 10 min, followed by 95°C denaturation for 30 s, 60°C annealing for 30 s, and 72°C extension for 30 s. After repeating the above denaturation, annealing, and extension process for a total of 40 times, a product W containing the amplified target gene CC3 and the internal reference gene GAPDH was obtained. The product W was then incubated at 95°C for 1 min, 55°C for 30 s, and 95°C for 30 s, and the melting curves of the target gene CC3 and the internal reference gene GAPDH in the product W were collected by real-time fluorescence quantitative PCR to obtain the Ct values ​​of the target gene CC3 and the internal reference gene GAPDH.

[0427] Table 8 Primer sequence information

[0428]

[0429] The comparative Ct (ΔΔCt) method was used to calculate the relative quantification of the target gene CC3 in each test group. The calculation method is as follows:

[0430] ΔCt(test group) = Ct(test group target gene) – Ct(test group reference gene)

[0431] ΔCt(control group) = Ct(control group target gene) – Ct(control group internal reference gene)

[0432] ΔΔCt(test group) = ΔCt(test group) - ΔCt(control group average)

[0433] ΔΔCt(control group) = ΔCt(control group) - ΔCt(control group average)

[0434] Wherein, ΔCt (control group average) is the arithmetic mean of the ΔCt (control group) of each of the two culture wells of the control group. Thus, each culture well of the test group and the control group corresponds to a ΔΔCt value.

[0435] The CC3 mRNA expression level of the test group was normalized based on the control group, and the CC3 mRNA expression level of the blank control group was defined as 100%.

[0436] Relative expression level of CC3 mRNA in the test group = 2 -ΔΔCt(测试组) ×100%

[0437] Test group CC3 mRNA inhibition rate = (1-test group CC3 mRNA relative expression level) × 100%

[0438] The inhibition rates of CC3 mRNA by conjugate 3 and conjugate 4 are summarized in Table 9. For the same test group of conjugates, the mRNA inhibition rate is the arithmetic mean of the test group CC3 mRNA inhibition rates measured in two culture wells.

[0439] Table 9 Inhibition efficiency of CC3 mRNA

[0440]

[0441] In this experimental example, after the hC3 transgenic mice were given conjugate 3 at a dose of 1 mg / kg mouse body weight (calculated as siRNA), the inhibition rate of CC3 mRNA in the mice could reach 70.1%. For the test group mice given conjugate 4 at doses of 1 mg / kg and 3 mg / kg, the inhibition rates of CC3 mRNA in the mice were 79.5% and 93.5%, respectively, both showing good CC3 mRNA inhibition effects.

[0442] In this experimental example, Human complement C3 ELISA Kit (ab108823) Abcam kit (purchased from Xi'an Zhuangzhi Biotechnology Co., Ltd.) was used at the same time. The expression of hCC3 protein in the serum of the three test groups of mice was detected on the 8th day, 15th day and 22nd day after administration according to the instructions, and the expression of hCC3 protein in the serum of the mice before the injection of the conjugate was normalized as 100%. Among them, the expression of hCC3 protein in hC3 transgenic mice after the injection of conjugate 3 and conjugate 4 is shown in Table 10.

[0443] Table 10hCC3 protein expression

[0444]

[0445]

[0446] As shown in Table 10, conjugate 3 and conjugate 4 at injection doses of 1 mg / kg and 3 mg / kg can significantly inhibit the expression of hCC3 protein in mouse serum, thereby effectively treating and / or preventing diseases and / or symptoms related to abnormal expression of hCC3 protein.

[0447] Experimental Example 6 Toxicity Effects of siRNA Conjugates in Rats

[0448] Conjugate 3 and conjugate 4 were dissolved in PBS to 6 mg / mL and 20 mg / mL solutions (calculated as siRNA conjugates), respectively. SD rats (half male and half female, 0.22-0.28 kg, 5-7 weeks old, purchased from Vital River) were randomly divided into groups, with 20 rats in each group. The first group of rats was injected with conjugate 3 solution, and the second group of rats was injected with conjugate 4 solution. Both injections were subcutaneous injections at the back of the neck. Specifically, 10 rats (half male and half female) in the first group of rats were injected with a solution of conjugate 3 at a concentration of 6 mg / mL, and the administration volume was 5 mL / kg (rat body weight), so the dosage was 30 mg / kg, and another 10 rats (half male and half female) were injected with a solution of conjugate 3 at a concentration of 20 mg / mL, and the administration volume was 5 mL / kg (rat body weight), so the dosage was 100 mg / kg; 10 rats (half male and half female) in the second group of rats were injected with a solution of conjugate 4 at a concentration of 6 mg / mL, and the administration volume was 5 mL / kg (rat body weight), so the dosage was 30 mg / kg, and another 10 rats (half male and half female) were injected with a solution of conjugate 4 at a concentration of 20 mg / mL, and the administration volume was 5 mL / kg (rat body weight), so the dosage was 100 mg / kg. The administration frequency was q2w*3, that is, once every two weeks, for four consecutive weeks, a total of 3 times, the day of administration was recorded as day 1 (D1), and the administration was performed on D15 and D29 respectively.

[0449] After the end of the administration period, on the 30th day, the rats were killed and autopsied, and preserved in 10% neutral buffered formalin fixative and pathological sections were made. No significant abnormalities were found in the gross autopsy; the severity of hepatic fatty degeneration and inflammation in the pathological sections was evaluated and graded and relatively compared.

[0450] In the rats given a 30 mg / kg dose of conjugate 3, no abnormalities were found in liver cell pathology. Among the five female rats given a 100 mg / kg dose of conjugate 3, only one rat was found to have mild fatty degeneration, which was manifested by dense arrangement of liver cells with clear boundaries, mild round vacuoles in the cytoplasm, and no obvious liver inflammation was found in any of the rats. Among the five female rats given a 30 mg / kg and 100 mg / kg dose of conjugate 4, only two rats were found to have mild fatty degeneration, mild round vacuoles in the cytoplasm, and no obvious liver inflammation was found in any of the rats.

[0451] The above results indicate that even at a dosage of up to 100 mg / kg, the siRNA conjugates described in the application or method provided by the present disclosure hardly show hepatotoxic reactions due to off-target effects, and therefore show high safety in the preparation of drugs for the treatment and / or prevention of diseases or symptoms related to CC3 mRNA expression. The application or method provided by the present disclosure has excellent application prospects.

[0452] Experimental Example 7 Therapeutic effect of siRNA conjugates in periodontitis model mice

[0453] Conjugate 4 was dissolved in PBS into 0.2 mg / mL, 0.6 mg / mL and 1.8 mg / mL solutions (in terms of siRNA). Reference conjugate NC was dissolved in PBS into 0.6 mg / mL solution (in terms of siRNA). Periodontitis model mice (all female, 6 weeks old, purchased from Beijing Stomatological Hospital) were randomly divided into groups, with 6 mice in each group. The second administration time was marked as the first day of the experiment. The above-mentioned 0.2 mg / mL and 0.6 mg / mL concentrations of conjugate 4 solution or reference conjugate NC solution were subcutaneously injected into the abdomen on the -7th day of the experiment (i.e., the 7th day after this administration was marked as the first day of the experiment), the 1st day and the 7th day. The dosage of each administration was 5 mL / kg of mouse body weight, and they were marked as 1 mg / kg dosage group, 3 mg / kg dosage group and negative control group in turn. The above-mentioned 1.8 mg / mL conjugate 4 was subcutaneously injected into the abdomen on the -7th day, marked as the 9 mg / kg dosage group. In addition, 3 normal mice were selected and marked as blank control group.

[0454] On the 14th day of the experiment (i.e., the 7th day after the administration on the 7th day above), mice in each dose group or control group were selected to observe the alveolar bone resorption in the oral cavity; cervical lymph node tissues of mice in each control group and 3mg / kg and 9mg / kg dose groups were taken to detect the relative expression levels of inflammatory factors IL-17a, IL6 and Tnf-a according to the method of Experimental Example 5. The periodontal tissues of mice in the control group and 3mg / kg dose group were taken to detect the relative expression level of Tnfsf11 mRNA according to the method of Experimental Example 5. See the results. Figures 1A-1E as well as Figures 2A-2D .

[0455] Figures 1A-1E The stereomicroscope photos show the alveolar bone resorption of mice in the blank control group, negative control group and after administration of different doses of siRNA conjugates. Figure 1B and 1A By comparison, it can be seen that the alveolar bone of mice given siRNA in the negative control group was significantly absorbed, and the gap at the tooth root was obvious; in comparison, the alveolar bone absorption of mice in each dosage group given siRNA disclosed in the present invention was significantly inhibited, and the gap at the tooth root was significantly reduced, indicating that the symptoms of periodontitis were significantly improved.

[0456] Figures 2A-2DThe bar graphs show the relative expression levels of inflammatory factors IL-17a, IL6, Tnf-a in lymph node tissues of each group of mice and Tnfsf11 mRNA in periodontal tissues. Blank represents the blank control group, Control represents the negative control group, and GalNAc-siRNA represents the 3 mg / kg dose group. Figure 2A-2C The results show that, compared with the blank control group (denoted as relative level 1), the relative level of inflammatory factors in the negative control group mice that were only given the reference conjugate NC increased significantly to more than 4-6 times; in comparison, in the mice in each dose group given the siRNA conjugate described in the application or method of the present invention, the relative level of inflammatory factors decreased significantly, which was basically equivalent to or even lower than that of the blank control group, indicating that the application or method provided by the present invention can effectively inhibit the expression of inflammatory factors by using an RNAi agent containing siRNA conjugates, thereby inhibiting the symptoms of periodontitis. Considering that conjugate 3 is a siRNA conjugate designed to specifically target and deliver to hepatocytes, these inhibitions on periodontitis are unexpectedly excellent effects. As an activator of NF-κB. Tnfsf11 (RANKL) Therefore, RANKL is essential for osteoclast maturation, bone modeling and bone remodeling, and the development of lymph nodes (LN). RANKL is a factor that induces osteoclast differentiation. It can bind to NF-κB and induce monocyte / macrophage lineage cells to differentiate into osteoclasts, further leading to the maturation of osteoclast precursors. And by Figure 2D It can be seen from the results that the application or method provided by the present disclosure can effectively reduce the relative expression level of Tnfsf11 mRNA, thereby inhibiting the differentiation of local Th17 in periodontitis into RANKL.

[0457] The above results show that in periodontitis model mice, compared with the negative control group, subcutaneous administration of the siRNA conjugate described in the application or method provided by the present disclosure can unexpectedly significantly inhibit the degree of alveolar bone resorption in periodontitis model mice, and can inhibit the expression of inflammatory factors in the periodontitis model mice to the same level as that of normal mice. In addition, the siRNA conjugate described in the application or method provided by the present disclosure can also inhibit the expression of inflammatory factors in periodontitis and inhibit local Th17 differentiation in periodontitis, thereby inhibiting the expression of RANKL and osteoclast differentiation, and ultimately inhibiting bone homeostasis imbalance and bone loss, indicating that the application or method provided by the present disclosure can effectively treat periodontitis.

[0458] Some embodiments of the present disclosure are described in detail above; however, the present disclosure is not limited to the specific details in the above embodiments. Within the technical concept of the present disclosure, a variety of simple modifications can be made to the technical solution of the present disclosure, and these simple modifications all fall within the protection scope of the present disclosure.

[0459] It should also be noted that the various specific technical features described in some of the above embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present disclosure will not further explain various possible combinations.

[0460] In addition, various embodiments of the present disclosure may be arbitrarily combined, and as long as they do not violate the concept of the present disclosure, they should also be regarded as the contents disclosed by the present disclosure.

Claims

1. Use of an RNAi agent in the preparation of a drug for treating and / or preventing periodontitis and periodontitis-related diseases, wherein the RNAi agent contains a pharmaceutically active component, wherein the pharmaceutically active component is a siRNA conjugate and / or a pharmaceutically acceptable salt thereof, in, The siRNA conjugate comprises a siRNA group and a conjugated group covalently linked to the siRNA group, wherein the siRNA group is formed by removing one or more atoms or functional groups from a siRNA capable of inhibiting CC3 mRNA, and the conjugated group contains one or more targeting groups having affinity for mammalian asialoglycoprotein receptor (ASGPR).

2. A method for treating and / or preventing periodontitis and periodontitis-related diseases, the method comprising administering an effective amount of an RNAi agent to a subject in need thereof, wherein the RNAi agent contains a pharmaceutically active component, wherein the pharmaceutically active component is a siRNA conjugate and / or a pharmaceutically acceptable salt thereof, in, The siRNA conjugate comprises a siRNA group and a conjugated group covalently linked to the siRNA group, wherein the siRNA group is formed by removing one or more atoms or functional groups from a siRNA capable of inhibiting CC3 mRNA, and the conjugated group contains one or more targeting groups having affinity for mammalian asialoglycoprotein receptor (ASGPR).

3. The use according to claim 1 or the method according to claim 2, in, The siRNA group comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand are at least partially reverse complementary to form a double-stranded region, and each nucleotide in the sense strand and the antisense strand is independently a modified or unmodified nucleotide; the antisense strand comprises a nucleotide sequence that is completely reverse complementary to at least 15 consecutive nucleotides in CC3 mRNA; Alternatively, the sense strand contains a nucleotide sequence I, the antisense strand contains a nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II are both 19 nucleotides in length, the nucleotide sequence I and the nucleotide sequence II are at least partially reverse-complementary to form a double-stranded region, the nucleotide sequence II is completely reverse-complementary to at least 15 consecutive nucleotides of the first nucleotide sequence, and the first nucleotide sequence is a nucleotide sequence of 19 nucleotides in length in CC3 mRNA; Alternatively, the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary or completely reverse complementary, and the nucleotide sequence II is completely reverse complementary to 16, 17, 18 or 19 consecutive nucleotides in the first nucleotide sequence.

4. The use or method according to claim 3, in, (1) the sense strand further comprises nucleotide sequence III, and the antisense strand nucleotide sequence further comprises nucleotide sequence IV; and / or (2) the sense strand further comprises nucleotide sequence VI; and / or (3) the antisense strand further comprises a nucleotide sequence V; Wherein, the nucleotide sequence III and the nucleotide sequence IV are respectively composed of 1-4 nucleotides, the nucleotide sequence IV and the nucleotide sequence III are equal in length and are substantially reverse complementary or completely reverse complementary, the nucleotide sequence III is connected to the 5' end of the nucleotide sequence I, the nucleotide sequence IV is connected to the 3' end of the nucleotide sequence II, and the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to the second nucleotide sequence, and the second nucleotide sequence refers to a nucleotide sequence in CC3 mRNA that is adjacent to the 5' end of the first nucleotide sequence and has the same length as the nucleotide sequence IV; The nucleotide sequence V and the nucleotide sequence VI are each independently composed of 1-3 nucleotides, the nucleotide sequence V is connected to the 3' end of the nucleotide sequence II or the nucleotide sequence IV, and the nucleotide sequence VI is connected to the 3' end of the nucleotide sequence I or the nucleotide sequence III, respectively constituting the 3' overhang of the antisense strand or the sense strand; Alternatively, the sense strand consists of nucleotide sequence I, and the antisense strand consists of nucleotide sequence II and nucleotide sequence V.

5. The use or method according to claim 4, in, The first nucleotide sequence is the nucleotide sequence shown in SEQ ID NO: 1, and there are no more than 3 nucleotide differences between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 1; the nucleotide sequence II is completely reverse complementary to the nucleotide sequence shown in SEQ ID NO: 1 in 16-19 consecutive nucleotides: 5'-UCAUGGUGUUCGUGACGAA-3' (SEQ ID NO: 1); Alternatively, the first nucleotide sequence is the nucleotide sequence shown in SEQ ID NO:46, and there are no more than 3 nucleotide differences between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:46; and the nucleotide sequence II is completely reverse complementary to the nucleotide sequence shown in SEQ ID NO:46 in 16-19 consecutive nucleotides: 5'-CCUACGUGGUCAAGGUCUU-3' (SEQ ID NO: 46); Alternatively, the first nucleotide sequence is the nucleotide sequence shown in SEQ ID NO: 91, and there are no more than 3 nucleotide differences between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 91; and the nucleotide sequence II is completely reverse complementary to the nucleotide sequence shown in SEQ ID NO: 91 in 16-19 consecutive nucleotides: 5'-UGGCCCUACUGCAGCUAAA-3' (SEQ ID NO:91); Alternatively, the first nucleotide sequence is the nucleotide sequence shown in SEQ ID NO: 136, and there are no more than 3 nucleotide differences between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 136; and the nucleotide sequence II is completely reverse complementary to the nucleotide sequence shown in SEQ ID NO: 136 in a continuous 16-19 nucleotide sequence: 5'-GGAACACCCUCAUCAUCUA-3' (SEQ ID NO: 136); Alternatively, from the 5' end to the 3' end, at least the nucleotides 3 to 19 of the nucleotide sequence II are completely reverse complementary to the nucleotides 1 to 17 of the first nucleotide sequence; Alternatively, nucleotide sequence II is completely reverse complementary to the first nucleotide sequence; Alternatively, in the direction from the 5' end to the 3' end, the nucleotides at positions 2 to 19 of the nucleotide sequence II are completely reverse complementary to the nucleotides at positions 1 to 18 of the first nucleotide sequence; Alternatively, in the direction from the 5' end to the 3' end, the nucleotides at positions 1 and 3-19 of the nucleotide sequence II are completely reverse complementary to the nucleotides at positions 1-17 and 19 of the first nucleotide sequence.

6. The use or method according to claim 4, in, The nucleotide sequence V consists of 2 nucleotides, and in the direction from the 5' end to the 3' end, the nucleotide sequence V is 2 consecutive thymine deoxyribonucleotides, 2 consecutive uracil ribonucleotides, or is completely reverse complementary to the third nucleotide sequence; the third nucleotide sequence refers to a nucleotide sequence in CC3 mRNA that is adjacent to the 5' end of the first nucleotide sequence or the 5' end of the second nucleotide sequence and has a length of 2 nucleotides.

7. The use or method according to claim 4, in, The nucleotide sequence VI consists of 2 nucleotides, and in the direction from the 5' end to the 3' end, the nucleotide sequence VI is 2 consecutive thymine deoxyribonucleotides, 2 consecutive uracil ribonucleotides, or is completely the same as the fourth nucleotide sequence, and the fourth nucleotide sequence refers to a nucleotide sequence in CC3 mRNA that is adjacent to the 3' end of the first nucleotide sequence or the 3' end of the second nucleotide sequence and has a length of 2 nucleotides.

8. The use or method according to claim 4, in, The nucleotide sequence I, nucleotide sequence II, nucleotide sequence III and nucleotide sequence IV are selected from one of the following 1) to 4): 1) The first nucleotide sequence is the nucleotide sequence shown in SEQ ID NO: 1, and there are no more than 3 nucleotide differences between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 1; the nucleotide sequence II is identical to the nucleotide sequence shown in SEQ ID NO: 2 and 16 to 19 consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO: 2: 5'-UCAUGGUGUUCGUGACGAA-3' (SEQ ID NO: 1); 5'-UUCGUCACGAACACCAUGA-3'(SEQ ID NO:2), The length of nucleotide sequences III and IV is 1 nucleotide each, the base of nucleotide sequence III is C, and the base of nucleotide sequence IV is G; or, the length of nucleotide sequences III and IV is 2 nucleotides each, the base composition of nucleotide sequence III is CC, and the base composition of nucleotide sequence IV is GG; or, the length of nucleotide sequences III and IV is 3 nucleotides each, the base composition of nucleotide sequence III is ACC, and the base composition of nucleotide sequence IV is GGU; or, the length of nucleotide sequences III and IV is 4 nucleotides each, the base composition of nucleotide sequence III is GACC, and the base composition of nucleotide sequence IV is GGUC; 2) The first nucleotide sequence is the nucleotide sequence shown in SEQ ID NO:46, and there are no more than 3 nucleotide differences between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:46; the nucleotide sequence II is completely reverse complementary to the nucleotide sequence shown in SEQ ID NO:47 in terms of 16 to 19 consecutive nucleotides: 5'-CCUACGUGGUCAAGGUCUU-3' (SEQ ID NO: 46); 5'-AAGACCUUGACCACGUAGG-3'(SEQ ID NO:47), The length of the nucleotide sequences III and IV is 1 nucleotide each, the base of the nucleotide sequence III is G, and the base of the nucleotide sequence IV is C; or, the length of the nucleotide sequences III and IV is 2 nucleotides each, the base composition of the nucleotide sequence III is CG, and the base composition of the nucleotide sequence IV is CG; or, the length of the nucleotide sequences III and IV is 3 nucleotides each, the base composition of the nucleotide sequence III is CCG, and the base composition of the nucleotide sequence IV is CGG; or, the length of the nucleotide sequences III and IV is 4 nucleotides each, the base composition of the nucleotide sequence III is ACCG, and the base composition of the nucleotide sequence IV is CGGU; 3) The first nucleotide sequence is the nucleotide sequence shown in SEQ ID NO:91, and there are no more than 3 nucleotide differences between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:91; the nucleotide sequence II is completely reverse complementary to the nucleotide sequence shown in SEQ ID NO:92 in a continuous 16-19 nucleotides: 5'-UGGCCCUACUGCAGCUAAA-3' (SEQ ID NO:91); 5'-UUUAGCUGCAGUAGGGCCA-3'(SEQ ID NO:92), The length of the nucleotide sequences III and IV is 1 nucleotide each, the base of the nucleotide sequence III is U, and the base of the nucleotide sequence IV is A; or, the length of the nucleotide sequences III and IV is 2 nucleotides each, the base composition of the nucleotide sequence III is CU, and the base composition of the nucleotide sequence IV is AG; or, the length of the nucleotide sequences III and IV is 3 nucleotides each, the base composition of the nucleotide sequence III is UCU, and the base composition of the nucleotide sequence IV is AGA; or, the length of the nucleotide sequences III and IV is 4 nucleotides each, the base composition of the nucleotide sequence III is CUCU, and the base composition of the nucleotide sequence IV is AGAG; 4) The first nucleotide sequence is the nucleotide sequence shown in SEQ ID NO: 136, and there are no more than 3 nucleotide differences between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 136; the nucleotide sequence II is completely reverse complementary to the nucleotide sequence shown in SEQ ID NO: 137 in a continuous 16-19 nucleotide sequence: 5'-GGAACACCCUCAUCAUCUA-3' (SEQ ID NO: 136); 5'-UAGAUGAUGAGGGUGUUCC-3'(SEQ ID NO:137), The length of the nucleotide sequences III and IV is 1 nucleotide each, the base of the nucleotide sequence III is G, and the base of the nucleotide sequence IV is C; or, the length of the nucleotide sequences III and IV is 2 nucleotides each, the base composition of the nucleotide sequence III is UG, and the base composition of the nucleotide sequence IV is CA; or, the length of the nucleotide sequences III and IV is 3 nucleotides each, the base composition of the nucleotide sequence III is CUG, and the base composition of the nucleotide sequence IV is CAG; or, the length of the nucleotide sequences III and IV is 4 nucleotides each, the base composition of the nucleotide sequence III is ACUG, and the base composition of the nucleotide sequence IV is CAGU.

9. The use or method according to any one of claims 1 to 8, in, The sense strand and antisense strand have the same or different lengths, the sense strand has a length of 19-23 nucleotides, and the antisense strand has a length of 19-26 nucleotides.

10. The use or method according to any one of claims 3 to 9, in, Each nucleotide group in the siRNA group is a modified nucleotide group. In the direction from the 5' end to the 3' end, in the sense strand, the nucleotide groups at positions 7, 8, 9 or positions 5, 7, 8, 9 of the nucleotide sequence I are fluorinated modified nucleotide groups, and the nucleotide groups at the remaining positions in the sense strand are non-fluorinated modified nucleotide groups; in the antisense strand, the nucleotide groups at positions 2, 6, 14, 16 or positions 2, 6, 8, 9, 14, 16 of the nucleotide sequence II are fluorinated modified nucleotide groups, and the nucleotide groups at the remaining positions in the antisense strand are non-fluorinated modified nucleotide groups; Wherein, each of the non-fluorinated modified nucleotide groups is independently selected from one of nucleotide groups or nucleotide analog groups formed by replacing the hydroxyl group at the 2' position of the ribose group of the nucleotide group with a non-fluorinated group; Alternatively, in the direction from the 5' end to the 3' end, at least one of the 3rd to 5th nucleotide groups of the nucleotide sequence II is a stabilizing modified nucleotide group, wherein the stabilizing modified nucleotide group refers to a nucleotide group formed by replacing the 2'-hydroxyl group of the ribose of the nucleotide group with a stabilizing modified group, and, compared with an siRNA in which the nucleotide group at the corresponding position is an unmodified nucleotide, the thermal stability of the siRNA comprising the stabilizing modified nucleotide group is increased, and the steric hindrance of the stabilizing modified group is greater than that of the methoxy group; Alternatively, except for the stabilizing modified nucleotides, no more than 5 of the non-fluorinated modified nucleotides are ribose 2'-deoxynucleotides, and each of the remaining non-fluorinated modified nucleotides is a methoxy-modified nucleotide; or, each of the non-fluorinated modified nucleotides is a methoxy-modified nucleotide; Alternatively, each of the stabilizing modifying groups is 2'-O-methoxyethyl.

11. The use or method according to any one of claims 3 to 10, in, The siRNA group is a group formed by removing one atom or functional group from one of the following siRNAs: siCC3a1-M1S, siCC3a1-M2S, siCC3a5-M1S, siCC3a5-M2S, siCC3b1-M1S, siCC3b1-M2S, siCC3b5-M1S, siCC3b5-M2S, siCC3c1-M1S, siCC3c1-M2S, siCC3c5-M1S, siCC3c5-M2S, siCC3d1-M1S, siCC3d1-M2S, siCC3d5-M1S or siCC3d5-M2S.

12. The use or method according to claim 1 or 2, in, The pharmaceutically active component is a siRNA conjugate having a structure represented by formula (308) and / or a pharmaceutically acceptable salt thereof: in, n1 is an integer selected from 1-3, n3 is an integer selected from 0-4; Each m1, m2 or m3 is independently an integer selected from 2-10; R 10 , R 11 , R 12 , R 13 , R 14 or R 15 Each is independently H, or is selected from the group consisting of: C 1 -C 10 Alkyl, C 1 -C 10 Haloalkyl and C 1 -C 10 Alkoxy; R 3 It has the structure shown in formula A59: (A59) Among them, E 1 OH, SH or BH 2 , Nu represents the siRNA group; R 2 is a straight chain alkylene group having a length of 1 to 20 carbon atoms, wherein one or more carbon atoms are optionally replaced by any one or more selected from the group consisting of: C(O), NH, O, S, CH=N, S(O) 2 , C 2 -C 10 Alkenylene, C 2 -C 10 Alkynylidene, C 6 -C 10 Arylene, C 3 -C 18 Heterocyclylene and C 5 -C 10 heteroarylene; and wherein R 2 It may optionally have any one or more substituents selected from the group consisting of: C 1 -C 10 Alkyl, C 6 -C 10 Aryl, C 5 -C 10 Heteroaryl, C 1 -C 10 Haloalkyl, -OC 1 -C 10 Alkyl, -OC 1 -C 10 Alkylphenyl, -C 1 -C 10 Alkyl-OH, -OC 1 -C 10 Halogenated alkyl, -SC 1 -C 10 Alkyl, -SC 1 -C 10 Alkylphenyl, -C 1 -C 10 Alkyl -SH, -SC 1 -C 10 Haloalkyl, halogen substituent, -OH, -SH, -NH 2 , -C 1 -C 10 Alkyl-NH 2 、-N(C 1 -C 10 Alkyl)(C 1 -C 10 Alkyl), -NH(C 1 -C 10 Alkyl), -N(C 1 -C 10 Alkyl)(C 1 -C 10 Alkylphenyl), -NH(C 1 -C 10 alkylphenyl), cyano, nitro, -CO 2 H、-C(O)O(C 1 -C 10 Alkyl), -CON(C 1 -C 10 Alkyl)(C 1 -C 10 alkyl), -CONH(C 1 -C 10 Alkyl), -CONH 2 、-NHC(O)(C 1 -C 10 alkyl), -NHC(O)(phenyl), -N(C 1 -C 10 Alkyl)C(O)(C 1 -C 10 Alkyl), -N(C 1 -C 10 alkyl)C(O)(phenyl), -C(O)C 1 -C 10 Alkyl, -C(O)C 1 -C 10 Alkylphenyl, -C(O)C 1 -C 10 Haloalkyl, -OC(O)C 1 -C 10 Alkyl, -SO 2 (C 1 -C 10 Alkyl), -SO 2 (phenyl), -SO 2 (C 1 -C 10 Haloalkyl), -SO 2 NH 2 、-SO 2 NH(C 1 -C 10 Alkyl), -SO 2 NH(phenyl), -NHSO 2 (C 1 -C 10 Alkyl), -NHSO 2 (phenyl) and -NHSO 2 (C 1 -C 10 haloalkyl); Each L 1 is independently a straight chain alkylene group having a length of 1 to 70 carbon atoms, wherein one or more carbon atoms are optionally replaced by any one or more selected from the group consisting of: C(O), NH, O, S, CH=N, S(O) 2 , C 2 -C 10 Alkenylene, C 2 -C 10 Alkynylidene, C 6 -C 10 Arylene, C 3 -C 18 Heterocyclylene and C 5 -C 10 heteroarylene; and wherein L 1 It may optionally have any one or more substituents selected from the group consisting of: C 1 -C 10 Alkyl, C 6 -C 10 Aryl, C 5 -C 10 Heteroaryl, C 1 -C 10 Haloalkyl, -OC 1 -C 10 Alkyl, -OC 1 -C 10 Alkylphenyl, -C 1 -C 10 Alkyl-OH, -OC 1 -C 10 Halogenated alkyl, -SC 1 -C 10 Alkyl, -SC 1 -C 10 Alkylphenyl, -C 1 -C 10 Alkyl -SH, -SC 1 -C 10 Haloalkyl, halogen substituent, -OH, -SH, -NH 2 , -C 1 -C 10 Alkyl-NH 2 、-N(C 1 -C 10 Alkyl)(C 1 -C 10 Alkyl), -NH(C 1 -C 10 Alkyl), -N(C 1 -C 10 Alkyl)(C 1 -C 10 Alkylphenyl), -NH(C 1 -C 10 alkylphenyl), cyano, nitro, -CO 2 H、-C(O)O(C 1 -C 10 Alkyl), -CON(C 1 -C 10 Alkyl)(C 1 -C 10 alkyl), -CONH(C 1 -C 10 Alkyl), -CONH 2 , -NHC(O)(C 1 -C 10 alkyl), -NHC(O)(phenyl), -N(C 1 -C 10 alkyl)C(O)(C 1 -C 10 Alkyl), -N(C 1 -C 10 alkyl)C(O)(phenyl), -C(O)C 1 -C 10 Alkyl, -C(O)C 1 -C 10 Alkylphenyl, -C(O)C 1 -C 10 Haloalkyl, -OC(O)C 1 -C 10 Alkyl, -SO 2 (C 1 -C 10 Alkyl), -SO 2 (phenyl), -SO 2 (C 1 -C 10 Haloalkyl), -SO 2 NH 2 、-SO 2 NH(C 1 -C 10 Alkyl), -SO 2 NH(phenyl), -NHSO 2 (C 1 -C 10 Alkyl), -NHSO 2 (phenyl) and -NHSO 2 (C 1 -C 10 haloalkyl); represents the site of covalent attachment of a group; Each M 1 Independently selected from one of the targeting groups having affinity for ASGPR; Alternatively, each of the targeting groups having affinity for ASGPR is independently selected from one of asialoglycoprotein, lactose, galactose or N-acetylgalactosamine.

13. The use or method according to claim 12, in, The pharmaceutically active ingredient is a siRNA conjugate having a structure as shown in Formula (403), (404), (405), (406), (407), (408), (409), (410), (411), (412), (413), (414), (415), (416), (417), (418), (419), (420), (421) or (422) and / or a pharmaceutically acceptable salt thereof; Alternatively, the pharmaceutically active component is an alkali metal salt or a partial alkali metal salt of a siRNA conjugate having a structure represented by formula (403).

14. The use according to claim 1 or the method according to claim 2, in, The RNAi agent further contains a pharmaceutically acceptable excipient.

15. The use or method according to any one of claims 1 to 14, in, The periodontitis-related diseases are one or more of diabetes, stroke, myocardial infarction, atherosclerosis, hypertension, osteoporosis and premature birth caused or promoted by periodontitis.

16. The use or method according to any one of claims 1 to 15, in, The amount of the RNAi agent in the medicament or the amount used in the method makes the maximum inhibition rate of CC3 mRNA in the liver of the subject greater than 50%.

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