Double-stranded oligonucleotide targeting CRTH2 mRNA and application thereof

Abstract

The invention provides double-stranded oligonucleotide targeting CRTH2 mRNA and application of the double-stranded oligonucleotide, and relates to the technical field of biology. The positive-sense strand nucleotide sequence of the double-strand oligonucleotide comprises sequences which have no more than 8 nucleotides different from the sequences shown in SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7 and the like; the antisense strand nucleotide sequence contains a sequence which has no more than 8 nucleotides different from the sequences shown in SEQ ID NO.2, 4, 6, 8 and the like; and the positive-sense strand and the antisense strand are at least partially reversely complementary to form a double-strand body region. The double-stranded oligonucleotide can inhibit CRTH2 mRNA and reduce CRTH2 protein expression of a subject, and can be applied to preparation of drugs for treating and / or preventing diseases related to CRTH2.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a double-stranded oligonucleotide targeting CRTH2 mRNA and its applications. Background Technology

[0002] The following statements are provided only as background information in relation to the present invention and do not necessarily constitute prior art.

[0003] The Th2 cell chemotactic receptor homolog (CRTH2) has been shown to mediate chemotaxis in eosinophils, basophils, and Th2 T lymphocytes. The major mast cell product prostaglandin (PG)D2 is considered the major ligand for CRTH2. CRTH2 is the most reliable marker for detecting circulating human type 2 Th and type 2 T cytotoxic cells in both healthy and diseased states. The CRTh2 receptor is expressed on eosinophils, basophils, T helper cells 2, macrophages, and neutrophils, participating in the chemotaxis and activation of these cells, thus forming key events that trigger inflammatory responses in allergic diseases. Studies have shown that CRTH2, as a receptor for prostaglandin D2 (PGD2), plays a crucial role in various allergic and inflammatory diseases. In autoimmune diseases, CRTH2 may exacerbate inflammatory responses by promoting the activation and recruitment of Th2 cells; in atopic dermatitis, CRTH2 activation exacerbates skin inflammation, itching, and epidermal barrier dysfunction, driving disease progression; CRTH2 can promote eosinophil chemotaxis and survival, leading to esophageal and gastrointestinal inflammation; CRTH2 is highly expressed in airway inflammation in asthma and is associated with asthma; in addition, CRTH2 is also associated with allergic rhinitis, conjunctivitis, and the eosinophilic form of chronic obstructive pulmonary disease (COPD).

[0004] RNA interference (RNAi) is a gene silencing phenomenon induced by double-stranded RNA (dsRNA). By specifically degrading homologous mRNAs of dsRNAs synthesized in vitro or produced in vivo, RNAi interferes with the expression of target genes. RNAi can rapidly, easily, effectively, and specifically regulate gene expression in cells. It is not only a powerful tool for studying gene function but also provides a new technical means for specific gene therapy, with broad application prospects. Using double-stranded oligonucleotides that can target and bind to CRTH2 mRNA to induce CRTH2 gene silencing can block or reduce CRTH2 activity, thereby alleviating diseases related to abnormal CRTH2 expression. Therefore, developing a double-stranded oligonucleotide that can effectively target CRTH2 mRNA to inhibit CRTH2 activity in subjects is currently in demand in the market.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] The purpose of this invention is to provide an antisense oligonucleotide that targets CRTH2 mRNA in order to inhibit or block CRTH2 in subjects.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: In a first aspect, a double-stranded oligonucleotide targeting CRTH2 mRNA is provided, the double-stranded oligonucleotide comprising a sense strand and an antisense strand; the nucleotide sequence of the sense strand comprises a sequence differing from the sequence shown in SEQ ID NO. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55 or 57 by no more than 8 nucleotides; the nucleotide sequence of the antisense .... The sequences shown in NO.2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or 58 are sequences differing by no more than 8 nucleotides; and the sense and antisense strands are at least partially anticomplementary to form a double-stranded region.

[0008] In a second aspect, a complex of a double-stranded oligonucleotide targeting CRTH2 mRNA is provided, the complex comprising: (a) the double-stranded oligonucleotide targeting CRTH2 mRNA as described in the first aspect; and (b) one or more targeting delivery ligands linked to (a).

[0009] Thirdly, the double-stranded oligonucleotide targeting CRTH2 mRNA as described in the first aspect, or the complex as described in the second aspect, is provided for use in any of the following: (I) Inhibition of CRTH2 mRNA and / or reduction of CRTH2 protein expression in subjects for non-diagnostic and non-therapeutic purposes.

[0010] (II) Prepare drugs that inhibit CRTH2 mRNA and / or reduce CRTH2 protein expression in subjects.

[0011] (III) Prepare medicines for treating, preventing and / or alleviating pathological conditions or diseases caused by CRTH2 in the subject.

[0012] Fourthly, a pharmaceutical composition is provided, the pharmaceutical composition comprising the double-stranded oligonucleotide targeting CRTH2 mRNA as described in the first aspect, or the complex as described in the second aspect.

[0013] Fifthly, a method for inhibiting CRTH2 in a subject for non-diagnostic and therapeutic purposes is provided, the method comprising contacting the subject with a double-stranded oligonucleotide targeting CRTH2 mRNA as described in the first aspect, or a complex as described in the second aspect, or a pharmaceutical composition as described in the fourth aspect.

[0014] Compared with the prior art, the present invention has the following beneficial effects: The double-stranded oligonucleotides targeting CRTH2 mRNA provided by this invention can inhibit CRTH2 mRNA and reduce CRTH2 protein expression in subjects. The double-stranded oligonucleotides targeting CRTH2 mRNA provided by this invention, as well as complexes and pharmaceutical compositions containing them, may help treat and / or prevent CRTH2-related diseases. Attached Figure Description

[0015] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0016] Figure 1 It is the psiCHECK-2-CRTH2 vector. Detailed Implementation

[0017] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0018] In this document, the terms “comprising” or “including” are open-ended expressions used in this disclosure to mean the phrase “including but not limited to”, and are used interchangeably with it, meaning that they include the contents specified in this disclosure, but do not exclude other contents.

[0019] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may or may not occur as described below, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0020] In this document, “and / or” is used to indicate that one or both of the situations described may occur, for example, A and / or B includes (A and B) and (A or B).

[0021] In this document, unless otherwise stated, any numbering is used to distinguish one entity or behavior from another, and is not required to require or imply any actual relationship, order, or importance between these entities or behaviors, such as numbering i, ii; first, second, etc.

[0022] In this document, the terms “comprising” or “including” mean that the stated elements, integers or steps are included, but do not exclude any other elements, integers or steps.

[0023] In this article, the term "double-stranded oligonucleotide" refers to a double-stranded structure formed by two oligonucleotides through partial or complete base pairing. The two oligonucleotides include a sense strand and an antisense strand, which may or may not be the same length. Double-stranded oligonucleotides include ribonucleotides, deoxyribonucleotides, or nucleotides containing purine and pyrimidine bases or other naturally occurring, chemically or biochemically modified, non-natural, or derived nucleotides.

[0024] In this article, the term "small interfering RNA (siRNA)" refers to a double-stranded RNA of 17 to 25 nucleotides in length, containing a sense strand and an antisense strand. siRNA mediates the targeted cleavage of RNA transcripts via the RISC pathway by forming an RNA-induced silencing complex (RISC).

[0025] In this article, when "connection" refers to the connection of two molecules, the two molecules can be directly connected (e.g., directly connected by chemical bonds) or connected by additional molecules. When two molecules are directly connected, they can be covalently or non-covalently connected. Covalent connection refers to the connection of two molecules through the formation of covalent bonds, which are chemical bonds formed between atoms by sharing electron pairs. Non-covalent connection refers to connection methods other than covalent connection, such as connection through hydrogen bonds, van der Waals forces, electrostatic interactions, hydrophobic interactions, and metal coordination bonds.

[0026] In this document, the terms "subject" or "patient" refer to a mammalian subject or patient, and organs, tissues, or cells derived from them. The mammals include, but are not limited to, humans, rhesus monkeys, rats, mice, guinea pigs, rabbits, dogs, cats, hamsters, nude mice, ferrets, pigs, sheep, goats, cattle, horses, donkeys, dogs, cynomolgus monkeys, macaques, baboons, gibbons, golden monkeys, long-tailed macaques, marmosets, squirrel monkeys, pig-tailed macaques, or chimpanzees. In some embodiments, the subject is a human, rat, mouse, guinea pig, rhesus monkey, or cynomolgus monkey.

[0027] In this document, the terms “treatment,” “relief,” or “improvement” are used interchangeably. These terms refer to methods of achieving beneficial or desired outcomes, including, but not limited to, treatment benefits. A “treatment benefit” means the eradication or improvement of one or more diseases, conditions, or circumstances associated with the underlying barrier being treated.

[0028] In this article, the terms “prevention” and “avoidance” are used interchangeably to refer to methods for obtaining beneficial or desired outcomes, including but not limited to preventive benefits. To obtain a “preventive benefit,” a drug may be given to a subject at risk of developing a specific disease, or to a subject who reports one or more physiological symptoms of a disease, even if a diagnosis of the disease may not have been made.

[0029] In this document, the term “inhibition” may be used interchangeably with “reduction,” “silencing,” “downregulation,” “blocking,” and other similar terms, and includes any level of inhibition. Inhibition can be assessed by a reduction in the absolute or relative level of one or more of these variables compared to a control level. This control level can be any type of control level used in the art, such as a baseline level before administration or a level determined from an untreated or controlled (e.g., a buffer-only control or an inert agent control) subject, cell, or sample.

[0030] Firstly, a double-stranded oligonucleotide targeting CRTH2 mRNA is provided.

[0031] The double-stranded oligonucleotide comprises a sense strand and an antisense strand; The positive strand nucleotide sequence comprises a sequence that differs from the sequence shown in SEQ ID NO. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, or 57 by no more than 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides; the antisense strand nucleotide sequence comprises a sequence that differs from the sequence shown in SEQ ID NO. 1, 3, 5, 7, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, The sequences shown in NO.2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or 58 differ by no more than 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides; and the sense and antisense strands are at least partially anticomplementary to form a double-stranded region.

[0032] In optional embodiments, the sense and antisense strands of the double-stranded oligonucleotide each comprise sequences that differ from the sequences shown in SEQ ID NO. 1 and 2 by no more than 8 nucleotides; or, each comprise sequences that differ from the sequences shown in SEQ ID NO. 3 and 4 by no more than 8 nucleotides; or, each comprise sequences that differ from the sequences shown in SEQ ID NO. 5 and 6 by no more than 8 nucleotides; or, each comprise sequences that differ from the sequences shown in SEQ ID NO. 7 and 8 by no more than 8 nucleotides; or, each comprise sequences that differ from the sequences shown in SEQ ID NO. 9 and 10 by no more than 8 nucleotides; or, each comprise sequences that differ from the sequences shown in SEQ ID NO. 11 and 12 by no more than 8 nucleotides; or, each comprise sequences that differ from the sequences shown in SEQ ID NO. 13 and 14 by no more than 8 nucleotides; or, each comprise sequences that differ from the sequences shown in SEQ ID NO. 15 and 16 by no more than 8 nucleotides; or, each comprise sequences that differ from the sequences shown in SEQ ID NO. 17 and 18 ...1 and 12 by The sequences shown in SEQ ID NO. 19 and 20 differ by no more than 8 nucleotides; or, respectively, contain sequences that differ by no more than 8 nucleotides from the sequences shown in SEQ ID NO. 21 and 22; or, respectively, contain sequences that differ by no more than 8 nucleotides from the sequences shown in SEQ ID NO. 23 and 24; or, respectively, contain sequences that differ by no more than 8 nucleotides from the sequences shown in SEQ ID NO. 25 and 26; or, respectively, contain sequences that differ by no more than 8 nucleotides from the sequences shown in SEQ ID NO. 27 and 28; or, respectively, contain sequences that differ by no more than 8 nucleotides from the sequences shown in SEQ ID NO. 29 and 30; or, respectively, contain sequences that differ by no more than 8 nucleotides from the sequences shown in SEQ ID NO. 31 and 32; or, respectively, contain sequences that differ by no more than 8 nucleotides from the sequences shown in SEQ ID NO. 33 and 34; or, respectively, contain sequences that differ by no more than 8 nucleotides from the sequences shown in SEQ ID NO. 35 and 36; or, respectively, contain sequences that differ by no more than 8 nucleotides from the sequences shown in SEQ ID NO. 29 and 20; or, respectively, contain sequences that differ by no more than 8 nucleotides from the sequences shown in SEQ ID NO. 21 and 2 ... The sequences shown in SEQ ID NO. 37 and 38 differ by no more than 8 nucleotides; or, each contains a sequence that differs by no more than 8 nucleotides from the sequences shown in SEQ ID NO. 39 and 40; or, each contains a sequence that differs by no more than 8 nucleotides from the sequences shown in SEQ ID NO. 41 and 42; or, each contains a sequence that differs by no more than 8 nucleotides from the sequences shown in SEQ ID NO. 43 and 44; or, each contains a sequence that differs by no more than 8 nucleotides from the sequences shown in SEQ ID NO. 45 and 46; or, each contains a sequence that differs by no more than 8 nucleotides from the sequences shown in SEQ ID NO. 47 and 48; or, each contains a sequence that differs by no more than 8 nucleotides from the sequences shown in SEQ ID NO. 39 and 40 ...The sequences shown in SEQ ID NOs 49 and 50 may differ by no more than 8 nucleotides; or, respectively, may contain sequences differing by no more than 8 nucleotides from the sequences shown in SEQ ID NOs 51 and 52; or, respectively, may contain sequences differing by no more than 8 nucleotides from the sequences shown in SEQ ID NOs 53 and 54; or, respectively, may contain sequences differing by no more than 8 nucleotides from the sequences shown in SEQ ID NOs 55 and 56; or, respectively, may contain sequences differing by no more than 8 nucleotides from the sequences shown in SEQ ID NOs 57 and 58.

[0033] In an optional implementation, the differentially expressed nucleotides in the positive strand that correspond to the sequences shown in SEQ ID NO. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, or 57 originate from mutations at one or more sites; and / or from sequences shown in SEQ ID NO. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, or 57 that correspond to 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides adjacent to the 5' and / or 3' ends of the fragment containing the CRTH2 mRNA.

[0034] In an optional embodiment, the differentially expressed nucleotides in the antisense strand that correspond to the sequences shown in SEQ ID NO.2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or 58 originate from mutations at one or more sites; and / or from the sequences shown in SEQ ID NO.2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or 58 that correspond to 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides adjacent to the 5' and / or 3' ends of the fragment containing the CRTH2 mRNA.

[0035] In an optional embodiment, the sense strand nucleotide sequence of the double-stranded oligonucleotide is a sequence containing no more than 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides different from the sequence shown in SEQ ID NO. 1, and the antisense strand nucleotide sequence contains a sequence containing no more than 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides different from the sequence shown in SEQ ID NO. 2. Further optionally, the sense strand nucleotide sequence of the double-stranded oligonucleotide is the sequence shown in SEQ ID NO. 1, and the antisense strand nucleotide sequence is the sequence shown in SEQ ID NO. 2.

[0036] In an optional embodiment, the sense strand nucleotide sequence of the double-stranded oligonucleotide comprises a sequence that differs from the sequence shown in SEQ ID NO. 5 by no more than 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides, and the antisense strand nucleotide sequence comprises a sequence that differs from the sequence shown in SEQ ID NO. 6 by no more than 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides. Further optionally, the sense strand nucleotide sequence of the double-stranded oligonucleotide is the sequence shown in SEQ ID NO. 3, and the antisense strand nucleotide sequence is the sequence shown in SEQ ID NO. 4.

[0037] In an optional embodiment, the sense strand nucleotide sequence of the double-stranded oligonucleotide comprises a sequence that differs from the sequence shown in SEQ ID NO. 9 by no more than 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides, and the antisense strand nucleotide sequence comprises a sequence that differs from the sequence shown in SEQ ID NO. 10 by no more than 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides. Further optionally, the sense strand nucleotide sequence of the double-stranded oligonucleotide is the sequence shown in SEQ ID NO. 9, and the antisense strand nucleotide sequence is the sequence shown in SEQ ID NO. 10.

[0038] In an optional embodiment, at least one nucleoside internucleotide bond of the double-stranded oligonucleotide is a modified nucleoside internucleotide bond.

[0039] In an optional embodiment, at least one nucleoside inter-bond is a phosphate thioside inter-bond.

[0040] In optional embodiments, the first, second, or third internucleotide bond, calculated from the 5' end of the sense strand, is a thiophosphate internucleotide bond; and / or, the first, second, or third internucleotide bond, calculated from the 3' end of the sense strand, is a thiophosphate internucleotide bond; and / or, the first, second, or third internucleotide bond, calculated from the 5' end of the antisense strand, is a thiophosphate internucleotide bond; and / or, the first, second, or third internucleotide bond, calculated from the 3' end of the antisense strand, is a thiophosphate internucleotide bond.

[0041] In an optional embodiment, the double-stranded oligonucleotide contains at least one modified nucleotide.

[0042] In optional embodiments, the modified nucleotide includes one or more of the following: 2'-O-methoxyethyl modified nucleotides, 5'-methyl modified nucleotides, 2'-(S)-restricted ethyl modified nucleotides, 2'-O-methyl modified nucleotides, 2'-deoxy-2'-fluorinated modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, non-locked nucleotides, configuration-restricted nucleotides, restricted ethyl nucleotides, base-free nucleotides, 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-hydroxy-modified nucleotides, 2'-O-alkyl-modified nucleotides, morpholinyl nucleotides, bridging nucleic acids, peptide nucleic acids, locked nucleic acids, and non-locked nucleic acids. The modified nucleotide can be one or more modified nucleotides.

[0043] In an optional embodiment, the modified nucleotide includes at least one of 2'-F modified nucleotide, 2'-O-methylated modified nucleotide, and 5'-(E)-VP modified nucleotide.

[0044] In an optional implementation, the first nucleotide at the 5' end of the antisense strand is a nucleotide modified with 5'-(E)-VP.

[0045] In an optional embodiment, the double-stranded oligonucleotide is modified with DV18, wherein the DV18 modification includes: from the 5' to 3' direction, the nucleotides at positions 1-6, 8, and 12-21 of the sense strand are 2'-O-methylated nucleotides, and the nucleotides at positions 7 and 9-11 are 2'-deoxy-2'-fluorinated nucleotides; from the 5' to 3' direction, the nucleotides at positions 1, 3-5, 7, 10-13, 15, and 17-21 of the antisense strand are 2'-O-methylated nucleotides, and the nucleotides at positions 2, 6, 8, 9, 14, and 16 are 2'-deoxy-2'-fluorinated nucleotides; further optionally, the first nucleotide at the 5' end of the antisense strand is a 5'-(E)-VP modified nucleotide.

[0046] In an optional embodiment, the double-stranded oligonucleotide is modified with DV22, wherein the DV22 modification includes: from the 5' to 3' direction, the nucleotides at positions 1-6, 8, and 12-21 of the sense strand are 2'-O-methylated nucleotides, and the nucleotides at positions 7 and 9-12 are 2'-deoxy-2'-fluorinated nucleotides; from the 5' to 3' direction, the nucleotides at positions 1, 3-5, 7-13, 15, and 17-21 of the antisense strand are 2'-O-methylated nucleotides, and the nucleotides at positions 2, 6, 14, and 16 are 2'-deoxy-2'-fluorinated nucleotides; further optionally, the first nucleotide at the 5' end of the antisense strand is a 5'-(E)-VP modified nucleotide.

[0047] In an optional embodiment, the double-stranded oligonucleotide is ESC modified, wherein the ESC modification includes: from the 5' to 3' direction, the nucleotides at positions 2, 4, 6, 8, 12, 14, 16, 18, and 20 of the sense strand are 2'-O-methylated nucleotides, and the nucleotides at positions 1, 3, 5, 7, 9-11, 13, 15, 17, 19, and 21 of the antisense strand are 2'-deoxy-2'-fluorinated nucleotides; from the 5' to 3' direction, the nucleotides at positions 1, 3, 5, 7, 9, 11-13, 15, 17, 19, and 21 of the antisense strand are 2'-O-methylated nucleotides, and the nucleotides at positions 2, 4, 6, 8, 10, 14, 16, 18, and 20 of the antisense strand are 2'-deoxy-2'-fluorinated nucleotides; further optionally, the first nucleotide at the 5' end of the antisense strand is a 5'-(E)-VP modified nucleotide.

[0048] In an optional implementation, the double-stranded oligonucleotide is siRNA.

[0049] In a second aspect, a complex of a double-stranded oligonucleotide targeting CRTH2 mRNA is provided, the complex comprising: (a) the double-stranded oligonucleotide targeting CRTH2 mRNA as described in the first aspect; and (b) one or more targeting delivery ligands linked to (a), the targeting delivery ligands being, for example, but not limited to, nucleic acid aptamers, targeting peptides, or compound drugs.

[0050] Thirdly, a double-stranded oligonucleotide targeting CRTH2 mRNA, or the complex described in the second aspect, is provided for use in any of the following: (I) Inhibition of CRTH2 mRNA and / or reduction of CRTH2 protein expression in subjects for non-diagnostic and non-therapeutic purposes.

[0051] (II) Prepare drugs that inhibit CRTH2 mRNA and / or reduce CRTH2 protein expression in subjects.

[0052] (III) To prepare drugs for the treatment, prevention and / or relief of pathological conditions or diseases caused by CRTH2 in the subject; In optional embodiments, the pathological conditions or diseases caused by the subject's CRTH2 include, but are not limited to, inflammation, respiratory diseases, cardiovascular diseases, hypersensitivity reactions, or skin diseases. Optionally, the pathological conditions or diseases caused by the subject's CRTH2 include, but are not limited to, asthma (including allergic asthma, eosinophilic asthma, persistent asthma, severe asthma), chronic sinusitis with nasal polyps, nasal polyps, eosinophilia, end-stage renal disease, chronic obstructive pulmonary disease, rhinitis (including allergic rhinitis), conjunctivitis (including allergic conjunctivitis, allergic conjunctivitis), atopic dermatitis, androgenetic alopecia, colds, eosinophilic esophagitis, rhinovirus infection, or chronic urticaria.

[0053] Fourthly, a pharmaceutical composition is provided, the pharmaceutical composition comprising the double-stranded oligonucleotide targeting CRTH2 mRNA as described in the first aspect, or the complex as described in the second aspect.

[0054] In an optional embodiment, the pharmaceutical composition further comprises pharmaceutically acceptable optional excipients. The acceptable excipients may be any excipients known in the art and conventionally used. Examples of excipients include, but are not limited to, any physiologically compatible solvents, dispersion media, coatings, antibacterial agents, antifungal agents, pH adjusters, lyophilization protectants, and emulsifiers.

[0055] In an optional embodiment, the pharmaceutical composition comprises a double-stranded oligonucleotide for delivering CRTH2 mRNA or a delivery system for delivering the complex. The delivery cells include, but are not limited to, lipid nanoparticles, liposomes, nanoparticles, cationic lipids, cationic polymers, metal nanopolymers, nanorods, micelles, microvesicles, cell-penetrating peptides, viral particles, protein coats, or lipoglobules.

[0056] Fifthly, a method for inhibiting CRTH2 in a subject for non-diagnostic and therapeutic purposes is provided, the method comprising contacting the subject with a double-stranded oligonucleotide targeting CRTH2 mRNA as described in the first aspect, or a complex as described in the second aspect, or a pharmaceutical composition as described in the fourth aspect.

[0057] In an optional implementation, the subjects are selected from humans, rhesus monkeys, cynomolgus monkeys, or guinea pigs. After homology comparison with species such as humans, rats, mice, guinea pigs, cynomolgus monkeys, and rhesus monkeys, CRTH2si-1 and CRTH2si-3 can be matched with species such as humans, rats, mice, guinea pigs, rhesus monkeys, and cynomolgus monkeys with ≤3 mismatches.

[0058] In an optional implementation, the subject is a cell, such as 293T (human renal epithelial cells) or HuH-7 cells.

[0059] In an optional implementation, the subject is a cell, and the contact includes delivering the double-stranded oligonucleotide targeting CRTH2 mRNA or the complex into the cell using any method known in the art.

[0060] In an optional implementation, the subject is a cell, and the working concentration of the double-stranded oligonucleotide targeting CRTH2 mRNA can be, for example, but not limited to, 10 nM, 20 nM, 30 nM, 40 nM or 50 nM.

[0061] In an optional implementation, the subject is an individual organism, and the contact includes administering the drug to the subject via any suitable route known in the art, including but not limited to: oral administration, intravenous administration, intramuscular administration, subcutaneous administration, transdermal administration, airway administration (aerosol), pulmonary administration, nasal administration, ocular administration, or rectal administration.

[0062] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0063] Preparation Example 1: CRTH2 siRNA Synthesis Steps 1. Synthesis preparation: Nucleosides, including T, A, C, G and U, were synthesized using standard phosphoramide structural units and solid-phase supports. All monomers used were in 0.05 M acetonitrile solutions in phosphoramide.

[0064] 2. Synthesis: A 200 nmol synthesis column made of CPG solid support was packed into an Instrument Platinum 192 synthesizer, and the specified sequence was synthesized using a phosphorusamide coupling method. For the coupling step, the phosphorusamide monomer was added in an amount approximately 70 times greater than the loading on the solid support. A 0.3 M BTT solution in acetonitrile was used as the activator in the coupling step, and phosphorusamide condensation was carried out for 4.5 min. All other steps were performed according to the manufacturer's standard protocol. Dimethoxytriphenylmethyl (DMT) was removed using a 3% trichloroacetic acid solution in dichloromethane. Thiophosphate bonds were introduced by sulfurization for 4 min with 0.05 M DDTT in a pyridine:acetonitrile = 2:3 (v / v) solution.

[0065] 3. Ammonolysis and Deprotection: After synthesizing the specified sequence, the specified sequence bound to the solid-phase support was placed in a 7M ammonia-methanol solution and ammonolysed at 90°C for 105 min. The solid-phase support was then filtered off, and the ammonia was removed under reduced pressure. If the sample contained TBDMS-protected native RNA, the resulting sample needed to be dissolved in DMSO, and a solution of triethylamine:triethylamine trifluoride = 2:1 (v / v) was added. TBDMS protection was then removed at 65°C for 150 min. After deprotection, 1.8 mL of ethanol was added to the sample, and the mixture was thoroughly vortexed and placed in a -20°C freezer for 2.5 h. The sample was then removed and centrifuged at 4°C and 12500 rpm for 10 min. The supernatant was removed, and the residue was treated with reduced pressure to remove residual ethanol.

[0066] 4. Purification: Add 0.2 mL of water to the sample, shake thoroughly to dissolve, filter the sample using a 0.22 μm membrane, and then purify by high performance liquid chromatography to prepare single-stranded siRNA.

[0067] 5. Quantification: The purified sample was quantified using an ultra-micro spectrophotometer; 6. Annealing and packaging: Mix the sense and antisense strands of siRNA in equal molar amounts as required, let stand in a 90℃ oven for 15 min, remove and let stand at room temperature for more than 1 h until it returns to room temperature, and then dry the resulting sample.

[0068] Example 1 1. siRNA sequence used: Table 1: 29 CRTH2 siRNA sequences

[0069] Note: In the table, the nucleic acid sequences from the sense and antisense strands of hCRTH2 si-1 to the sense and antisense strands of hCRTH2 si-29 are SEQ ID NO.1-58, respectively.

[0070] 2. Experimental Procedure: (1) Construct the psiCHECK-2-CRTH2 vector, such as Figure 1 As shown.

[0071] (2) CRTH2 target gene siRNA transfection (10 nM): 1) Cell count: 293T; 24 hours before transfection, the cell suspension was diluted with fresh, preheated culture medium and plated. 2) Formation of the Lipomaster 3000 Reagent / DNA / T3000 Enhancer Reagent / siRNA / T3000 complex: ① Add opti-MEM serum-free culture medium and Lipomaster3000 Transfection Reagent to a 1.5mL sterile centrifuge tube at a ratio of 1:0.03 and mix well; ② In a 1.5 mL sterile centrifuge tube, add opti-MEM and siRNA of various concentrations at a ratio of 5:1 and mix well; in a new 1.5 mL sterile tube, add 2.5 μL opti-MEM and 0.02 μg plasmid psiCHECK-CRTH2, mix well, then add 0.2 μL T3000 Enhancer Reagent, mix well, and then add siRNA / opti-MEM to DNA / T3000Enhancer Reagent / opti-MEM; ③ Add DNA / T3000 Enhancer Reagent / siRNA / opti-MEM to the Lipomaster 3000 Reagent / opti-MEM in step ①, mix well, and let stand at room temperature for 10 minutes before transfection; 3) Transfection: The above Lipomaster 3000 Reagent / DNA / T3000 Enhancer Reagent / siRNA complex mixture was added dropwise to the cells, dispersed, and then incubated overnight at 37°C for 48 hours. 4) Collect cells and run them on an ELISA reader.

[0072] (3) CRTH2 target gene siRNA dual-luciferase detection: The siRNA was added to the firefly luciferase assay kit and the kidney luciferase assay kit for detection. The knockdown efficiency of siRNA on CRTH2 was statistically analyzed, and the results are shown in Table 2.

[0073] Table 2: Knockdown efficiency statistics of 29 CRTH2 siRNAs

[0074] Example 2 Cell Screening Validation 1. Validation of the knockdown efficiency of CRTH2 siRNA sequence in HuH-7 cells: (1) Cell count: HuH-7 cells; (2) Cell plating (48-well plate): Dilute the cell suspension with fresh, preheated culture medium and then plate the cells; (3) Transfection: Prepare the corresponding concentration of transfection complex using the kit (Dona Pharmaceuticals #DN003-05), add 25 μL of the prepared siRNA transfection complex to each cell well, shake gently to mix, and incubate in a 37℃ cell culture incubator; after 24 h, collect the cells, add lysis buffer and store at -80℃.

[0075] (4) RNA extraction, reverse transcription to cDNA and qPCR reaction: RNA was extracted using a fully automated nucleic acid extractor (Wuhan Nanomagnetic: #S48) and matching nucleic acid extraction reagents (Wuhan Nanomagnetic: #NMR0211), cDNA was synthesized, and the relative expression level of CRTH2 mRNA in cells of each experimental group was detected by qPCR.

[0076] 2. Experimental Results: The knockdown efficiency of the CRTH2 target siRNA sequence was verified in HuH-7 cells using transfection reagent (Dona Pharmaceuticals #DN003-05). The experimental results are shown in Table 3. Table 3: Validation of the knockdown efficiency of 29 CRTH2 siRNAs in HuH-7 cells

[0077] The knockdown efficiency of CRTHsi-1, CRTHsi-2, CRTHsi-3, CRTHsi-18, and CRTHsi-25 was further verified on HuH-7 target cells at a concentration of 50 nM. The experimental results are shown in Table 4. Table 4: Validation of the knockdown efficiency of the 6 CRTH2 siRNA sequences in HuH-7 cells

[0078] The main reagents and consumables used in the above embodiments are shown in Table 5.

[0079] Table 5: Reagents and Consumables

[0080] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A double-stranded oligonucleotide targeting CRTH2 mRNA, characterized in that, The double-stranded oligonucleotide comprises a sense strand and an antisense strand; The positive strand nucleotide sequence comprises a sequence that differs from the sequence shown in SEQ ID NO. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55 or 57 by no more than 8 nucleotides; The antisense strand nucleotide sequence comprises a sequence that differs from the sequence shown in SEQ ID NO.2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56 or 58 by no more than 8 nucleotides; Furthermore, the justice chain and the antisense chain are at least partially complementary in opposite directions, forming a bichain region.

2. The double-stranded oligonucleotide targeting CRTH2 mRNA according to claim 1, characterized in that, The sense strand nucleotide sequence contains a sequence that differs from the sequence shown in SEQ ID NO.1 by no more than 8 nucleotides, and the antisense strand nucleotide sequence contains a sequence that differs from the sequence shown in SEQ ID NO.2 by no more than 8 nucleotides. Alternatively, the sense strand nucleotide sequence may contain a sequence that differs from the sequence shown in SEQ ID NO.5 by no more than 8 nucleotides, and the antisense strand nucleotide sequence may contain a sequence that differs from the sequence shown in SEQ ID NO.6 by no more than 8 nucleotides. Alternatively, the sense strand nucleotide sequence may contain a sequence that differs from the sequence shown in SEQ ID NO. 9 by no more than 8 nucleotides, and the antisense strand nucleotide sequence may contain a sequence that differs from the sequence shown in SEQ ID NO. 10 by no more than 8 nucleotides. Optionally, the sense strand nucleotide sequence is the sequence shown in SEQ ID NO.1 and the antisense strand nucleotide sequence is the sequence shown in SEQ ID NO.2; or, the sense strand nucleotide sequence is the sequence shown in SEQ ID NO.5 and the antisense strand nucleotide sequence is the sequence shown in SEQ ID NO.6; or, the sense strand nucleotide sequence is the sequence shown in SEQ ID NO.9 and the antisense strand nucleotide sequence is the sequence shown in SEQ ID NO.

10.

3. The double-stranded oligonucleotide targeting CRTH2 mRNA according to claim 1, characterized in that, At least one internucleotide bond is a modified internucleotide bond; Optionally, at least one nucleoside inter-bond is a phosphate thioside inter-bond; Optionally, the first to third internucleotide bonds, counting from the 5' end of the positive chain, are thiophosphate internucleotide bonds; and / or, the first to third internucleotide bonds, counting from the 3' end of the positive chain, are thiophosphate internucleotide bonds; and / or, the first to third internucleotide bonds, counting from the 5' end of the antisense chain, are thiophosphate internucleotide bonds; and / or, the first to third internucleotide bonds, counting from the 3' end of the antisense chain, are thiophosphate internucleotide bonds.

4. The double-stranded oligonucleotide targeting CRTH2 mRNA according to any one of claims 1 to 3, characterized in that, The double-stranded oligonucleotide contains at least one modified nucleotide; Optionally, the modified nucleotides include one or more of the following: 2'-O-methoxyethyl modified nucleotides, 5'-methyl modified nucleotides, 2'-(S)-restricted ethyl modified nucleotides, 2'-O-methyl modified nucleotides, 2'-deoxy-2'-fluorinated modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, non-locked nucleotides, configuration-restricted nucleotides, restricted ethyl nucleotides, base-free nucleotides, 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-hydroxy-modified nucleotides, 2'-O-alkyl-modified nucleotides, morpholinyl nucleotides, bridging nucleic acids, peptide nucleic acids, locked nucleic acids, and non-locked nucleic acids. Optionally, the modified nucleotide includes at least one of 2'-F modified nucleotides and 2'-O-methylated modified nucleotides; Optionally, the 5' end of the antisense chain is modified with 5'-(E)-vinyl phosphate.

5. The double-stranded oligonucleotide targeting CRTH2 mRNA according to claim 4, characterized in that, The double-stranded oligonucleotide is modified with DV18, wherein the DV18 modification includes: from the 5' to 3' direction, the nucleotides at positions 1-6, 8, and 12-21 of the sense strand are 2'-O-methylated nucleotides, and the nucleotides at positions 7 and 9-11 are 2'-deoxy-2'-fluorinated nucleotides; from the 5' to 3' direction, the nucleotides at positions 1, 3-5, 7, 10-13, 15, and 17-21 of the antisense strand are 2'-O-methylated nucleotides, and the nucleotides at positions 2, 6, 8, 9, 14, and 16 are 2'-deoxy-2'-fluorinated nucleotides; further optionally, the 5' end of the antisense strand is modified with 5'-(E)-vinyl phosphate; Alternatively, the double-stranded oligonucleotide is modified with DV22, wherein the DV22 modification comprises: from the 5' to 3' direction, the nucleotides at positions 1-6, 8, and 12-21 of the sense strand are 2'-O-methylated nucleotides, and the nucleotides at positions 7 and 9-12 are 2'-deoxy-2'-fluorinated nucleotides; from the 5' to 3' direction, the nucleotides at positions 1, 3-5, 7-13, 15, and 17-21 of the antisense strand are 2'-O-methylated nucleotides, and the nucleotides at positions 2, 6, 14, and 16 are 2'-deoxy-2'-fluorinated nucleotides; further optionally, the 5' end of the antisense strand is modified with 5'-(E)-vinyl phosphate; Alternatively, the double-stranded oligonucleotide is ESC modified, wherein the ESC modification includes: from the 5' to 3' direction, the nucleotides at positions 2, 4, 6, 8, 12, 14, 16, 18, and 20 of the sense strand are 2'-O-methylated nucleotides, and the nucleotides at positions 1, 3, 5, 7, 9-11, 13, 15, 17, 19, and 21 of the antisense strand are 2'-deoxy-2'-fluorinated nucleotides; from the 5' to 3' direction, the nucleotides at positions 1, 3, 5, 7, 9, 11-13, 15, 17, 19, and 21 of the antisense strand are 2'-O-methylated nucleotides, and the nucleotides at positions 2, 4, 6, 8, 10, 14, 16, 18, and 20 of the antisense strand are 2'-deoxy-2'-fluorinated nucleotides; further optionally, the 5' end of the antisense strand is modified with 5'-(E)-vinyl phosphate; Optionally, the double-stranded oligonucleotide is siRNA.

6. A complex of double-stranded oligonucleotides targeting CRTH2 mRNA, characterized in that, The complex contains: (a) a double-stranded oligonucleotide targeting CRTH2 mRNA as described in any one of claims 1 to 5; and (b) one or more targeted delivery ligands linked to (a).

7. The double-stranded oligonucleotide targeting CRTH2 mRNA according to any one of claims 1 to 5, or the complex according to claim 6, used in any one of the following: (I) Inhibition of CRTH2 mRNA and / or reduction of CRTH2 protein expression in subjects for non-diagnostic and non-therapeutic purposes; (II) Prepare drugs that inhibit CRTH2 mRNA and / or reduce CRTH2 protein expression in subjects; (III) Prepare medicines for treating, preventing and / or alleviating pathological conditions or diseases caused by CRTH2 in the subject.

8. The application according to claim 7, characterized in that, The pathological conditions or diseases caused by the subject's CRTH2 include inflammation, respiratory diseases, cardiovascular diseases, hypersensitivity reactions, or skin diseases; Optionally, the pathological conditions or diseases caused by the subject's CRTH2 include asthma, chronic sinusitis with nasal polyps, nasal polyps, eosinophilia, end-stage renal disease, chronic obstructive pulmonary disease, rhinitis, conjunctivitis, atopic dermatitis, androgenetic alopecia, common cold, eosinophilic esophagitis, rhinovirus infection, or chronic urticaria.

9. A pharmaceutical composition, characterized in that, Includes the double-stranded oligonucleotide targeting CRTH2 mRNA as described in any one of claims 1 to 5, or the complex as described in claim 6.

10. A method for inhibiting CRTH2 in a subject for non-diagnostic and non-therapeutic purposes, characterized in that, This includes contacting the subject with the double-stranded oligonucleotide targeting CRTH2 mRNA as described in any one of claims 1 to 5, or the complex as described in claim 6, or the pharmaceutical composition as described in claim 9.

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