Antisense oligonucleotide for inhibiting trpv1 gene expression and application thereof

Abstract

The invention provides antisense oligonucleotide for inhibiting trpv1 gene expression and application of the antisense oligonucleotide.

Description

Antisense oligonucleotide for inhibiting TRPV1 gene expression and its application Technical Field

[0001] The present invention relates to the field of biomedicine and an antisense oligonucleotide for inhibiting the expression of the TRPV1 gene and its application. Background Art

[0002] Antisense oligonucleotides (ASOs) are single-stranded oligonucleotides that are complementary to the target gene's mRNA, typically containing 15-30 nucleotides. In 1978, Harvard University scientists Zamecni and Stephenson designed and synthesized a short RNA complementary to the Rous sarcoma virus gene, which inhibited Rous sarcoma virus replication and cellular transformation, thus proposing the concept of antisense nucleic acids. The launch of the first antisense oligonucleotide drug, fomivirsen, in 1998 marked the beginning of the era of clinical application of ASOs.

[0003] Based on the mechanism of action, antisense oligonucleotides can be divided into two categories: those involving RNA degradation and those inhibiting or regulating RNA expression through steric hindrance. Specifically: ① The double-stranded ASO-mRNA binding site acts as a substrate to recruit RNase H1, which then degrades the target transcript. ② ASO interacts with RISC (RNA-induced silencing complex), then binds to Ago2, directing RISC to the target mRNA for degradation. ③ ASO binds to pre-mRNA to change the polyadenylation site and reduce mRNA stability and expression levels. ④ ASO binds to the 5' cap region of mRNA, preventing the binding of translation initiation factors and inhibiting mRNA translation. ⑤ ASO inhibits the function of miRNA to increase the expression of its target mRNA. ⑥ ASO binds to the upstream open reading frame (uORF) and increases the amount of protein translated by the downstream ORF. It can be seen that the mechanism of action of antisense oligonucleotides is very diverse and there is huge room for development.

[0004] Transient receptor potential cation channel subfamily V member 1 (TRPV1) is a calcium-permeable, nonselective cation channel expressed in the peripheral and central terminals of small-diameter sensory neurons. Highly selective chemical ablation of peripheral nerve terminals containing TRPV1 or entire TRPV1-expressing neurons can be used to control chronic pain. Administration of potent TRPV1 antagonists to the perineuronal or nerve terminal regions inhibits calcium cytotoxicity and the noxious primary afferent population expressing TRPV1. This selective nerve ablation, termed "molecular neurosurgery," has the advantage of preserving motor, proprioceptive, and other somatosensory functions, which are crucial for coordinating movement, performing activities of daily living, and maintaining quality of life. Therefore, it holds promise as a potential therapeutic target for analgesic and immunoinflammatory conditions, such as dry eye, arthritis and joint pain, cancer pain, and intractable pain.

[0005] Existing treatments for chronic pain are often inadequate or can produce debilitating side effects. Ablating certain nociceptive sensory neurons while preserving all other sensory modalities and motor function represents a novel therapeutic approach to manage severe pain while avoiding off-target side effects. Therefore, developing a highly effective inhibitor that silences TRPV1 would provide an effective means for long-term treatment of pain, immune inflammation, and its complications with improved efficacy, specificity, stability, targeting, or tolerability.

[0006] TRPV1 has attracted widespread attention primarily because it plays a crucial role in the development of nociceptive heat perception and pathological heat hyperalgesia. Trpv1 knockout mice show significantly reduced reactivity to noxious heat stimuli, particularly in models of inflammatory pain, where heat hyperalgesia is significantly attenuated.

[0007] Currently, there are more than 70 drugs based on the TRPV1 target, most of which are small molecule drugs. There is currently no ASO drug, and patients need more diverse treatment options.

[0008] Summary of the Invention

[0009] Based on the defects of the prior art, the applicant of the present application obtained an antisense oligonucleotide that can effectively inhibit the expression of TRPV1 gene mRNA and protein. The modified antisense oligonucleotide of the present application has high stability while maintaining high inhibitory activity.

[0010] Specifically, this application relates to the following aspects:

[0011] An antisense oligonucleotide comprising a modified oligonucleotide formed by 12 to 50 nucleosides linked by internucleoside bonds, and having a nucleobase sequence comprising at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases of the nucleobase sequence shown in any one of SEQ ID NOs: 1-11. In some embodiments, the antisense oligonucleotide consists of 12 to 50 nucleosides linked by internucleoside bonds, at least one of the nucleosides is chemically modified, and has a nucleobase sequence comprising at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases of the nucleobase sequence shown in any one of SEQ ID NOs: 1-11.

[0012] Furthermore, the nucleobase sequence of the antisense oligonucleotide is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleobase sequence shown in any one of SEQ ID NOs: 1-11.

[0013] Furthermore, the modified oligonucleotide comprises or consists of a gap body, and the gap body has the following structure from 5' to 3':

[0014] 5' wing segment-gap segment-3' wing segment, wherein the nucleosides in the wing segment and the gap segment have different ribose sugars;

[0015] The combination of the number of nucleotides in the 5' wing segment - the number of nucleotides in the gap segment - the number of nucleotides in the 3' wing segment is selected from any one of the following:

[0016] 5-10-5, 4-8-4, 4-12-3, 4-12-4, 3-14-3, 2-13-5, 2-16-2, 1-18-1, 3-10-3, 2-10-2, 1-10-1, 2-8-2, 6-8-6 and 5-8-5.

[0017] Further, at least one internucleoside linkage is a modified internucleoside linkage.

[0018] Furthermore, the modified internucleoside bond is a phosphorothioate internucleoside bond.

[0019] Further, each internucleoside bond or each internucleoside bond in the gap matrix is ​​a phosphorothioate internucleoside bond.

[0020] Furthermore, the sugar ring of at least one nucleoside comprises a 2'-O-methoxyethyl modification and / or the sugar ring of at least one nucleoside is deoxyribose.

[0021] Furthermore, the sugar ring of each nucleoside in the 5'-wing segment and the 3'-wing segment comprises a 2'-O-methoxyethyl modification, and the sugar ring of each nucleoside in the gapmer is deoxyribose.

[0022] Furthermore, the modified oligonucleotide comprises 5-methylcytidine.

[0023] Furthermore, each cytidine, or each cytidine in the gap matrix is ​​5-methylcytidine.

[0024] Furthermore, the modified oligonucleotide comprises or consists of an oligonucleotide selected from any one of the following:

[0025] 1) Tes Ges Ges mCes Ges Ads Tds Gds Tds Gds mCds Ads Gds Tds Gds mCes Tes Ges Tes mCe (SEQ ID NO: 12);

[0026] 2) Aes Tes Ges Ges mCes Gds Ads Tds Gds Tds Gds mCds Ads Gds Tds Ges mCes Tes Ges Te (SEQ ID NO: 13);

[0027] 3) Aes Aes Ges Aes Aes Gds Tds mCds mCds mCds mCds Ads Tds Gds Gds Ges mCes mCes Ges mCe (SEQ ID NO: 14);

[0028] 4) Aes Aes Aes Ges Aes Ads Gds Tds mCds mCds mCds Cds Ads Tds Gds Ges Ges mCes mCes Ge (SEQ ID NO: 15);

[0029] 5) Tes Aes Aes Aes Ges Ads Ads Gds Tds mCds mCds mCds mCds Ads Tds Ges Ges Ges mCes mCe (SEQ ID NO: 16);

[0030] 6) Tes Tes Aes Aes Aes Gds Ads Ads Gds Tds mCds mCds mCds mCds Ads Tes Ges Ges Ges mCe (SEQ ID NO: 17);

[0031] 7) mCes Tes Tes Aes Aes Ads Gds Ads Ads Gds Tds mCds mCds mCds mCds Aes Tes Ges Ges Ge (SEQ ID NO: 18);

[0032] 8) Tes mCes Tes Tes Aes Ads Ads Gds Ads Ads Gds Tds mCds mCds mCds mCes Aes Tes Ges Ge (SEQ ID NO: 19);

[0033] 9) Tes Tes mCes Tes Tes Ads Ads Ads Gds Ads Ads Gds Tds mCds mCds mCes mCes Aes Tes Ge (SEQ ID NO: 20);

[0034] 10) Ges Aes Tes Ges Ges mCds Gds Ads Tds Gds Tds Gds mCds Ads Gds Tes Ges mCes Tes Ge (SEQ ID NO: 21);

[0035] 11) mCes Ges Aes Tes Ges Gds mCds Gds Ads Tds Gds Tds Gds mCds Ads Ges Tes Ges mCes Te (SEQ ID NO: 22);

[0036] in,

[0037] A = adenine nucleobase,

[0038] mC=5-methylcytosine nucleobase,

[0039] G = guanine nucleobase,

[0040] T = thymine nucleobase,

[0041] e=2'-O-MOE modified sugar,

[0042] d=2'-deoxyribose,

[0043] s = phosphorothioate internucleoside linkage.

[0044] A pharmaceutical composition comprising any one of the above-mentioned antisense oligonucleotides and a pharmaceutically acceptable carrier.

[0045] Use of any one of the above antisense oligonucleotides in the preparation of a medicament for preventing or treating a disease mediated by TRPV1 expression.

[0046] Furthermore, the disease mediated by TRPV1 expression is TRPV1-mediated pain, preferably arthritis or joint pain.

[0047] A method for preventing or treating a disease mediated by TRPV1 expression, comprising administering to a subject a therapeutically effective amount of any one of the above-mentioned antisense oligonucleotides or any one of the above-mentioned pharmaceutical compositions.

[0048] Furthermore, the disease mediated by TRPV1 expression is TRPV1-mediated pain, preferably arthritis or joint pain.

[0049] This application also involves the following aspects:

[0050] An antisense oligonucleotide comprising a modified oligonucleotide consisting of 12 to 50 linked nucleosides and having a nucleobase sequence comprising at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 linked nucleobases of any one of the nucleobase sequences of SEQ ID NOs: 1-11.

[0051] Furthermore, the antisense oligonucleotide comprises a nucleotide sequence that is at least 90%, preferably at least 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 1-11.

[0052] Further, at least one internucleoside linkage is a modified internucleoside linkage.

[0053] Further, each internucleoside linkage is a phosphorothioate internucleoside linkage.

[0054] Furthermore, at least one nucleoside of the modified oligonucleotide comprises a modified sugar.

[0055] Further, at least one modified sugar comprises a 2'-O-methoxyethyl group.

[0056] Further, the modified oligonucleotide comprises: a gap segment consisting of 10 linked deoxynucleosides, a 5' wing segment consisting of 5 linked nucleosides, and a 3' wing segment consisting of 5 linked nucleosides;

[0057] wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment, each nucleoside of each wing segment comprises a 2'-O-methoxyethyl modified sugar, each internucleoside linkage is a phosphorothioate linkage, and wherein each cytosine of the modified oligonucleotide is a 5-methylcytosine.

[0058] Furthermore, the modified oligonucleotide is selected from any one of the following oligonucleotides:

[0059] 1) Tes Ges Ges mCes Ges Ads Tds Gds Tds Gds mCds Ads Gds Tds Gds mCes Tes Ges Tes mCe (SEQ ID NO: 12);

[0060] 2) Aes Tes Ges Ges mCes Gds Ads Tds Gds Tds Gds mCds Ads Gds Tds Ges mCes Tes Ges Te (SEQ ID NO: 13);

[0061] 3) Aes Aes Ges Aes Aes Gds Tds mCds mCds mCds mCds Ads Tds Gds Gds Ges mCes mCes Ges mCe (SEQ ID NO: 14);

[0062] 4) Aes Aes Aes Ges Aes Ads Gds Tds mCds mCds mCds Cds Ads Tds Gds Ges Ges mCes mCes Ge (SEQ ID NO: 15);

[0063] 5) Tes Aes Aes Aes Ges Ads Ads Gds Tds mCds mCds mCds mCds Ads Tds Ges Ges Ges mCes mCe (SEQ ID NO: 16);

[0064] 6) Tes Tes Aes Aes Aes Gds Ads Ads Gds Tds mCds mCds mCds mCds Ads Tes Ges Ges Ges mCe (SEQ ID NO: 17);

[0065] 7) mCes Tes Tes Aes Aes Ads Gds Ads Ads Gds Tds mCds mCds mCds mCds Aes Tes Ges Ges Ge (SEQ ID NO: 18);

[0066] 8) Tes mCes Tes Tes Aes Ads Ads Gds Ads Ads Gds Tds mCds mCds mCds mCes Aes Tes Ges Ge (SEQ ID NO: 19);

[0067] 9) Tes Tes mCes Tes Tes Ads Ads Ads Gds Ads Ads Gds Tds mCds mCds mCes mCes Aes Tes Ge (SEQ ID NO: 20);

[0068] 10) Ges Aes Tes Ges Ges mCds Gds Ads Tds Gds Tds Gds mCds Ads Gds Tes Ges mCes Tes Ge (SEQ ID NO: 21);

[0069] 11) mCes Ges Aes Tes Ges Gds mCds Gds Ads Tds Gds Tds Gds mCds Ads Ges Tes Ges mCes Te (SEQ ID NO: 22);

[0070] in,

[0071] A = adenine nucleobase,

[0072] mC=5-methylcytosine nucleobase,

[0073] G = guanine nucleobase,

[0074] T = thymine nucleobase,

[0075] e=2'-O-MOE modified sugar,

[0076] d=2'-deoxyribose,

[0077] s = phosphorothioate internucleoside linkage.

[0078] Use of any one of the above antisense oligonucleotides in the preparation of a medicament for preventing / treating diseases mediated by TRPV1 expression.

[0079] Furthermore, the diseases include analgesic and immune inflammatory diseases such as dry eye, cough, respiratory diseases, arthritis and joint pain, cancer pain, and intractable pain.

[0080] A pharmaceutical composition comprises a pharmacologically effective amount of any one of the above antisense oligonucleotides and other pharmaceutically acceptable components.

[0081] A method comprising administering any one of the above-mentioned antisense oligonucleotides or pharmaceutical compositions to an animal.

[0082] Furthermore, the animal is a human.

[0083] Furthermore, the method further comprises administering a second therapeutic agent to the animal. Beneficial effects

[0084] The antisense oligonucleotides of the present application have high stability and high inhibitory activity; while maintaining high inhibitory activity and stability, the antisense oligonucleotides of the present application can adopt a single-dose local administration treatment strategy to reduce the impact on other tissues or organs and reduce the amount of antisense oligonucleotides used, thereby achieving the purpose of reducing toxicity and reducing costs; the antisense oligonucleotides of the present application can enter target cells and target tissues without the need for transfection reagents, reducing the negative effects of transfection reagents, such as cell or tissue toxicity, thereby providing the possibility for targeted therapy; in vivo pharmacodynamic experiments in rats have shown that compared with the clinical Class I drug celecoxib, the antisense oligonucleotides of the present application have a faster onset of action and a longer TRPV1 analgesia time, with significant effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] Figure 1: High-throughput screening of TRPV1 candidate sequence gene expression results.

[0086] Figure 2A-2D: IC values ​​calculated for TRPV1-ASO candidate sequences in 293T cells 50 Figure 2A: Y3, Figure 2B: Y5, Figure 2C: Y8, Figure 2D: Y11.

[0087] Figure 3: Gene expression results of candidate sequences in articular cartilage of SD rats.

[0088] Figure 4: PWT effect of the drug in a behavioral efficacy study in SD rats. Two-way ANOVA analysis was used. *** indicates P < 0.001 compared with the model group.

[0089] Figure 5: PWT change rate in the behavioral efficacy test in SD rats. Two-way ANOVA analysis was used. * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001 compared with the model group.

[0090] Figure 6: Histopathological examination results.

[0091] Figure 7: Mankin score. DETAILED DESCRIPTION

[0092] The present application is further described below in conjunction with the implementation methods. It should be understood that the implementation methods are only used to further describe and illustrate the present application and are not used to limit the present application.

[0093] Unless otherwise defined, technical and scientific terms used in this specification have the same meanings as those commonly understood by those skilled in the art. Although methods and materials similar or identical to those described herein may be used in experiments or practical applications, the materials and methods are described herein below. In the event of a conflict, the present specification, including definitions, will prevail. In addition, the materials, methods, and examples are provided for illustrative purposes only and are not intended to be limiting. The following description further illustrates the present application in conjunction with specific embodiments, but is not intended to limit the scope of the present application.

[0094] definition

[0095] "TRPV1" refers to transient receptor potential cation channel subfamily V member 1 (TRPV1), also known as vanilloid receptor 1 (VR-1), a capsaicin-responsive ligand-gated cation channel protein. An example of a TRPV1 mRNA sequence is shown in GenBank Accession No. NM__018727.5.

[0096] "G", "C", "A", "U", and "T" generally represent nucleotides comprising guanine, cytosine, adenine, uracil, and thymine as bases, respectively. It should be understood that the term "ribonucleotide" or "nucleotide" or "deoxyribonucleotide" may also refer to a modified nucleotide or a natural nucleotide. The skilled artisan is well aware that certain portions of guanine, cytosine, adenine, uracil, and thymine may be substituted without substantially altering the base pairing properties of an oligonucleotide (including a nucleotide having such substituted portions). Examples of the present application include sequences of nucleotides having such substituted portions.

[0097] "Antisense oligonucleotides," also known as ASOs, are single-stranded oligonucleotides that are complementary to mRNA through base pairing, including ASOs that recruit RNase H1 to cleave target RNA. Antisense oligonucleotides can direct sequence-specific degradation of mRNA, for example, by degrading TRPV1 mRNA, thereby inhibiting TRPV1 expression in cells, such as cells in a subject (e.g., a mammalian subject).

[0098] "Nucleobase" refers to the heterocyclic portion of a nucleotide that can pair with the base of another nucleotide. Therefore, as used in this application, the term "nucleobase sequence" refers to the order of arrangement of nucleotides defined by the base sequence, and depending on the context, it can also refer to a nucleic acid molecule or nucleic acid fragment composed of nucleotides arranged in this order. Unless otherwise specified, the nucleobase sequence is the order of arrangement of nucleotides from 5' to 3'. Among them, the bases can be bases present in natural nucleotides, or modified bases, such as 5'methylated cytosine; the sugar ring (or "ribose") in the nucleotide can be a sugar ring present in natural DNA or RNA, or a modified sugar ring, such as a 2'-O-MOE modified sugar ring.

[0099] "Oligonucleotide" refers to a polymer composed of nucleosides linked by internucleoside bonds. Each nucleoside may be modified or unmodified, and the modifications of each nucleoside are independent of the others. "Internucleoside bond" refers to the chemical bond between nucleosides, for example, a phosphate bond or a substituted phosphate bond, such as a phosphorothioate bond.

[0100] "2'-modification" refers to the replacement of the H or OH group at the 2' position of the furanose ring of a nucleoside or nucleotide with another group. As used herein, the term "nucleotide furanose ring" refers to the sugar ring in a natural nucleotide. In this application, a "nucleotide furanose ring" or a chemically modified or engineered "nucleotide furanose ring" may be referred to as the "sugar ring" of a nucleoside, as long as it still forms a nucleoside after being linked to a base without losing its base-pairing function and the nucleic acid molecule in which it is located still has the corresponding transcription, translation, or regulatory function.

[0101] "2'-O-methoxyethyl," "2'-O-MOE," or "2'-OCH2CH2-OCH3" refers to an O-methoxyethyl modification at the 2' position of the furanose ring. A 2'-O-methoxyethyl-modified sugar is a modified sugar. "2'-O-MOE nucleoside / nucleotide" or "2'-O-methoxyethyl nucleoside / nucleotide" refers to a nucleoside / nucleotide containing a 2'-MOE-modified sugar moiety.

[0102] "5-methylcytosine" refers to a cytosine having a methyl modification attached to the 5' position.

[0103] "Inhibit" refers to a reduction or blocking of the expression or activity of a target nucleic acid or protein, and does not necessarily indicate complete elimination of target expression or activity.

[0104] "Complementarity" or "complementarity" refers to the ability of a nucleobase of a first nucleic acid chain to base pair with a nucleobase of a second nucleic acid chain, mediated by hydrogen bonding (e.g., Watson-Crick, Hodgkin's, or anti-Hodgkin's hydrogen bonding) between the corresponding nucleobases. For example, in DNA, adenine (A) is complementary to thymine (T); while guanosine (G) is complementary to cytosine (C). For example, in RNA, adenine (A) is complementary to uracil (U); while guanosine (G) is complementary to cytosine (C). In certain embodiments, complementary nucleobases refer to the ability of a nucleobase of an antisense oligonucleotide to pair with a nucleobase of its target nucleic acid (e.g., mRNA). For example, if a nucleobase at a certain position of an antisense oligonucleotide is able to form a hydrogen bond with a nucleobase at a certain position of a target nucleic acid, then the position at which hydrogen bonds are formed between the oligonucleotide and the target nucleic acid is considered to be complementary at that complementary base pair. Nucleobases comprising certain modifications can maintain the ability to pair with corresponding nucleobases and are therefore still capable of nucleobase complementarity.

[0105] "Effective amount" refers to an amount sufficient to achieve a beneficial or desired result. For example, a therapeutic amount is an amount that achieves a desired therapeutic effect. This amount may be the same as or different from a prophylactic effective amount, which is the amount necessary to prevent the onset of a disease or disease symptoms. An effective amount can be administered in one or more administrations, applications, or dosages. A "therapeutically effective amount" (i.e., an effective dose) of a therapeutic compound (such as the antisense oligonucleotide of the present application) depends on the therapeutic compound selected. Administration of the therapeutic compound may range from one or more times per day to one or more times per week; including every other day. It will be understood by those skilled in the art that certain factors may affect the dosage and time required to effectively treat a subject, including but not limited to the severity of the subject's disease or condition, previous treatment, overall health and / or age, and other diseases present.

[0106] The terms "homology" or "identity" refer to the identity of subunit sequences between two polymeric molecules, for example, between two nucleic acid molecules such as two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both molecules is occupied by the same monomeric subunit, for example, if a position in each of the two DNA molecules is occupied by adenine, then they are homologous or identical at that position. The homology between two sequences is a direct function of the number of matching or homologous positions; for example, if half of the positions in the two sequences (e.g., five positions in a 10-subunit polymer) are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 out of 10) are matching or homologous, the two sequences are 90% homologous. The percentage of "sequence identity" can be determined by comparing two optimally aligned sequences over a comparison window, where for optimal alignment of the two sequences, a segment of the amino acid sequence in the comparison window may contain additions or deletions (e.g., gaps or overhangs) relative to the reference sequence (which does not contain additions or deletions). This percentage can be calculated as follows: the number of matching positions is generated by determining the number of positions at which the identical amino acid residue occurs in both sequences, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying by 100 to generate the percentage of sequence identity. The output is the percentage identity of the subject sequence relative to the query sequence. As used herein, a percentage of "identity," such as 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5% identity, refers to a degree of similarity between amino acid sequences or between nucleotide sequences determined by sequence alignment of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%. For example, the ratio of the number of positions with identical bases or amino acid residues to the total number of positions determined after two sequences have identical residues at as many positions as possible by introducing gaps or the like. The percentage of "identity" can be determined using software programs known in the art. Preferably, the comparison is performed using default parameters. A preferred comparison program is BLAST. Preferred programs are BLASTN and BLASTP. Details of these programs can be found on the corresponding pages of the NCBI website. It should be noted that when describing the identity of two nuclear base sequences, it is only necessary that the bases of the two nucleosides are the same, or that the bases of the natural nucleosides before modification are the same, to determine that the two nucleosides are nucleosides with the same base, or to consider that the two nucleosides have identity.

[0107] The purpose of "treating" is to prevent or slow down an undesirable physiological change or disorder. For the purposes of this application, beneficial or desired clinical results include, but are not limited to, detectable or undetectable: alleviation of symptoms, reduction in disease severity, stabilization (i.e., non-worsening) of the disease state, delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (partial or complete), such as relief of inflammation, raising the pain threshold, etc. "Treatment" may also refer to prolonging survival compared to expected survival if not receiving treatment.

[0108] "Pharmaceutically acceptable" refers to compounds, materials, compositions and / or dosage forms that are suitable for use in contact with human or animal tissues without excessive toxicity, irritation, allergic response, other problems or complications, and with a reasonable benefit / risk ratio. In some embodiments, pharmaceutically acceptable compounds, materials, compositions and / or dosage forms are those approved by regulatory agencies (such as the U.S. Food and Drug Administration, the China National Medical Products Administration, the European Medicines Agency) or listed in recognized pharmacopoeias (such as the U.S. Pharmacopoeia, the Chinese Pharmacopoeia, the European Pharmacopoeia) for use in animals, particularly humans.

[0109] "Pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, that is used to carry or transport the antisense oligonucleotides of the present invention from one location, body fluid, tissue, organ (internal or external), or body part to another location, body fluid, tissue, organ, or body part. A pharmaceutically acceptable carrier can be a vehicle, diluent, excipient, or other material that can be used to contact animal tissue without excessive toxicity or adverse reactions. Exemplary pharmaceutically acceptable carriers include, but are not limited to, sugars, starches, cellulose, malt, tragacanth gum, gelatin, Ringer's solution, alginic acid, isotonic saline, buffers, and the like.

[0110] "Subject" refers to an animal, human or non-human, to whom the treatment described in the methods of the present invention is provided. Veterinary and non-veterinary applications are contemplated. The term includes, but is not limited to, mammals such as humans, other primates, pigs, rodents such as mice and rats, rabbits, guinea pigs, hamsters, cattle, horses, cats, dogs, sheep, and goats. Typical subjects include humans, farm animals, and domestic pets such as cats and dogs.

[0111] The purpose of this application is to provide a method, compound and composition for inhibiting the expression of TRPV1 gene mRNA and protein.

[0112] antisense oligonucleotides

[0113] In a first aspect, the present application relates to an antisense compound that regulates the expression levels of TRPV1 mRNA and protein.

[0114] In some embodiments, the antisense compound is an antisense oligonucleotide.

[0115] In some embodiments, the antisense oligonucleotides of the present application include single-stranded RNA that interacts with a target RNA sequence, such as a TRPV1 target mRNA sequence, to guide cleavage of the target RNA.

[0116] In some embodiments, the antisense oligonucleotide can be a single-stranded RNA introduced into a cell or organism to inhibit the target mRNA. In some embodiments, the single-stranded RNA recruits RNase H1 through the double-stranded structure of the ASO-mRNA binding, and then degrades the target transcript, such as the mRNA.

[0117] In some embodiments, the "modulation" can occur in a cell or tissue; in certain embodiments, the cell or tissue is in an animal, and in certain embodiments, the animal is a human.

[0118] In some embodiments, the "modulation" includes upregulation or downregulation.

[0119] In some embodiments, trpv1 mRNA levels are reduced.

[0120] In some embodiments, the level of TRPV1 protein is reduced. The reduction can occur in a time-dependent or dose-dependent manner.

[0121] In some embodiments, the antisense oligonucleotide is capable of inhibiting TRPV1 gene expression in humans, monkeys, rabbits, or rats.

[0122] In some embodiments, the antisense oligonucleotide comprises a modified oligonucleotide consisting of 12 to 50 nucleosides linked by internucleoside linkages, wherein the nucleobase sequence of the modified oligonucleotide is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to an equal length portion of a trpv1 nucleic acid, and wherein the modified oligonucleotide comprises at least one modification selected from a modified sugar and a modified internucleoside linkage.

[0123] In some embodiments, the antisense oligonucleotide comprises a modified oligonucleotide formed by 12 to 50 nucleosides linked by internucleoside bonds, and has a nucleobase sequence comprising at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases of the nucleobase sequence shown in any one of SEQ ID NOs: 1-11.

[0124] Those skilled in the art will appreciate that any of SEQ ID NOs: 1-11 may represent a DNA sequence or an RNA sequence, i.e., "T" in SEQ ID NOs: 1-11 may represent either a T in a DNA sequence or a U in an RNA sequence.

[0125] In some embodiments, the antisense oligonucleotide is a modified oligonucleotide formed by 12 to 50 nucleosides linked by internucleoside bonds, and has a nucleobase sequence comprising at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases of the nucleobase sequence shown in any one of SEQ ID NOs: 1-11.

[0126] The modified oligonucleotides include but are not limited to modified sugar rings and modified internucleoside bonds. The antisense compounds of the present application (e.g., antisense oligonucleotides) may optionally include one or more nucleosides, wherein the sugar group has been modified. Such sugar-modified nucleosides can impart enhanced nuclease stability, increased binding affinity, or some other beneficial biological properties to the antisense compounds.

[0127] In certain embodiments, the nucleoside comprises a chemically modified ribofuranose ring portion. Examples of chemical modifications of the ribofuranose ring include, but are not limited to, the addition of substituent groups (including 5' and 2' substituent groups) to the sugar ring of a natural nucleoside, bridging of non-geminal ring atoms to form bicyclic nucleic acids (BNAs), replacement of the oxygen atoms of the sugar ring of a natural nucleoside with S, N(R), or C(R1)(R2) (R, R1, and R2 are each independently H, C1-C12 alkyl, or a protecting group), and combinations of the foregoing chemical modifications.

[0128] Examples of chemically modified sugars include 2′-F-5′-methyl substituted nucleosides or replacement of the ribosyl ring oxygen atom of a natural nucleoside with S and further substitution at the 2′-position or optionally 5′-substituted BNA (see PCT International Application WO 2007 / 134181 published on November 22, 2007, in which LNA is substituted with, for example, a 5′-methyl or 5′-vinyl group).

[0129] Examples of nucleosides with modified sugar moieties include, but are not limited to, nucleosides comprising 5′-vinyl, 5′-methyl (R or S), 4′-S, 2′-F, 2′-OCH 3 , 2′-OCH 2 CH 3 , 2′-OCH 2 CH 2 F and / or 2′-O(CH 2 ) 2 OCH 3 substituent groups. The substituent at the 2′ position may also be selected from alkyl, amino, azido, thio, O-alkyl, O—C 1 -C 10 alkyl, OCF 3 , OCH 2 F, O(CH 2 ) 2 SCH 3 , O(CH 2 ) 2 —ON(R m )(R n ), O—CH 2 —C(═O)—N(R m )(R n ) and O-CH2-C(=O)-N(R m )(R n ), where each R1, R m and R n and R is independently H or substituted or unsubstituted C1-C10 alkyl.

[0130] In some embodiments, the 2′-substituent group can also be selected from: C1-C12 alkyl, substituted alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkylaryl, aminoalkylamino, polyalkylamino, substituted silyl.

[0131] In some embodiments, the modified nucleoside comprises a 2'-O-MOE side chain.

[0132] In some embodiments, the sugar ring of at least one nucleoside comprises a 2'-O-methoxyethyl modification and / or the sugar ring of the at least one nucleoside is deoxyribose.

[0133] The naturally occurring internucleoside linkage of RNA and DNA is a 3' to 5' phosphodiester bond. Because of desirable properties such as increased cellular uptake, enhanced affinity for target nucleic acids, and increased stability in the presence of nucleases, antisense compounds having one or more modified, i.e., non-naturally occurring, internucleoside linkages are generally selected over antisense compounds having naturally occurring internucleoside linkages.

[0134] In some embodiments, the oligonucleotides have modified internucleoside bonds, including internucleoside bonds that retain phosphorus atoms and internucleoside bonds that do not have phosphorus atoms. Representative phosphorus-containing internucleoside bonds include, but are not limited to, phosphodiester bonds, phosphotriester bonds, methylphosphonate bonds, phosphoramidate bonds, and phosphorothioate bonds. Methods for preparing phosphorus-containing and non-phosphorus-containing bonds are well known. In some embodiments, antisense compounds targeted to Trpv1 (e.g., antisense oligonucleotides) comprise one or more modified internucleoside bonds.

[0135] In some embodiments, the modified internucleoside linkage is a phosphorothioate linkage.

[0136] In some embodiments, the modified oligonucleotide comprises or consists of a gapmer having the following structure from 5' to 3': 5' wing segment-gap segment-3' wing segment, the wing segment (i.e., the 5' wing segment or the 3' wing segment) having a different ribose sugar than the nucleosides in the gap segment.

[0137] "Gapmer" means an antisense oligonucleotide in which an internal segment having a plurality of nucleosides that support RNase H cleavage is positioned between external segments having one or more nucleosides, wherein the nucleosides comprising the internal segment are chemically different from the nucleosides comprising the external segments. The internal segment may be referred to as a "gap segment" and the external segments may be referred to as "wing segments."

[0138] Antisense oligonucleotides with gap body motifs are considered to be chimeric antisense oligonucleotides. In the gap body, there are multiple nucleotides or nucleosides supporting RNAeH (or referred to as RNA enzyme H) cutting connected to form an internal segment (i.e., a "gap segment"), the internal segment is located between the external segment (i.e., "wing segment"), and the nucleosides of the external segment are different from the nucleosides of the internal segment in chemical modification, such as having different ribose modifications. In the case of antisense oligonucleotides with gap body motifs, the gap segment is generally used as a substrate for endonuclease cutting, although the wing segment includes modified nucleosides. In certain embodiments, the segments of the gap body (e.g., gap segment and wing segment) are distinguished by the type of sugar moieties comprising each different segment. The types of sugar moieties used to distinguish the segments of the gap body may include, in some embodiments, β-D-ribonucleosides, β-D-deoxyribonucleosides, 2'-modified nucleosides (such 2'-modified nucleosides may further include 2'-MOE and 2'-O-CH3), and bicyclic sugar-modified nucleosides (such bicyclic sugar-modified nucleosides may include those with a 4'-(CH2)nO-2' bridge, where n=1 or n=2). Preferably, each different segment comprises a consistent sugar moiety, for example, each nucleoside in the gap segment comprises the same ribose, or, for example, each nucleoside in the 5' or 3' wing segment comprises the same ribose, or, for example, each nucleoside in the 5' and 3' wing segments comprises the same ribose, but the nucleosides in the gap segment and the wing segment comprise different riboses. Wing-gap-wing motif (i.e., 5' wing segment-gap segment-3' wing segment) is often referred to as "XYZ", where "X" represents the length of the 5' wing segment, "Y" represents the length of the gap segment, and "Z" represents the length of the 3' wing segment, wherein the length of the nucleic acid segment is the number of nucleotides therein (nt). As used herein, the gap matrix referred to as "XYZ" has such a configuration that the position of the gap segment is directly adjacent to the 5' wing segment and the 3' wing segment, respectively. Therefore, there is no nucleoside inserted between the 5' wing segment and the gap segment, or between the gap segment and the 3' wing segment. Any antisense oligonucleotide described herein may have a gap matrix. In some embodiments, X and Z are identical, in other embodiments, they are different. In some embodiments, Y is 8 to 15 nucleotides. X, Y or Z can be any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more nucleotides. Thus, the structure of the gap matrix includes, but is not limited to, for example, 5-10-5, 4-8-4, 4-12-3, 4-12-4, 3-14-3, 2-13-5, 2-16-2, 1-18-1, 3-10-3, 2-10-2, 1-10-1, 2-8-2, 6-8-6 or 5-8-5.

[0139] In some embodiments, the sugar ring of each nucleoside of the 5'-wing segment and the 3'-wing segment comprises a 2'-O-methoxyethyl modification, and the sugar ring of each nucleoside in the gapmer is deoxyribose.

[0140] In some embodiments, the modified oligonucleotide comprises 5-methylcytidine.

[0141] In some embodiments, each cytidine, or each cytidine in the mid-gapomer, is 5-methylcytidine.

[0142] In some embodiments, the modified oligonucleotide comprises: a gap segment consisting of 10 deoxynucleoside linkages, a 5'-wing segment consisting of 5 nucleoside linkages, and a 3'-wing segment consisting of 5 nucleoside linkages; wherein the gap segment is positioned between the 5'-wing segment and the 3'-wing segment, each nucleoside of each wing segment comprises a 2'-O-methoxyethyl modified sugar, each internucleoside linkage is a phosphorothioate linkage, and wherein each cytosine of the modified oligonucleotide is a 5-methylcytosine.

[0143] In some embodiments, the antisense oligonucleotide comprises:

[0144] (1) A nucleotide sequence that is at least 90%, preferably at least 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 1.

[0145] (2) A nucleotide sequence that is at least 90%, preferably at least 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 2.

[0146] (3) A nucleotide sequence that is at least 90%, preferably at least 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 3.

[0147] (4) A nucleotide sequence that is at least 90%, preferably at least 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 5.

[0148] (5) A nucleotide sequence that is at least 90%, preferably at least 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 5.

[0149] (6) A nucleotide sequence that is at least 90%, preferably at least 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 6.

[0150] (7) A nucleotide sequence that is at least 90%, preferably at least 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 7.

[0151] (8) A nucleotide sequence that is at least 90%, preferably at least 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 8.

[0152] (9) A nucleotide sequence that is at least 90%, preferably at least 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 9.

[0153] (10) A nucleotide sequence that is at least 90%, preferably at least 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 10.

[0154] (11) A nucleotide sequence that is at least 90%, preferably at least 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 11.

[0155] In some specific embodiments, the present application relates to an antisense oligonucleotide targeting the TRPV1 gene, the sequence of the antisense oligonucleotide being shown in any one of SEQ ID NOs: 1-11.

[0156] In some specific embodiments, the 5' end and the 3' end of the antisense oligonucleotide targeting the TRPV1 gene are modified with 1-5 2'-methoxyethyl nucleic acids, respectively.

[0157] In some specific embodiments, the nucleotide backbones of the antisense oligonucleotides targeting the TRPV1 gene are all phosphorothioate-modified nucleotides.

[0158] In some specific embodiments, the antisense oligonucleotide targeting the TRPV1 gene has the following structural formula: (X) a *(Z) b *(Y) c ,

[0159] Wherein, X and Y are 2'-methoxyethyl nucleic acids; Z is a deoxyribonucleotide, and the sequence of Z is the 6th to 15th nucleobases of any one of the sequences shown in SEQ ID NOs. 1-11; a, b, and c represent the nucleotide numbers of X, Z, and Y, respectively; a, b, and c are natural numbers, wherein 1≤a≤5, 1≤c≤5; 10<a+b+c<30; * indicates that the nucleotides in the backbone of the antisense oligonucleotide are phosphorothioate-modified nucleotides.

[0160] In some embodiments, the structure of the 2′-methoxyethyl nucleic acid of the antisense oligonucleotide targeting the TRPV1 gene is as shown in formula (I);

[0161] Wherein, Base is adenine, guanine, cytosine, thymine, uracil or 5-methylcytosine.

[0162] In some embodiments, the structure of the deoxyribonucleotide of the antisense oligonucleotide targeting the TRPV1 gene is as shown in formula (II);

[0163] Wherein, Base is adenine, guanine, cytosine, thymine, uracil or 5-methylcytosine.

[0164] In some preferred embodiments, the present application provides an antisense oligonucleotide comprising a modified oligonucleotide according to the following formula:

[0165] Y1:

[0166] Tes Ges Ges mCes Ges Ads Tds Gds Tds Gds mCds Ads Gds Tds Gds mCes Tes Ges Tes mCe (SEQ ID NO: 12); wherein,

[0167] A = adenine nucleobase,

[0168] mC=5-methylcytosine nucleobase,

[0169] G = guanine nucleobase,

[0170] T = thymine nucleobase,

[0171] e=2'-O-MOE modified sugar,

[0172] d=2'-deoxyribose,

[0173] s = phosphorothioate internucleoside linkage,

[0174] Y2:

[0175] Aes Tes Ges Ges mCes Gds Ads Tds Gds Tds Gds mCds Ads Gds Tds Ges mCes Tes Ges Te (SEQ ID NO: 13); wherein,

[0176] A = adenine nucleobase,

[0177] mC=5-methylcytosine nucleobase,

[0178] G = guanine nucleobase,

[0179] T = thymine nucleobase,

[0180] e=2'-O-MOE modified sugar,

[0181] d=2'-deoxyribose,

[0182] s = phosphorothioate internucleoside linkage,

[0183] Y3:

[0184] Aes Aes Ges Aes Aes Gds Tds mCds mCds mCds mCds Ads Tds Gds Gds Ges mCes mCes Ges mCe (SEQ ID NO: 14); where,

[0185] A = adenine nucleobase,

[0186] mC=5-methylcytosine nucleobase,

[0187] G = guanine nucleobase,

[0188] T = thymine nucleobase,

[0189] e=2'-O-MOE modified sugar,

[0190] d=2'-deoxyribose,

[0191] s = phosphorothioate internucleoside linkage,

[0192] Y4:

[0193] Aes Aes Aes Ges Aes Ads Gds Tds mCds mCds mCds Cds Ads Tds Gds Ges Ges mCes mCes Ge (SEQ ID NO: 15); where,

[0194] A = adenine nucleobase,

[0195] mC=5-methylcytosine nucleobase,

[0196] G = guanine nucleobase,

[0197] T = thymine nucleobase,

[0198] e=2'-O-MOE modified sugar,

[0199] d=2'-deoxyribose,

[0200] s = phosphorothioate internucleoside linkage,

[0201] Y5:

[0202] Tes Aes Aes Aes Ges Ads Ads Gds Tds mCds mCds mCds mCds Ads Tds Ges Ges Ges mCes mCe (SEQ ID NO: 16); wherein,

[0203] A = adenine nucleobase,

[0204] mC=5-methylcytosine nucleobase,

[0205] G = guanine nucleobase,

[0206] T = thymine nucleobase,

[0207] e=2'-O-MOE modified sugar,

[0208] d=2'-deoxyribose,

[0209] s = phosphorothioate internucleoside linkage,

[0210] Y6:

[0211] Tes Tes Aes Aes Aes Gds Ads Ads Gds Tds mCds mCds mCds mCds Ads Tes Ges Ges Ges mCe (SEQ ID NO: 17); wherein,

[0212] A = adenine nucleobase,

[0213] mC=5-methylcytosine nucleobase,

[0214] G = guanine nucleobase,

[0215] T = thymine nucleobase,

[0216] e=2'-O-MOE modified sugar,

[0217] d=2'-deoxyribose,

[0218] s = phosphorothioate internucleoside linkage,

[0219] Y7:

[0220] mCes Tes Tes Aes Aes Ads Gds Ads Ads Gds Tds mCds mCds mCds mCds Aes Tes Ges Ges Ge (SEQ ID NO: 18); wherein,

[0221] A = adenine nucleobase,

[0222] mC=5-methylcytosine nucleobase,

[0223] G = guanine nucleobase,

[0224] T = thymine nucleobase,

[0225] e=2'-O-MOE modified sugar,

[0226] d=2'-deoxyribose,

[0227] s = phosphorothioate internucleoside linkage,

[0228] Y8:

[0229] Tes mCes Tes Tes Aes Ads Ads Gds Ads Ads Gds Tds mCds mCds mCds mCes Aes Tes Ges Ge (SEQ ID NO: 19); wherein,

[0230] A = adenine nucleobase,

[0231] mC=5-methylcytosine nucleobase,

[0232] G = guanine nucleobase,

[0233] T = thymine nucleobase,

[0234] e=2'-O-MOE modified sugar,

[0235] d=2'-deoxyribose,

[0236] s = phosphorothioate internucleoside linkage,

[0237] Y9:

[0238] Tes Tes mCes Tes Tes Ads Ads Ads Gds Ads Ads Gds Tds mCds mCds mCes mCes Aes Tes Ge (SEQ ID NO: 20); where,

[0239] A = adenine nucleobase,

[0240] mC=5-methylcytosine nucleobase,

[0241] G = guanine nucleobase,

[0242] T = thymine nucleobase,

[0243] e=2'-O-MOE modified sugar,

[0244] d=2'-deoxyribose,

[0245] s = phosphorothioate internucleoside linkage,

[0246] Y10:

[0247] Ges Aes Tes Ges Ges mCds Gds Ads Tds Gds Tds Gds mCds Ads Gds Tes Ges mCes Tes Ge (SEQ ID NO: 21); wherein,

[0248] A = adenine nucleobase,

[0249] mC=5-methylcytosine nucleobase,

[0250] G = guanine nucleobase,

[0251] T = thymine nucleobase,

[0252] e=2'-O-MOE modified sugar,

[0253] d=2'-deoxyribose,

[0254] s = phosphorothioate internucleoside linkage,

[0255] Y11:

[0256] mCes Ges Aes Tes Ges Gds mCds Gds Ads Tds Gds Tds Gds mCds Ads Ges Tes Ges mCes Te (SEQ ID NO: 22); wherein,

[0257] A = adenine nucleobase,

[0258] mC=5-methylcytosine nucleobase,

[0259] G = guanine nucleobase,

[0260] T = thymine nucleobase,

[0261] e=2'-O-MOE modified sugar,

[0262] d=2'-deoxyribose,

[0263] s = phosphorothioate internucleoside linkage.

[0264] In some embodiments, the antisense oligonucleotide is composed of ribonucleosides and deoxyribonucleosides, wherein except for the cytosine which is 5-methylcytosine, the other nucleosides do not contain base modifications, the chemical modification of the ribose in the ribonucleoside (RNA) only contains 2'-O-MOE, the ribose in all deoxyribonucleosides (DNA) is not chemically modified, and all internucleoside linkages are phosphorothioate internucleoside linkages.

[0265] Pharmaceutical compositions and reagents

[0266] In another aspect, the present application relates to a pharmaceutical composition comprising any one or more antisense oligonucleotides described above and a pharmaceutically acceptable carrier. The pharmaceutical composition may further comprise another therapeutic agent for treating diseases mediated by TRPV1 expression.

[0267] In some embodiments, the pharmaceutical composition comprises a pharmacologically effective amount of the antisense oligonucleotide of the present application and other pharmaceutically acceptable components.

[0268] In some embodiments, the pharmaceutically acceptable carrier can be selected from water, saline, glucose, buffer (such as PBS), excipients, diluents, disintegrants, binders, lubricants, sweeteners, flavorings, preservatives, or combinations thereof.

[0269] The form of the pharmaceutical composition depends on a number of criteria, including, for example, the route of administration, the extent of the disease, or the dose to be administered.

[0270] In some embodiments, the pharmaceutical composition is an injection. The injection includes a sterile aqueous solution or dispersion, suspension or emulsion. In all cases, the injection should be sterile and should be liquid for easy injection. It should remain stable under production and storage conditions and should be resistant to contamination by microorganisms (e.g., bacteria and fungi). The carrier can be a solvent or dispersion medium, which includes, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and their appropriate mixtures and / or vegetable oils. The injection should maintain appropriate fluidity, and appropriate fluidity can be maintained in a variety of ways, for example, by using coatings such as lecithin, using surfactants, etc. Antimicrobial contamination can be achieved by adding various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.).

[0271] On the other hand, the present application also relates to a DNA molecule capable of producing the above-mentioned antisense oligonucleotide, a vector capable of expressing the double-stranded antisense oligonucleotide, and a reagent or kit containing the antisense oligonucleotide or the DNA molecule or the vector.

[0272] In another aspect, the present application also provides a cell comprising the above antisense oligonucleotide.

[0273] Treatment methods, therapeutic uses

[0274] On the other hand, the present application relates to the use of the antisense oligonucleotide targeting the TRPV1 gene in the preparation of a TRPV1 gene expression inhibitor.

[0275] On the other hand, the present application relates to the use of the antisense oligonucleotide targeting the TRPV1 gene in the preparation of a medicament for preventing / treating diseases mediated by TRPV1 expression.

[0276] In this application, "diseases mediated by TRPV1 expression" are intended to include any disease associated with the TRPV1 gene or protein. Such a disease can be any disease caused by abnormal activation of the TRPV1 channel.

[0277] In some embodiments, the disease mediated by TRPV1 expression is TRPV1-mediated pain, such as arthritis or arthralgia.

[0278] On the other hand, the present application also provides a method for inhibiting TRPV1 expression in a cell, the method comprising: (a) contacting the cell with the above-mentioned antisense oligonucleotide or pharmaceutical composition; (b) maintaining the cell produced in step (a) for a period of time sufficient to obtain degradation of the mRNA transcript of the TRPV1 gene, thereby inhibiting the expression of the TRPV1 gene in the cell.

[0279] "Contacting a cell with an antisense oligonucleotide or a pharmaceutical composition" includes contacting a cell with an antisense oligonucleotide or a pharmaceutical composition by any possible means. Contacting a cell with an antisense oligonucleotide includes contacting the cell with the antisense oligonucleotide in vitro or contacting the cell with the antisense oligonucleotide in vivo. The contact can be performed directly or indirectly. Thus, for example, the antisense oligonucleotide may be in physical contact with the cell by the individual performing the method, or alternatively, the antisense oligonucleotide may enter a situation that permits or causes it to subsequently contact the cell. "Inhibiting TRPV1 expression in a cell" includes inhibiting the expression of any TRPV1 gene (e.g., a mouse TRPV1 gene, a rat TRPV1 gene, a monkey TRPV1 gene, or a human TRPV1 gene) and a variant (e.g., a naturally occurring variant) or mutant of a TRPV1 gene. Thus, the TRPV1 gene may be a wild-type TRPV1 gene, a mutant TRPV1 gene, or a transgenic TRPV1 gene in the context of a genetically manipulated cell, cell group, or organism.

[0280] The cells are mammalian cells expressing TRPV1, such as primate cells or human cells. Preferably, the cells express the TRPV1 gene at a high level. More preferably, the cells are derived from the brain, eye, salivary gland, heart, spleen, lung, liver, kidney, intestine, or tumor.

[0281] "Inhibiting TRPV1 gene expression" includes any level of inhibition of the TRPV1 gene, such as at least partial inhibition of TRPV1 gene expression, such as inhibition of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0282] In another aspect, the present application also provides the use of the aforementioned antisense oligonucleotide or pharmaceutical composition thereof for inhibiting TRPV1 gene expression or preparing a product for inhibiting TRPV1 gene expression, wherein the inhibition of TRPV1 gene expression is to inhibit or reduce the expression level of the TRPV1 gene in human, monkey, rabbit, rat, or mouse cells in vivo or in vitro. The cells are mammalian cells expressing TRPV1, such as primate cells or human cells. Preferably, the cells express the TRPV1 gene at a high level.

[0283] In some embodiments, the cell is derived from the brain, eye, salivary gland, heart, spleen, lung, liver, kidney, intestine, or tumor. In some embodiments, the cell is a neural cell, such as a neuronal cell.

[0284] On the other hand, the present application also provides a method for preventing or treating a disease mediated by TRPV1 expression, comprising administering a therapeutically effective amount of the above-mentioned antisense oligonucleotide or pharmaceutical composition to a subject.

[0285] In some embodiments, the antisense oligonucleotide and pharmaceutical composition thereof can be administered by any suitable means, such as parenteral administration, such as intra-articular administration, enteral administration including intramuscular, intravenous, intra-arterial, peritoneal, or subcutaneous injection. The administration mode includes but is not limited to single administration or multiple administration.

[0286] In some embodiments, the subject is a human.

[0287] On the other hand, the present application also provides the use of the oligomeric compound antisense oligonucleotide or its pharmaceutical composition in the preparation of a product for reducing the concentration of TRPV1 in bone and joint tissue.

[0288] In some embodiments, the method of reducing the concentration of TRPV1 in bone and joint tissues is to reduce the expression level of TRPV1 in rat bone and joint tissues.

[0289] In some embodiments, the expression level of TRPV1 in the rat bone and joint tissue is reduced by at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.

[0290] In some embodiments, a single dose of the pharmaceutical composition can be long-lasting, with the reduction in TRPV1 expression lasting for at least 1, 3, 5, 7, 14, 21, 28 days or longer.

[0291] The innovation of this application is reflected in the following aspects: 1. The antisense oligonucleotides screened by high-throughput screening have high inhibitory activity; 2. The modified antisense oligonucleotides have high stability and high inhibitory activity. 3. While maintaining high inhibitory activity and stability, the ligand-modified antisense oligonucleotides can adopt a single-dose local administration treatment strategy to reduce the impact on other tissues or organs and reduce the amount of antisense oligonucleotides used, thereby achieving the purpose of reducing toxicity and reducing costs; 4. The modified antisense oligonucleotides can enter target cells and target tissues without the need for transfection reagents, reducing the negative effects of transfection reagents, such as cell or tissue toxicity, thereby providing the possibility for targeted therapy; 5. In vivo pharmacodynamic experiments in rats have shown that the antisense oligonucleotides of this application are more effective in terms of analgesia than the clinical class I drug celecoxib, with a faster onset and longer TRPV1 persistence.

[0292] It should be noted that, although many modifications can be attempted to improve the performance of oligomeric compounds, these attempts are generally difficult to demonstrate both RNA interference mediation and improved stability at the site of local administration (e.g., increased resistance to nucleases and / or extended duration). The modified oligomeric compounds of the present application have high stability while maintaining high inhibitory activity.

[0293] Example

[0294] Example 1. Screening of TRPV1 antisense oligonucleotide activity

[0295] 1. Antisense Oligonucleotide (ASO) Design

[0296] Antisense oligonucleotide (ASO) sequences were designed targeting the human TRPV1 mRNA sequence (transcripts are shown in Table 1 ) (as shown in Table 2 , the targeted region was highly homologous to rat TRPV1 mRNA), and sequence similarity software was used to align the sequences, which showed the lowest homology with all other non-target gene sequences.

[0297] Table 1 Target genes

[0298] Table 2 High-throughput screening sequences

[0299] 2. Synthesis of antisense oligonucleotides (natural RNA / modified RNA)

[0300] 2.1 Synthesis steps

[0301] The nucleotide monomers in the antisense oligonucleotides of the present application are deoxyribonucleotides and / or 2'-O-methoxyethyl nucleic acids, and the bases of all cytosine nucleoside monomers are 5-mC. The synthesis is completed according to the theoretical yield of 1 μmol, and 1 μmol of the universal Frit vector ( All oligonucleotides were prepared on an LK-192X synthesizer (Biocomma Bio). All nucleoside phosphoramidite monomers (Shanghai Zhaowei, PR3-007) were diluted 1:30 (g / mL) in anhydrous acetonitrile. Coupling was performed three times for 12 minutes. Deprotection was performed using 3% TCA, activation was performed using 0.6 M benzylthiotetrazolyl in acetonitrile, and capping and oxidation were performed using CAPA / CAPB and 50 mM I2 solutions, respectively. After trityl-off synthesis, the solid support was transferred to a 2 mL centrifuge tube, 1.2 mL of ammonia was added, and the tube was heated in a 65°C oven for 4 hours to remove the protecting groups. Then cool to room temperature and vacuum concentrate for 30 minutes. Filter the solution through a 0.22 μm filter membrane into a sample injection bottle and use a semi-preparative reverse phase purification instrument for single-chain purification. The elution gradient is 7% to 30% (ACN: 100 mM TEAA) for 10 minutes; the flow rate is 5 mL / min. After purification, vacuum concentrate and spin dry at room temperature. Finally, dissolve the sample in water and desalt each solution on a GE Hi-Trap desalting column to elute the final oligonucleotide product. All characteristics and purity are confirmed by ESI-MS and IEX HPLC respectively. The concentration is determined by ultraviolet light using a microplate reader, and the antisense oligonucleotide of the required mass is divided into new shipping tubes. Finally, a vacuum concentrator is used to spin dry at room temperature to obtain the final product.

[0302] 3. TRPV1 ASO in vitro activity detection-qPCR screening

[0303] 1. TRPV1 ASO transfection into 293T cells

[0304] All cells were obtained from the collection of the Chinese Academy of Sciences or from other publicly available sources; other reagents were commercially available.

[0305] Table 3 Cell names and types

[0306] 293T cells were cultured in DMEM medium containing 10% fetal bovine serum in a 5% CO2, 37°C constant temperature incubator. When the cells were in the logarithmic growth phase and in good condition (70% confluence), they were plated for transfection. The cell density was adjusted to 1.5×10 cells per well. 5Cells were plated into 24-well plates. Prepare transfection complex: Mix 250 μL Opti-MEM and the ASO in Table 2 (final concentrations of 1 nM, 10 nM, and 100 nM, respectively), mix 250 μL Opti-MEM and 2.5 μL Lipofectamine 2000 transfection reagent, let stand for 5 minutes, then mix the above two mixtures and let stand for 20 minutes. Add the above transfection complex to a 24-well plate and incubate in a 5% CO2, 37°C constant temperature incubator for 6 hours. Aspirate the supernatant, add 1 mL of complete culture medium to each well, and continue culturing for 24 hours. In addition to the experimental group, the following control group is also set up for each cell transfection: the NC group is a negative control (no oligonucleotide added).

[0307] 2. Real-time fluorescence quantitative PCR analysis:

[0308] 24 hours after transfection, cells were lysed and total RNA was extracted using a column extraction kit (Novagen). Real-time quantitative PCR was performed using a CFX96 fluorescence quantitative PCR instrument (Bio-Rad) using a Taqman probe, using the β-actin gene as an internal reference gene. Primer information is shown in Table 4.

[0309] Table 4 Primer sequence information

[0310] 3. Data Analysis

[0311] After the PCR reaction, the Ct error of 9 replicates of a sample (3 transfection replicates and 3 qPCR replicates for each sample) was within ±0.5, and 2 –ΔΔCt The target gene expression levels in the ASO-transfected cells were quantitatively analyzed using the Livak method (Lipofectamine 2000; Thermo) with the target gene expression level in the NC group (the relative mRNA expression level in the NC group was set as 1) as the standard. As shown in Tables 5 and 6 and Figure 1, 11 candidate ASOs were screened for TRPV1 cell activity in 293T cells. At a low transfection concentration of 1 nM, Y3, Y5, Y8, and Y11 still showed relatively significant inhibition of TRPV1 expression, with target gene TRPV1 mRNA expression levels of 59.87%, 72.61%, 66.85%, and 63.04%, respectively. This indicates that Y3, Y5, Y8, and Y11 exhibited high inhibitory activity in 293T cells.

[0312] Table 5 Real-time quantitative PCR detection results

[0313] Table 6 Inhibitory activity of 11 antisense oligonucleotide sequences

[0314] Example 2, Optimization of TRPV1-ASO

[0315] 1. IC transfection of 293T cells 50 value

[0316] Y3, Y5, Y8, and Y11 were diluted with Nuclease-Free Water (Invitrogen) to 7 different concentrations, with the highest final concentration set at 200 nM, and transfected into 293T cells. The transfection and quantitative PCR analysis steps were as described in Example 1, and IC values ​​were calculated using Graphpad Prism software. 50 As shown in Figures 2A, 2B, 2C, and 2D, the IC values ​​of Y3, Y5, Y8, and Y11 in 293T cells were 50 The values ​​were 22.96 nM, 25.57 nM, 46.23 nM and 30.46 nM, respectively.

[0317] Example 3: In vivo efficacy test

[0318] 3.1. Rat articular cartilage mRNA expression levels

[0319] Thirty-six male, 6- to 8-week-old SPF-grade Sprague-Dawley rats (Jiangsu Jinzhihe Biotechnology Co., Ltd.) were randomly divided into five groups of three rats each. The drug was administered intra-articularly at a dose of 1 mg / kg (see Table 7 for the experimental protocol). All animals were euthanized on day 7 after administration, and joint tissue was harvested.

[0320] qPCR experimental steps

[0321] The entire experiment must be performed in an RNA-specific fume hood to prevent RNA degradation caused by RNase contamination. All consumables used are RNase-free products. The steps are as follows:

[0322] (1) Preparation

[0323] Prepare 0.1% DEPC water and 0.1% DEPC in PBS in advance and mix thoroughly using a magnetic stirrer. Soak all laboratory instruments in 0.1% DEPC water to sterilize the instruments, 0.1% DEPC water, and 0.1% DEPC in PBS. Dry the instruments in an oven until ready for use.

[0324] (2) RNA extraction

[0325] Wipe the laboratory bench with alcohol and exogenous RNase remover. Pre-cool the centrifuge and high-throughput tissue grinder to 4°C. Remove the knee cartilage tissue from -80°C freezer, add magnetic beads and Trizol, and grind in a tissue grinder. Add chloroform (one-fifth the volume of Trizol) and vigorously shake up and down to mix thoroughly. After standing at room temperature, place the suspension in a pre-cooled centrifuge and centrifuge at 12,000g for 15 minutes. Aspirate the supernatant and transfer it to a fresh EP tube. Add an equal volume of isopropanol, let stand at room temperature for 15 minutes, and centrifuge at 12,000g for 10 minutes. The resulting white flocculent material is the RNA precipitate. Discard the supernatant. Prepare a 75% ethanol solution using DEPC water and anhydrous ethanol. Add the 75% ethanol solution to the precipitate and mix vigorously on a shaker. Resuspend the precipitate at the bottom of the tube. Centrifuge at 8,000g for 5 minutes and discard the supernatant. Resuspend the pellet in 75% ethanol as described above. Aspirate the supernatant, uncap the EP tube, and air-dry in a clean hood. Once the EP tube is dry, add an appropriate amount of Takara RNase-Free HO and gently pipette to fully dissolve the RNA pellet. Assess RNA concentration and purity, and aliquot a small amount for reverse transcription and Q-PCR (same procedures as in Example 1).

[0326] Table 7 Experimental plan for intra-articular injection of ASO drugs in rats

[0327] Table 8 Trpv1 mRNA expression levels in SD rat knee cartilage normalized to PBS group after 7 days of administration

[0328] Table 9 Trpv1 mRNA expression in SD rat knee cartilage normalized to that of PBS group after 7 days of administration

[0329] The experimental results of knee joint cartilage mRNA detection seven days after administration (D7) showed (Table 8, Table 9, Figure 3) that Y3, Y5, Y8, and Y11 still had a relatively significant inhibitory effect on trpv1 expression in vivo, with the expression levels after inhibition being 59.42%, 75.52%, 37.39%, and 29.38%, respectively.

[0330] 3.2 In vivo pharmacodynamic behavioral experiments

[0331] Thirty-eight SPF male Sprague-Dawley rats (Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd.) aged 6-8 weeks were randomly divided into three groups based on baseline behavioral results: a normal group (n=6), a model group (MIA (sodium iodoacetate)-induced osteoarthritis disease model group) (n=8), and a drug-treated group (MIA (sodium iodoacetate)-induced osteoarthritis disease model group administered with the test drug) (n=8). The drug was administered via a single intra-articular injection of 1 mg / kg (dosage group, see Table 10; dosing schedule, see Table 11). Mechanical allodynia was assessed using a plantar allergy test 4 hours, 1 day, 3 days, 7 days, 14 days, 21 days, and 28 days after administration.

[0332] Table 10 Experimental plan for intra-articular injection of ASO drugs in SD rats

[0333] Table 11 Dosage regimen of Example 3.2

[0334] Mechanical pain threshold measurement (PWT)

[0335] Before modeling, animals were acclimated to the plantar analgesia test, and preoperative baseline data were collected to screen out animals with abnormal pain thresholds. Pre-dose mechanical pain thresholds were measured 3, 7, and 14 days after modeling. Mechanical pain thresholds (von Frey filaments) were measured in the center of the plantar area for each animal three times, with 3-5 minute intervals between each measurement, and the average value was taken.

[0336] The experimental results show (Table 12, Table 13, Figure 4, Figure 5) that 4h after administration, the PWT mechanical pain thresholds of the test drug groups Y8, Y11, and celecoxib were 26.98g, 25.40g, and 24.44g, respectively, which were significantly higher than 17.99g in the model group. As time went on, on D14 after administration, the PWT mechanical pain thresholds of the test drug groups Y8, Y11, and celecoxib were 26.82g, 26.70g, and 28.23g, respectively, which were significantly higher than 21.05g in the model group, close to 4W (4 weeks) after administration; the model group rebounded to a baseline level close to the modeling (the baseline level was the mean PWT test value 1 day before administration (D-1); the experimental endpoint was D28, the model group recovered on its own, and the test ended). The results of the PWT rate of change experiment showed that the PWT rates of change of the test drug groups Y8 and Y11 were 41.46% and 31.65% respectively 4 hours after administration, which was better than the -4.41% of the model group and also better than the current clinical first-line drug celecoxib (25.48%). The experimental results showed that compared with the model group, Y8 and Y11 could achieve a significantly faster pain relief effect. The experimental results of the PWT and PWT rate of change 14 days after administration (D14) also showed that Y8 and Y11 could achieve a pain relief effect, and the analgesic effect was better than celecoxib.

[0337] PWT change rate = (PWT (time point after administration) - PWT (D-1)) / PWT (D-1)

[0338] Table 12 PWT test results of in vivo experiment in Example 3 (mechanical pain threshold measurement)

[0339] Table 13 Example 3 (Mechanical pain threshold measurement) In vivo experiment PWT change rate test results

[0340] 3.3 Histopathological examination

[0341] Organs that need to be fixed and preserved: Ipsilateral knee joint

[0342] Tissue fixation method: 10% formalin

[0343] Preparation and staining: The affected knee joint was sampled, paraffin-embedded, and sectioned according to the standard histopathological techniques.

[0344] Animals to be tested: All animals that survived step 3.2

[0345] Detection time: End point of the trial (28 days after administration)

[0346] Staining methods: knee joints were stained by H&E and Safranin O.

[0347] Slide scanning: Hamamatsu NanoZoomer Digital Pathology (S210)

[0348] Analysis method: Mankin score;

[0349] Immunostaining (Figure 6; long arrows indicate cartilage tissue structure; short arrows indicate chondrocytes) and scoring (Figure 7) indicate that the test drug groups Y8, Y11, and celecoxib alleviated inflammation in the arthritis model. Compared with the model group, the Mankin scores of Y8 and Y11 were 32.14% and 23.21% lower, respectively. This demonstrates superior efficacy to celecoxib, currently a first-line clinical medication.

[0350] Based on the above, it can be seen that the preferred molecules Y8 and Y11 can effectively inhibit TRPV1 gene expression, significantly reduce pain and relieve inflammation; the Mankin scores of Y8 and Y11 are 32.14% and 23.21% lower than those of the model group, respectively, and their efficacy is better than that of the Yangshen drug celecoxib (Mankin scores are equivalent to those of the model group), and there are no obvious side effects, achieving excellent arthritis treatment effects.

[0351] The above description is only a preferred embodiment, which is only used as an example and does not limit the combination of features necessary for implementing the present application. The title provided is not intended to limit the various embodiments of the present application. Terms such as "comprising", "containing" and "including" are not intended to be limiting. In addition, unless otherwise indicated, plural forms are included when there is no numeral modification, and "or" and "or" mean "and / or". Unless otherwise defined herein, the meaning of all technical and scientific terms used herein is the same as that generally understood by those skilled in the art. All publications and patents mentioned in this application are incorporated herein by reference. Without departing from the scope and spirit of the present application, various modifications and variations of the described methods and compositions of the present application are obvious to those skilled in the art. Although the present application is described by specific preferred embodiments, it should be understood that the present application claimed for protection should not be unduly limited to these specific embodiments. In fact, various variations of the described modes for implementing the present application that are obvious to those skilled in the relevant art are intended to be included in the scope of the appended claims.

Claims

1. A modified antisense oligonucleotide, which consists of 12 to 50 nucleosides connected by internucleoside bonds, and has a nucleobase sequence comprising at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or at least 20 connected nucleobases of the nucleobase sequence shown in any one of SEQ ID NOs: 1-11.

2. The antisense oligonucleotide of claim 1, wherein the nucleobase sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleobase sequence as shown in any one of SEQ ID NOs: 1-11.

3. The antisense oligonucleotide according to claim 1 or 2, wherein the modified oligonucleotide comprises or consists of a gap body, wherein the gap body has the following structure from 5' to 3': 5' wing segment-gap segment-3' wing segment, the nucleosides in the wing segment and the gap segment have different ribose sugars; The combination of the number of nucleotides in the 5' wing segment - the number of nucleotides in the gap segment - the number of nucleotides in the 3' wing segment is selected from any one of the following: 5-10-5, 4-8-4, 4-12-3, 4-12-4, 3-14-3, 2-13-5, 2-16-2, 1-18-1, 3-10-3, 2-10-2, 1-10-1, 2-8-2, 6-8-6 and 5-8-5.

4. The antisense oligonucleotide according to any one of claims 1 to 3, wherein at least one internucleoside bond is a modified internucleoside bond.

5. The antisense oligonucleotide of claim 4, wherein the modified internucleoside linkage is a phosphorothioate internucleoside linkage.

6. The antisense oligonucleotide of claim 5, wherein each internucleoside bond or each internucleoside bond in the gapmer is a phosphorothioate internucleoside bond.

7. The antisense oligonucleotide according to any one of claims 1 to 6, wherein the sugar ring of at least one nucleoside comprises a 2'-O-methoxyethyl modification and / or the sugar ring of the at least one nucleoside is deoxyribose.

8. The antisense oligonucleotide according to any one of claims 3 to 7, wherein the sugar ring of each nucleoside of the 5' wing segment and the 3' wing segment comprises a 2'-O-methoxyethyl modification, and the sugar ring of each nucleoside in the gapmer is deoxyribose.

9. The antisense oligonucleotide of any one of claims 1-8, wherein the modified oligonucleotide comprises 5-methylcytidine.

10. The antisense oligonucleotide according to claim 9, wherein each cytidine, or each cytidine in the mid-gap moiety is 5-methylcytidine.

11. The antisense oligonucleotide according to any one of claims 1 to 10, wherein the modified oligonucleotide comprises or consists of an oligonucleotide selected from any one of the following: 1)Tes mCes Tes Tes Aes Ads Ads Gds Ads Ads Gds Tds mCds mCds mCds mCes Aes Tes Ges Ge(SEQ ID NO:19); 2)mCes Ges Aes Tes Ges Gds mCds Gds Ads Tds Gds Tds Gds mCds Ads Ges Tes Ges mCes Te(SEQ ID NO:22); 3)Tes Ges Ges mCes Ges Ads Tds Gds Tds Gds mCds Ads Gds Tds Gds mCes Tes Ges Tes mCe(SEQ ID NO:12); 4)Aes Tes Ges Ges mCes Gds Ads Tds Gds Tds Gds mCds Ads Gds Tds Ges mCes Tes Ges Te(SEQ ID NO:13); 5)Aes Aes Ges Aes Aes Gds Tds mCds mCds mCds mCds Ads Tds Gds Gds Ges mCes mCes Ges mCe(SEQ ID NO:14); 6)Aes Aes Aes Ges Aes Ads Gds Tds mCds mCds mCds Cds Ads Tds Gds Ges Ges mCes mCes Ge(SEQ ID NO:15); 7)Tes Aes Aes Aes Ges Ads Ads Gds Tds mCds mCds mCds mCds Ads Tds Ges Ges Ges mCes mCe(SEQ ID NO:16); 8)Tes Tes Aes Aes Aes Gds Ads Ads Gds Tds mCds mCds mCds mCds Ads Tes Ges Ges Ges mCe(SEQ ID NO:17); 9)mCes Tes Tes Aes Aes Ads Gds Ads Ads Gds Tds mCds mCds mCds mCds Aes Tes Ges Ges Ge(SEQ ID NO:18); 10)Tes Tes mCes Tes Tes Ads Ads Ads Gds Ads Ads Gds Tds mCds mCds mCes mCes Aes Tes Ge(SEQ ID NO:20); 11) Ges Aes Tes Ges Ges mCds Gds Ads Tds Gds Tds Gds mCds Ads Gds Tes Ges mCes Tes Ge (SEQ ID NO: 21); in, A = adenine nucleobase, mC=5-methylcytosine nucleobase, G = guanine nucleobase, T = thymine nucleobase, e=2'-O-MOE modified sugar, d = 2'-deoxyribose, s = phosphorothioate internucleoside linkage.

12. A pharmaceutical composition comprising the antisense oligonucleotide according to any one of claims 1 to 11 and a pharmaceutically acceptable carrier.

13. Use of the antisense oligonucleotide according to any one of claims 1 to 11 in the preparation of a medicament for preventing or treating a disease mediated by TRPV1 expression.

14. The use according to claim 13, wherein the disease mediated by TRPV1 expression is TRPV1-mediated pain, preferably arthritis or joint pain. 15 . A method for preventing or treating a disease mediated by TRPV1 expression, comprising administering a therapeutically effective amount of the antisense oligonucleotide according to any one of claims 1 to 11 or the pharmaceutical composition according to claim 12 to a subject.

16. The method according to claim 15, wherein the disease mediated by TRPV1 expression is TRPV1-mediated pain, preferably arthritis or arthralgia.

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