Antisense oligonucleotide which promotes TRPV1 gene exon 7 skipping, and use in preparing drug which treats chronic pain

Abstract

Provided are an antisense oligonucleotide which promotes exon 7 skipping at the splicing level of TRPV1 gene expression, and a use in preparing a drug which treats chronic pain. The antisense oligonucleotide is a single-stranded oligonucleotide. A monomeric glycosyl group undergoes 2'-O-methoxyethyl or 2'-O-methyl chemical modification, and one non-bridging oxygen among all phosphodiester bonds linking the monomers is replaced by a sulfur atom. The beneficial effects are reflected in the fact that the provided antisense oligonucleotide drug significantly achieves the effect of promoting Trpv1 gene exon 7 skipping in cells cultured in vitro and in C57BL / 6N mice, and in pain behavior experiments, the antisense oligonucleotide drug has been found to significantly improve thermal pain and mechanical pain thresholds of mice, and has strong application potential for preparing drugs which treat chronic pain disorders.

Description

Antisense oligonucleotides that promote exon 7 skipping in the TRPV1 gene and their application in the preparation of drugs for treating chronic pain Technical Field

[0001] This invention belongs to the technical field of biomedicine, and particularly relates to an antisense oligonucleotide that promotes exon 7 skipping at the RNA splicing level of TRPV1 gene expression and its application in the preparation of drugs for treating chronic pain. Background Technology

[0002] The International Association for the Study of Pain formally defines pain as "an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or in a manner that describes such damage." (1) This definition emphasizes the evolutionary adaptive function of pain, enabling us to coexist with our external environment. Acute pain, triggered by specific stimuli, functions as an important alarm system to protect the body's integrity and reduce the risk of injury. In contrast to the transient nature of acute pain, chronic pain typically exceeds the threshold of habituation, lasting longer than three months, and can profoundly impact an individual's physical, mental, and overall quality of life. (2) The causes of chronic pain are usually multifactorial, such as peripheral tissue damage, persistent inflammatory processes, or pathological changes in the peripheral and central nervous systems. In modern medical practice, pain has been considered the fifth vital sign, alongside respiration, pulse, blood pressure, and body temperature. (2) Therefore, chronic pain has gradually been examined and treated as an independent disease entity.

[0003] Opioid analgesics are the primary medications for treating severe and chronic pain. For decades, opioids have been widely used to treat pain. However, long-term use of opioids can lead to analgesia tolerance, increased pain sensitivity, addiction, and other side effects, severely impacting patient comfort and potentially causing serious public health and social problems. In the United States, over 70,000 people died from drug overdoses in 2019, and the mortality rate from synthetic opioid overdoses increased tenfold compared to 2013. (3) In China, the use of oral opioids such as acetaminophen / oxycodone, tramadol / trimethoprim, and diphenoxylate / trimethoprim tablets is generally on the rise, and their abuse is receiving increasing attention. Therefore, finding a feasible strategy to reduce side effects while ensuring analgesic efficacy is particularly important.

[0004] TRPV1 (transient receptor potential vanilloid 1) is the first member of the TRPV subfamily discovered in mammals and is the most widely studied and studied transient receptor potential channel, also known as the capsaicin receptor and vanilloid receptor 1. TRPV1 is a non-selective cation channel that can be activated by various physical and chemical stimuli, such as heat (temperature >43°C), protons (pH <5.9), osmotic pressure, and mechanical force. TRPV1 is expressed in both sensory neurons and some non-neuronal cells and is involved in the pathogenesis of various diseases, such as pain, inflammation, cardiovascular disease, cough, mental illness, and diabetes.

[0005] TRPV1 has been the most studied for its analgesic effects, with both its agonists and antagonists producing analgesia. The main drugs related to TRPV1 agonists are various formulations of capsaicin. Low-dose capsaicin creams, capsaicin patches (8%), and liquid capsaicin have been approved for marketing by the European Union and the US Food and Drug Administration (FDA). (4) In addition, several clinical trials are testing the efficacy of capsaicin gels and patches in neuropathic pain, acute back and neck pain, anti-cancer treatment, and diabetic neuropathy. Although preclinical studies have demonstrated analgesic effects of capsaicin and resiniferatoxin, potent agonists, in various animal models, adverse reactions to blood pressure, respiration, and other reflex pathways have occurred at high doses, making it difficult to distinguish between efficacy and toxicity. (5)Furthermore, it can cause burning and pain in the initial stages of use, leading to discomfort for patients with pain. Given the toxic side effects of agonists, pharmaceutical companies and institutions have focused their research on TRPV1 antagonists. The first discovered TRPV1 antagonist was capsaicin derivative capsaicin, however, capsaicin has poor selectivity, exhibiting strong blocking effects on nicotinic receptors, voltage-gated calcium channels, and TRPM8. Subsequently, many TRPV1 antagonists emerged, such as ABT-102, GRC-6211, and JTS-653. Their molecular structures are similar to capsaicin, comprising three regions: head, neck, and tail. The head structure of these antagonists is an aromatic group similar to the vanillyl group of capsaicin; the neck structure is a linking group, such as urea, thiourea, ester, and amide groups; and the tail structure is generally a lipophilic aliphatic group, with highly active compounds typically having a longer tail. Based on the differences in these three parts, various types of antagonists have been derived. While these antagonists have some analgesic effect, they often cause adverse reactions such as elevated body temperature and abnormal perception of noxious thermal stimuli. Developing a safe and effective analgesic drug that targets TRPV1 has been a hot research topic in this field.

[0006] Within the TRP gene family, multiple alternative splicing events exist, increasing the diversity of functionally distinct TRP proteins and providing tissue-specific regulation. Several TRPV1 splice variants have been reported in mice, rats, and humans. One such TRPV1 splice variant is TRPV1b, which involves exon 7 skipping. (6) Exon 7 encodes a 60-amino acid peptide containing a third ankyrin domain. This splice variant exhibits a dominant-negative effect on the responsiveness of the TRPV1 channel, an effect that intensifies with the progression of peripheral inflammation. Vos et al. found that TRPV1b significantly reduced sensitivity to capsaicin, protons, and heat in human cells. (7) When TRPV1b is co-expressed with TRPV1, it reduces TRPV1's response to channel activators in a ratio-dependent manner and also decreases TRPV1's sensitivity to heat, thus exhibiting a negative regulatory effect on the TRPV1 protein. Therefore, employing an exon skipping strategy to convert more TRPV1 to TRPV1b has the potential for analgesic effects.

[0007] Exon skipping therapy primarily employs two strategies: gene editing and antisense oligonucleotide (ASO) therapy. Gene editing aims to modify the splice site sequence to eliminate the exon's characteristic features in the target exon. However, this technology is currently immature, inefficient, and prone to off-target effects. ASOs are specially modified single-stranded oligonucleotides that bind to the RNA expressed by the target gene through base pairing, influencing protein synthesis at the post-transcriptional or translational level. Significant progress has been made in treating some neuromuscular diseases using ASO technology. In 2016, the FDA approved two ASO drugs: nusinersen for spinal muscular atrophy (SMA) and eteplirsen for Duchenne muscular dystrophy (DMD). Several other ASO drugs have been approved for marketing in recent years. Since the 1990s, antisense oligonucleotides have also been widely used to discover and validate pain targets. However, ideal drugs for treating chronic pain still lack sufficient reserves.

[0008] The specific references to the literature mentioned in the background section above are as follows:

[0009] 1. Raja SN, Carr DB, Cohen M, Finnerup NB, Flor H, Gibson S, et al. The revised International Association for the Study of Pain definition of pain: concepts, challenges, and compromises. Pain. 2020; 161(9): 1976-82.

[0010] 2. Levy N, Sturgess J, Mills P. "Pain as the fifth vital sign" and dependence on the "numerical pain scale" is being abandoned in the US: Why? British journal of anaesthesia.2018;120(3):435-8.

[0011] 3.Mattson CL,Tanz LJ,Quinn K,Kariisa M,Patel P,Davis NL.Trends and Geographic Patterns in Drug and Synthetic Opioid Overdose Deaths-United States,2013-2019.MMWR Morbidity and mortality weekly report.2021;70(6):202-7.

[0012] 4.Kolasinski SL,Neogi T,Hochberg MC,Oatis C,Guyatt G,Block J,et al.2019American College of Rheumatology / Arthritis Foundation Guideline for the Management of Osteoarthritis of the Hand,Hip,and Knee.Arthritis care&research.2020;72(2):149-62.

[0013] 5.Gavva NR,Tamir R,Qu Y,Klionsky L,Zhang TJ,Immke D,et al.AMG 9810[(E)-3-(4-t-butylphenyl)-N-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)acrylamide],a novel vanilloid receptor 1(TRPV1)antagonist with antihyperalgesic properties.The Journal of pharmacology and experimental therapeutics.2005;313(1):474-84.

[0014] 6.Lu G,Henderson D,Liu L,Reinhart PH,Simon SA.TRPV1b,a functional human vanilloid receptor splice variant.Mol Pharmacol.2005;67(4):1119-27.

[0015] 7. Vos MH, Neelands TR, McDonald HA, Choi W, Kroeger PE, Puttfarcken PS, et al. TRPV1b overexpression negatively regulates TRPV1 responsiveness to capsaicin, heat and low pH in HEK293 cells. J Neurochem. 2006;99(4):1088-102. Summary of the Invention

[0016] To address the aforementioned technical problem of the lack of ideal analgesic drugs, this invention provides an antisense oligonucleotide that promotes exon 7 skipping in the TRPV1 gene, a screening method thereof, and its application in the preparation of drugs for treating chronic pain. Some of these antisense oligonucleotides can promote exon 7 skipping at the pre-mRNA level after TRPV1 gene transcription, generating a truncated protein TRPV1b with a 60-amino acid deletion, which has the potential for use in the preparation of drugs for treating chronic pain.

[0017] The objective of this invention is achieved through the following solution:

[0018] An antisense oligonucleotide that promotes exon 7 jumping of the TRPV1 gene, wherein the antisense oligonucleotide targets exon 7 of the TRPV1 gene, or the junction of intron 6 and exon 7, or the junction of exon 7 and intron 7.

[0019] Preferably, the target site of the antisense oligonucleotide is the sequence of nucleotides 95 to 123 of exon 7 of the TRPV1 gene.

[0020] Preferably, the sequences of the antisense oligonucleotides are shown in SEQ ID NO.081, SEQ ID NO.085 and SEQ ID NO.095.

[0021] Preferably, the antisense oligonucleotide sequence uses 5' splice site decoy technology (5D-ASO technology) to improve drug efficacy.

[0022] Preferably, the sequence of the antisense oligonucleotide is as shown in SEQ ID NO.164, obtained by appending the 8-base sequence of SEQ ID NO.181 to the 5' end of the sequence SEQ NO.081 using 5D-ASO technology.

[0023] Preferably, a portion of the T in the sequence of the antisense oligonucleotide can be replaced by U, where the portion refers to one or more.

[0024] Preferably, the antisense oligonucleotide is a single-stranded oligonucleotide, wherein the monomeric glycosyl group is chemically modified with 2′-O-methoxyethyl or 2′-O-methyl, and the chemical modification includes, but is not limited to, morpholinoylation; and one non-bridging oxygen atom in all phosphodiester bonds linking the monomers is replaced by a sulfur atom. The antisense oligonucleotide is a single-stranded oligonucleotide, wherein the monomeric glycosyl group is composed of 2′-O-methoxyethyl RNA nucleotide residues, and wherein part or all of the phosphate ester portion of the main chain is replaced with thiophosphate.

[0025] Preferably, the antisense oligonucleotide targets the precursor mRNA of TRPV1 gene expression, promotes exon 7 jumping at the splicing level, and the resulting mature mRNA does not contain exon 7.

[0026] Preferably, the target site of the antisense oligonucleotide is located in the sequence of nucleotides 95 to 123 of exon 7 of the TRPV1 gene, and its sequence in the gene is SEQ ID NO.186: CCTCGCTGTACGACCTGTCCTGCATCGAC, and its corresponding precursor mRNA sequence is SEQ ID NO.187: CCUCGCUGUACGACCUGUCCUGCAUCGAC.

[0027] Preferably, the method for screening ASOs that promote exon 7 skipping at the precursor mRNA splicing level of TRPV1 gene expression includes the following steps:

[0028] S1. Initial screening of ASOs;

[0029] S2. Further screening of ASOs based on preliminary screening;

[0030] S3. Based on fine screening, ASO length is optimized.

[0031] The preliminary screening and optimization of S1 and S2 were performed using a step-by-step method. Both the preliminary screening and optimization of S1 and S2 used a buffer (without ASO added during cell transfection) and SEQ ID NO. 000 as two negative controls, and SEQ ID NO. 000 as an unrelated ASO with the sequence TGCATCTCATTGTAG.

[0032] Preferably, step S1 includes the following steps:

[0033] Thirteen 20-nt long alpha-splicing sites (ASOs) were designed targeting exon 7 and flanking introns of TRPV1. Each ASO targets a 200-nt sequence: the 3' end of intron 6 (10 nt), the entire 180-nt exon 7, and the 5' end of intron 7 (10 nt). Adjacent ASOs have a 5-nt overlap. ASOs were named SEQ ID NO. 001 to SEQ ID NO. 013 based on their target location, moving from the 5' end to the 3' end. Using Vazyme's ExFect Transfection Reagent at a concentration of 25 nM, ASOs were transfected into cultured 293T cells to observe their effect on the splicing of endogenous TRPV1 exon 7. RNA samples collected from cells were analyzed using semi-quantitative RT-PCR to determine the abundance of full-length transcripts and transcripts lacking exon 7, and the percentage of exon 7 inclusion was calculated. The two ASOs, SEQ ID NO. 005 and SEQ ID NO. 008, significantly reduced the inclusion percentage of exon 7. SEQ ID NO. 005 targets nucleotides 50 to 71 of exon 7, while SEQ ID NO. 008 targets nucleotides 96 to 115 of exon 7. This suggests the presence of splicing enhancer sequences in these two regions. The ASOs may inhibit splicing by affecting the binding of specific splicing activators to these splicing regulatory sequences through a occupancy effect.

[0034] The 13 ASO sequences mentioned above are as follows:

[0035] SEQ ID NO.001: AGGCCAAGACCTGCCCCCGG

[0036] SEQ ID NO.002:CCCGCTGGAGAATATAGGCC

[0037] SEQ ID NO.003: CGGGCTCCTGGATCTCCCGC

[0038] SEQ ID NO.004:ACAGGTGCCTGCACTCGGGC

[0039] SEQ ID NO.005: CGGTGAACTTCCTGGACAGG

[0040] SEQ ID NO.006: GCCCGTAGGCCCACTCGGTG

[0041] SEQ ID NO.007: GCGAGGAGTGCACGGGCCCG

[0042] SEQ ID NO.008: AGGACAGGTCGTACAGCGAG

[0043] SEQ ID NO.009: CGCAGGTGTCGATGCAGGAC

[0044] SEQ ID NO.010: GCACCGAGTTCTCTCGCAG

[0045] SEQ ID NO.011:AGGCGATCACCTCCAGCACC

[0046] SEQ ID NO.012: TCTCGCTGCTGCTGTAGGCG

[0047] SEQ ID NO.013: AGCCACTCACAGGGGTCTCG

[0048] Preferably, step S2 includes the following steps:

[0049] Based on the initial screening results, a fine screening experiment was designed for two regions. The fine screening target areas covered were nucleotides 41 to 80 of exon 7 and nucleotides 86 to 125 of exon 7. A walking method was used. According to the target position, ASO named them SEQ ID NO.014 to SEQ ID NO.053 from the 5' end to the 3' end. The inclusion percentage of SEQ ID NO.020 to SEQ ID NO.033 decreased significantly. SEQ ID NO.023, which targets nucleotides 50 to 69 of exon 7, and SEQ ID NO.024, which targets nucleotides 52 to 71 of exon 7, had lower inclusion percentages, and the target position of SEQ ID NO.005 was located in between them. Similarly, the percentage of entries in SEQ ID NO.035 to SEQ ID NO.053 also decreased significantly, as did the percentage of entries in the regions SEQ ID NO.036 to SEQ ID NO.37, SEQ ID NO.043 to SEQ ID NO.044, SEQ ID NO.046 to SEQ ID NO.47, and SEQ ID NO.049 to SEQ ID NO.52.

[0050] The 40 ASO sequences mentioned above are:

[0051] SEQ ID NO.014: CCTGGACAGGTGCCTGCACT

[0052] SEQ ID NO.015: TCCTGGACAGGTGCCTGCAC

[0053] SEQ ID NO.016: TTCCTGGACAGGTGCCTGCA

[0054] SEQ ID NO.017:CTTCCTGGACAGGTGCCTGC

[0055] SEQ ID NO.018: ACTTCCTGGACAGGTGCCTG

[0056] SEQ ID NO.019:AACTTCCTGGACAGGTGCCT

[0057] SEQ ID NO.020: GAACTTCCTGGACAGGTGCC

[0058] SEQ ID NO.021: TGAACTTCCTGGACAGGTGC

[0059] SEQ ID NO.022: GTGAACTTCCTGGACAGGTG

[0060] SEQ ID NO.023: GGTGAACTTCCTGGACAGGT

[0061] SEQ ID NO.024: TCGGTGAACTTCCTGGACAG

[0062] SEQ ID NO.025: CTCGGTGAACTTCCTGGACA

[0063] SEQ ID NO.026:ACTCGGTGAACTTCCTGGAC

[0064] SEQ ID NO.027:CACTCGGTGAACTTCCTGGA

[0065] SEQ ID NO.028:CCACTCGGTGAACTTCCTGG

[0066] SEQ ID NO.029:CCCACTCGGTGAACTTCCTG

[0067] SEQ ID NO.030:GCCCACTCGGTGAACTTCCT

[0068] SEQ ID NO.031:GGCCCACTCGGTGAACTTCC

[0069] SEQ ID NO.032:AGGCCCACTCGGTGAACTTC

[0070] SEQ ID NO.033:TAGGCCCCACTCGGTGAACTT

[0071] SEQ ID NO.034:GTACAGCGAGGAGTGCACGG

[0072] SEQ ID NO.035:CGTACAGCGAGGAGTGCACG

[0073] SEQ ID NO.036:TCGTACAGCGAGGAGTGCAC

[0074] SEQ ID NO.037:GTCGTACAGCGAGGAGTGCA

[0075] SEQ ID NO.038:GGTCGTACAGCGAGGAGTGC

[0076] SEQ ID NO.039:AGGTCGTACAGCGAGGAGTG

[0077] SEQ ID NO.040:CAGGTCGTACAGCGAGGAGT

[0078] SEQ ID NO.041:ACAGGTCGTACAGCGAGGAG

[0079] SEQ ID NO.042: GACAGGTCGTACAGCGAGGA

[0080] SEQ ID NO.043:GGACAGGTCGTACAGCGAGG

[0081] SEQ ID NO.044: CAGGACAGGTCGTACAGCGA

[0082] SEQ ID NO.045: GCAGGACAGGTCGTACAGCG

[0083] SEQ ID NO.046: TGCAGGACAGGTCGTACAGC

[0084] SEQ ID NO.047: ATGCAGGACAGGTCGTACAG

[0085] SEQ ID NO.048: GATGCAGGACAGGTCGTACA

[0086] SEQ ID NO.049: CGATGCAGGACAGGTCGTAC

[0087] SEQ ID NO.050: TCGATGCAGGACAGGTCGTA

[0088] SEQ ID NO.051: GTCGATGCAGGACAGGTCGT

[0089] SEQ ID NO.052: TGTCGATGCAGGACAGGTCG

[0090] SEQ ID NO.053: GTTGTCGATGCAGGACAGGTC

[0091] Preferably, step S3 includes the following steps:

[0092] S31. Due to the low inclusion percentage of SEQ ID NO.023 and SEQ ID NO.024 in S2 above, and the fact that the target position of SEQ ID NO.005 is located between them, 13 ASOs with lengths of 22nt, 21nt, 19nt, and 18nt were designed in the region of nucleotides 50 to 71 of exon 7, with adjacent ASOs shifted one nucleotide position. In the results of S2, the inclusion percentage of SEQ ID NO.035 to SEQ ID NO.053 showed a significant decrease, and several ASOs with lengths close to the average of SEQ ID NO.008 existed. Therefore, 80 ASOs with lengths of 30nt, 29nt, 28nt, 27nt, 26nt, 25nt, 24nt, 23nt, 22nt, 21nt, 19nt, and 18nt were further designed in the region of nucleotides 95 to 124 of exon 7, with adjacent ASOs shifted one nucleotide position. The ASOs were named SEQ ID NO.54 to SEQ ID NO.145, respectively. ASO (Exon 7 Splicing Agent) was transfected into 293T cells at a concentration of 25 nM. SEQ ID NO. 000 and buffer were used as negative controls, while SEQ ID NO. 005 and SEQ ID NO. 008 were used as positive controls. The effect of ASO on the splicing of exon 7 of the endogenous TRPV1 gene was observed. Approximately 30 hours after transfection, cells were collected, RNA was extracted, and the abundance changes of full-length mRNA and mRNA lacking exon 7 were analyzed using semi-quantitative fluorescent RT-PCR to calculate the inclusion percentage of exon 7. The ASO optimized for the length of SEQ ID NO. 005 did not show a significant improvement compared to the positive control. However, among the ASOs optimized for the length of SEQ ID NO. 008, SEQ ID NO. 081, SEQ ID NO. 085, and SEQ ID NO. 095, they showed stronger splicing inhibition effects.

[0093] The 92 ASO sequences mentioned above are as follows:

[0094] SEQ ID NO.054:TCGGTGAACTTCCTGGACAGGT

[0095] SEQ ID NO.055: CGGTGAACTTCCTGGACAGGT

[0096] SEQ ID NO.056: TCGGTGAACTTCCTGGACAGG

[0097] SEQ ID NO.057:GTGAACTTCCTGGACAGGT

[0098] SEQ ID NO.058: GGTGAACTTCCTGGACAGG

[0099] SEQ ID NO.059:CGGTGAACTTCCTGGACAG

[0100] SEQ ID NO.060: TCGGTGAACTTCCTGGACA

[0101] SEQ ID NO.061: TGAACTTCCTGGACAGGT

[0102] SEQ ID NO.062: GTGAACTTCCTGGACAGG

[0103] SEQ ID NO.063: GGTGAACTTCCTGGACAG

[0104] SEQ ID NO.064: CGGTGAACTTCCTGGACA

[0105] SEQ ID NO.065: TGGTGAACTTCCTGGAC

[0106] SEQ ID NO.066:TGTCGATGCAGGACAGGTCGTACAGCGAGG

[0107] SEQ ID NO.067:GTCGATGCAGGACAGGTCGTACAGCGAGG

[0108] SEQ ID NO.068:TGTCGATTGCAGGACAGGTCGTACAGCGAG

[0109] SEQ ID NO.069:TCGATGCAGGACAGGTCGTACAGCGAGG

[0110] SEQ ID NO.070:GTCGATGCAGGACAGGTCGTACAGCGAG

[0111] SEQ ID NO.071: TGTCGATGCAGGACAGGTCGTACAGCGA

[0112] SEQ ID NO.072:CGATGCAGGACAGGTCGTACAGCGAGG

[0113] SEQ ID NO.073: TCGATGCAGGACAGGTCGTACAGCGAG

[0114] SEQ ID NO.074: GTCGATGCAGGACAGGTCGTACAGCGA

[0115] SEQ ID NO.075:TGTCGATTGCAGGACAGGTCGTACAGCG

[0116] SEQ ID NO.076:GATGCAGGACAGGTCGTACAGCGAGG

[0117] SEQ ID NO.077:CGATGCAGGACAGGTCGTACAGCGAG

[0118] SEQ ID NO.078: TCGATGCAGGACAGGTCGTACAGCGA

[0119] SEQ ID NO.079:GTCGATGCAGGACAGGTCGTACAGCG

[0120] SEQ ID NO.080:TGTCGATGCAGGACAGGTCGTACAGC

[0121] SEQ ID NO.081: ATGCAGGACAGGTCGTACAGCGAGG

[0122] SEQ ID NO.082: GATGCAGGACAGGTCGTACAGCGAG

[0123] SEQ ID NO.083: CGATGCAGGACAGGTCGTACAGCGA

[0124] SEQ ID NO.084: TCGATGCAGGACAGGTCGTACAGCG

[0125] SEQ ID NO.085:GTCGATGCAGGACAGGTCGTACAGC

[0126] SEQ ID NO.086:TGTCGATTGCAGGACAGGTCGTACAG

[0127] SEQ ID NO.087: TGCAGGACAGGTCGTACAGCGAGG

[0128] SEQ ID NO.088: ATGCAGGACAGGTCGTACAGCGAG

[0129] SEQ ID NO.089: GATGCAGGACAGGTCGTACAGCGA

[0130] SEQ ID NO.090:CGATGCAGGACAGGTCGTACAGCG

[0131] SEQ ID NO.091: TCGATGCAGGACAGGTCGTACAGC

[0132] SEQ ID NO.092:GTCGATGCAGGACAGGTCGTACAG

[0133] SEQ ID NO.093:TGTCGATGCAGGACAGGTCGTACA

[0134] SEQ ID NO.094:GCAGGACAGGTCGTACAGCGAGG

[0135] SEQ ID NO.095: TGCAGGACAGGTCGTACAGCGAG

[0136] SEQ ID NO.096: ATGCAGGACAGGTCGTACAGCGA

[0137] SEQ ID NO.097: GATGCAGGACAGGTCGTACAGCG

[0138] SEQ ID NO.098:CGATGCAGGACAGGTCGTACAGC

[0139] SEQ ID NO.099: TCGATGCAGGACAGGTCGTACAG

[0140] SEQ ID NO.100: GTCGATGCAGGACAGGTCGTACA

[0141] SEQ ID NO.101:TGTCGATGCAGGACAGGTCGTAC

[0142] SEQ ID NO.102:CAGGACAGGTCGTACAGCGAGG

[0143] SEQ ID NO.103: GCAGGACAGGTCGTACAGCGAG

[0144] SEQ ID NO.104: TGCAGGACAGGTCGTACAGCGA

[0145] SEQ ID NO.105: ATGCAGGACAGGTCGTACAGCG

[0146] SEQ ID NO.106: GATGCAGGACAGGTCGTACAGC

[0147] SEQ ID NO.107:CGATGCAGGACAGGTCGTACAG

[0148] SEQ ID NO.108: TCGATGCAGGACAGGTCGTACA

[0149] SEQ ID NO.109:GTCGATGCAGGACAGGTCGTAC

[0150] SEQ ID NO.110: TGTCGATGCAGGACAGGTCGTA

[0151] SEQ ID NO.111:AGGACAGGTCGTACAGCGAGG

[0152] SEQ ID NO.112: CAGGACAGGTCGTACAGCGAG

[0153] SEQ ID NO.113: GCAGGACAGGTCGTACAGCGA

[0154] SEQ ID NO.114: TGCAGGACAGGTCGTACAGCG

[0155] SEQ ID NO.115: ATGCAGGACAGGTCGTACAGC

[0156] SEQ ID NO.116: GATGCAGGACAGGTCGTACAG

[0157] SEQ ID NO.117: CGATGCAGGACAGGTCGTACA

[0158] SEQ ID NO.118: TCGATGCAGGACAGGTCGTAC

[0159] SEQ ID NO.119: GTCGATGCAGGACAGGTCGTA

[0160] SEQ ID NO.120:TGTCGATGCAGGACAGGTCGT

[0161] SEQ ID NO.121:GACAGGTCGTACAGCGAGG

[0162] SEQ ID NO.122: GGACAGGTCGTACAGCGAG

[0163] SEQ ID NO.123: AGGACAGGTCGTACAGCGA

[0164] SEQ ID NO.124:CAGGACAGGTCGTACAGCG

[0165] SEQ ID NO.125: GCAGGACAGGTCGTACAGC

[0166] SEQ ID NO.126: TGCAGGACAGGTCGTACAG

[0167] SEQ ID NO.127: ATGCAGGACAGGTCGTACA

[0168] SEQ ID NO.128:GATGCAGGACAGGTCGTAC

[0169] SEQ ID NO.129: CGATGCAGGACAGGTCGTA

[0170] SEQ ID NO.130: TCGATGCAGGACAGGTCGT

[0171] SEQ ID NO.131:GTCGATGCAGGACAGGTCG

[0172] SEQ ID NO.132:TGTCGATGCAGGACAGTC

[0173] SEQ ID NO.133:ACAGGTCGTACAGCGAGG

[0174] SEQ ID NO.134: GACAGGTCGTACAGCGAG

[0175] SEQ ID NO.135: GGACAGGTCGTACAGCGA

[0176] SEQ ID NO.136: AGGACAGGTCGTACAGCG

[0177] SEQ ID NO.137:CAGGACAGGTCGTACAGC

[0178] SEQ ID NO.138: GCAGGACAGGTCGTACAG

[0179] SEQ ID NO.139: TGCAGGACAGGTCGTACA

[0180] SEQ ID NO.140: ATGCAGGACAGGTCGTAC

[0181] SEQ ID NO.141:GATGCAGGACAGGTCGTA

[0182] SEQ ID NO.142: CGATGCAGGACAGGTCGT

[0183] SEQ ID NO.143:TCGATGCAGGACAGGTCG

[0184] SEQ ID NO.144: GTCGATGCAGGACAGGTC

[0185] SEQ ID NO.145: TGTCGATGCAGGACAGGT

[0186] S32. Further dose-dependent response analysis was performed on the optimal ASO from S31. The percentage of full-length inclusion of exon 7 was analyzed by semi-quantitative fluorescent RT-PCR to confirm its promotion of exon 7 skipping. Four concentrations (0 nM, 5 nM, 10 nM, 25 nM) of ASO were transfected into 293T cells. At a dose of 5 nM, the percentages of full-length inclusion of SEQ ID NO.095, SEQ ID NO.081, and SEQ ID NO.085 were all lower than those of SEQ ID NO.080, and there was no significant difference between them. Therefore, these three ASOs were selected for further optimization analysis.

[0187] Preferably, for the ASO obtained after primary screening, secondary screening, and length optimization, the 5' splice site decoy (5D) technology, i.e., the 5D-ASO technology, is used to improve the efficiency of ASO in inhibiting splicing, including the following steps:

[0188] Initial screening of S1 and decoy sequences;

[0189] S2 and decoy sequence length optimization.

[0190] Preferably, step S1 includes the following steps:

[0191] Introns 6 and 7 flanking exon 7 of the TRPV1 gene are classic GT / AG type introns. The 5D-ASO technology constructs a bifunctional ASO by adding a near-perfect 5′ splice site sequence to one side of an ASO sequence of a target RNA. This 5′ splice site sequence is typically 6-11 nt and perfectly pairs with part or all of the sequence in the 5′ end of the U1 snRNA single-stranded free sequence, acting as a 5′ splice site decoy to disrupt the U1 snRNP's recognition of the true splice site, thereby inhibiting splicing. Based on the screened SEQ ID NO.095, SEQ ID NO.081, and SEQ ID NO.085, we first tested three 9-nt decoy sequences at the 5′ and 3′ ends of these three ASOs, naming them SEQ ID NO.178 to SEQ ID NO.180, respectively. Bifunctional ASO sequences designed for SEQ ID NO.095 were named SEQ ID NO.146 to SEQ ID NO.151, sequences designed for SEQ ID NO.081 were named SEQ ID NO.152 to SEQ ID NO.157, and sequences designed for SEQ ID NO.085 were named SEQ ID NO.158 to SEQ ID NO.163. These sequences were transfected into 293T cells at a concentration of 25 nM. A buffer and SEQ ID NO.000 were used as negative controls, and SEQ ID NO.095, SEQ ID NO.081, and SEQ ID NO.085 were used as positive controls. Compared to its positive control SEQ ID NO.081, SEQ ID NO.154 showed a significantly reduced percentage of exon 7 inclusion, indicating significant inhibition of normal splicing.

[0192] The 18 ASO sequences mentioned above are as follows:

[0193] SEQ ID NO.146:CAGGTAAGTTGCAGGACAGGTCGTACAGCGAG

[0194] SEQ ID NO.147:AGGTAAGTATGCAGGACAGGTCGTACAGCGAG

[0195] SEQ ID NO.148:GGTAAGTATTGCAGGACAGGTCGTACAGCGAG

[0196] SEQ ID NO.149:TGCAGGACAGGTCGTACAGCGAGCAGGTAAGT

[0197] SEQ ID NO.150:TGCAGGACAGGTCGTACAGCGAGAGGTAAGTA

[0198] SEQ ID NO.151:TGCAGGACAGGTCGTACAGCGAGGGTAAGTAT

[0199] SEQ ID NO.152:CAGGTAAGTATGCAGGACAGGTCGTACAGCGAGG

[0200] SEQ ID NO.153:AGGTAAGTAATGCAGGACAGGTCGTACAGCGAGG

[0201] SEQ ID NO.154:GGTAAGTATATGCAGGACAGGTCGTACAGCGAGG

[0202] SEQ ID NO.155:ATGCAGGACAGGTCGTACAGCGAGGCAGGTAAGT

[0203] SEQ ID NO.156:ATGCAGGACAGGTCGTACAGCGAGGAGGTAAGTA

[0204] SEQ ID NO.157:ATGCAGGACAGGTCGTACAGCGAGGGGTAAGTAT

[0205] SEQ ID NO.158:CAGGTAAGTGTCGATGCAGGACAGGTCGTACAGC

[0206] SEQ ID NO.159:AGGTAAGTAGTCGATGCAGGACAGGTCGTACAGC

[0207] SEQ ID NO.160:GGTAAGTATGTCGATGCAGGACAGGTCGTACAGC

[0208] SEQ ID NO.161:GTCGATGCAGGACAGGTCGTACAGCCAGGTAAGT

[0209] SEQ ID NO.162:GTCGATGCAGGACAGGTCGTACAGCAGGTAAGTA

[0210] SEQ ID NO.163:GTCGATGCAGGACAGGTCGTACAGCGGTAAGTAT

[0211] Furthermore, the three 9-nt 5' splice site decoy sequences mentioned above are:

[0212] SEQ ID NO.178: CAGGTAAGT

[0213] SEQ ID NO.179: AGGTAAGTA

[0214] SEQ ID NO.180: GGTAAGTAT

[0215] Preferably, step S2 includes the following steps:

[0216] SEQ ID NO. 154 was further optimized. SEQ ID NO. 154 consists of SEQ ID NO. 081 and its 5' end SEQ ID NO. 180. The optimization strategy, based on SEQ ID NO. 180, designed 5' splice site decoy sequences of 8-nt (SEQ ID NO. 181 to SEQ ID NO. 182), 7-nt (SEQ ID NO. 183 to SEQ ID NO. 185), and 6-nt, which were added to the 5' end of SEQ ID NO. 081. The bifunctional ASOs with the added 8-nt decoys were named SEQ ID NO. 164 to SEQ ID NO. 165, the bifunctional ASOs with the added 7-nt decoys were named SEQ ID NO. 166 to SEQ ID NO. 168, and the bifunctional ASOs with the added 6-nt decoys were named SEQ ID NO. 169 to SEQ ID NO. 172. Transfected into 293T cells at a concentration of 10 nM, with SEQ ID NO. 000 at 25 nM and buffer as negative controls, and SEQ ID NO. 156 at 25 nM and 10 nM as positive controls. At 10 nM, SEQ ID NO. 164 and SEQ ID NO. 166 showed stronger splicing inhibition than SEQ ID NO. 154, with a significantly reduced percentage of exon 7 inclusion.

[0217] The nine ASO sequences mentioned above are:

[0218] SEQ ID NO.164:GGTAAGTAATGCAGGACAGGTCGTACAGCGAGG

[0219] SEQ ID NO.165:GTAAGTATATGCAGGACAGGTCGTACAGCGAGG

[0220] SEQ ID NO.166:GGTAAGTATGCAGGACAGGTCGTACAGCGAGG

[0221] SEQ ID NO.167:GTAAGTAATGCAGGACAGGTCGTACAGCGAGG

[0222] SEQ ID NO.168:TAAGTATGCAGGACAGGTCGTACAGCGAGG

[0223] SEQ ID NO.169:GGTAAGATGCAGGACAGGTCGTACAGCGAGG

[0224] SEQ ID NO.170:GTAAGTATGCAGGACAGGTCGTACAGCGAGG

[0225] SEQ ID NO.171:TAGTAATGCAGGACAGGTCGTACAGCGAGG

[0226] SEQ ID NO.172:FATHERSGCAGGACAGGTCGTACAGCGAGG

[0227] Additionally, the 8-nt and 7-nt 5 manufacturers can use the following:

[0228] SEQ ID NO.181:GGTAAGTA

[0229] SEQ ID NO.182:GTAGTAT

[0230] SEQ ID NO.183:GGTAAGT

[0231] SEQ ID NO.184:GTAAGTA

[0232] SEQ ID NO.185:TAG

[0233] Preferably, the target region of SEQ ID NO. 081 on exon 7 of the human TRPV1 gene and the mouse Trpv1 gene, specifically nucleotides 95 to 119, showed three different bases in this sequence between humans and mice: position 99 (G in humans, C in mice), position 102 (G in humans, T in mice), and position 105 (C in humans, T in mice). Based on SEQ ID NO. 164 and SEQ ID NO. 166, two additional ASOs matching the mouse-derived gene were designed and named SEQ ID NO. 173 and SEQ ID NO. 174. These were administered to newborn C57BL / 6N mice via intraventricular injection (ICV), with a saline group serving as a negative control. 15 μg of SEQ ID NO.164, SEQ ID NO.173, SEQ ID NO.166, and SEQ ID NO.174 were administered to the lateral ventricle of newborn C57BL / 6N infants, once at 1 day of age (P1) and once at 3 days of age (P3). RNA was extracted from samples taken at 7 days of age (P7). SEQ ID NO.164, SEQ ID NO.173, SEQ ID NO.166, and SEQ ID NO.174 all significantly reduced the percentage of exon 7 of the Trpv1 gene in the brain and spinal cord, but had no significant effect in the dorsal root ganglion (DRG). SEQ ID NO.164 showed a stronger splicing inhibition effect in the brain compared to SEQ ID NO.166 and SEQ ID NO.174, with no significant difference compared to SEQ ID NO.173.

[0234] The two mouse-derived ASO sequences involved are:

[0235] SEQ ID NO.173:GGTAAGTAATGCAGGACAGGTCATAAAGGGAGG

[0236] SEQ ID NO.174:GGTAAGTATGCAGGACAGGTCATAAAGGGAGG

[0237] Preferred, the screened SEQ ID NO. 164 was injected into the lateral ventricle of adult C57BL / 6N mice, and changes in pain behavior were observed. Using the saline group and the mismatch sequence SEQ ID NO. 175 (GGCAATTACTGTAGTACCGGCCGCACCGCTAGT) designed for SEQ ID NO. 164 as negative controls, it was found that SEQ ID NO. 164 significantly increased the thermal pain threshold and mechanical pain threshold in mice, and significantly inhibited the normal splicing of the Trpv1 gene in the mouse brain and spinal cord, with a significant decrease in the percentage of exon 7 inclusion.

[0238] Preferably, the application of ASO that promotes TRPV1 gene exon 7 skipping at the pre-mRNA splicing level of TRPV1 gene expression in the preparation of drugs for chronic pain syndromes, wherein the ASO binds to the pre-mRNA of TRPV1 gene expression, promotes exon 7 skipping, and translates into a functionally defective truncated protein with 60 amino acids missing in the middle, so as to relieve chronic pain.

[0239] The beneficial effects of this invention are reflected in the fact that the antisense oligonucleotide drug proposed in this invention significantly promotes the skipping of exon 7 of the Trpv1 gene in in vitro cultured cells and C57BL / 6N mice, and in pain behavior experiments, it was found that it significantly increased the pain threshold of thermal pain and mechanical pain in mice, and has strong application potential for the preparation of drugs for the treatment of chronic pain. Attached Figure Description

[0240] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0241] Figure 1: Schematic diagram of the mechanism of TRPV1 exon 7 skipping therapy.

[0242] Figure 2 shows the RT-PCR results of the preliminary screening of 20-nt ASO (MOE / PS modification) of exon 7 of TRPV1 in Example 1 of the present invention.

[0243] Figure 3 illustrates the effect of the fine screening ASO (SEQ ID NO.014-SEQ ID NO.053) designed based on the target regions of SEQ ID NO.005 and SEQ ID NO.008 in the initial screening results on TRPV1 exon 7 skipping in Embodiment 1 of the present invention.

[0244] Figure 4 illustrates the effect of different lengths of optimized ASOs (SEQ ID NO.054-SEQ ID NO.065; SEQ ID NO.094-SEQ ID NO.101; SEQ ID NO.076-SEQ ID NO.086) designed to inhibit normal splicing of exon 7 in 293T cells under 25 nM treatment, targeting SEQ ID NO.005 and SEQ ID NO.008 at nucleotides 50-71 and 95-124 of exon 7.

[0245] Figure 5 illustrates the effect of SEQ ID NO.095, SEQ ID NO.081, SEQ ID NO.085 and SEQ ID NO.080 at different concentrations in Example 2 of the present invention on inhibiting normal splicing of exon 7 in the TRPV1 gene in 293T cells.

[0246] Figure 6 illustrates the initial screening and length screening of 5D sequences in a second-generation ASO redesigned using the 5' splice site decoy (5D) technique, based on SEQ ID NO.095, SEQ ID NO.081, and SEQ ID NO.085 screened in Example 2, in Embodiment 3 of the present invention.

[0247] Figure 7 illustrates the splicing inhibition effect of human ASO SEQ ID NO.164 and its corresponding mouse ASO SEQ ID NO.173, human ASO SEQ ID NO.166 and its corresponding mouse ASO SEQ ID NO.174 on exon 7 of the Trpv1 gene in the brain, spinal cord and dorsal root ganglion of newborn mice in Example 4 of the present invention.

[0248] Figure 8 illustrates the effect of SEQ ID NO.164 on the body weight of adult C57BL / 6N mice in Example 5 of the present invention, which significantly increased the pain thresholds for thermal and mechanical pain in the mice.

[0249] Figure 9 shows that SEQ ID NO.164 significantly inhibited the normal splicing of exon 7 of the Trpv1 gene in the brain and spinal cord of adult C57BL / 6N mice in Example 5 of the present invention, but had no significant effect in the dorsal root ganglion. Detailed Implementation

[0250] This invention provides an antisense oligonucleotide that promotes exon 7 skipping at the TRPV1 gene precursor mRNA splicing level, resulting in a truncated protein with a 60-amino acid deletion in the middle, and its application. The mechanism of TRPV1 exon 7 skipping therapy described in this invention is shown in Figure 1.

[0251] The ASO used in the embodiments of the present invention has three modifications: 2′-O-methoxyethyl, thiophosphate ester bond and 5-methylcytosine modification at the glycosyl site.

[0252] Cell culture

[0253] The 293T cells used in this patent were purchased from the Cell Bank of the Chinese Academy of Sciences. All adherent 293T cells were cultured in DMEM complete medium (containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin). All cell cultures were conducted at 37°C and 5% carbon dioxide.

[0254] Laboratory animal husbandry

[0255] SPF-grade male C57BL / 6N mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. The animals were kept under 12-hour light / dark cycles, with free access to food and water, and the room temperature was maintained at 22±2℃, with indoor humidity at approximately 60%-80%. The laboratory animals were raised and used strictly in accordance with the relevant provisions and clauses of the "Regulations on the Management of Laboratory Animals" (Order No. 2 of the State Science and Technology Commission of the People's Republic of China, 1988).

[0256] ASO transfection

[0257] (1) Before dissolving ASO in this patent, the ASO dry powder is centrifuged at 6000 rpm for 5 min, DEPC water is added, and the mixture is sonicated for 3 min. The storage concentration is 25 μM.

[0258] (2) On the first day, seed cells at an appropriate density (~2.5 × 10⁻⁶) in each well of a 12-well plate. 5 Add 1 mL of complete culture medium to each well and incubate overnight until the cell density reaches 80%.

[0259] (3) Prepare the transfection mixture. Solution A: Add an appropriate amount of ASO to 50 μL of serum-free DMEM medium and mix. Solution B: Add 1.5 μL of Transfection Reagent (Vazyme) to 50 μL of serum-free DMEM medium, mix well, and let stand at room temperature for 5 min.

[0260] (4) Mix solution A and solution B, gently tap with your finger to mix, centrifuge briefly, and let stand at room temperature for 20 minutes.

[0261] (5) Remove the old culture medium from each well and quickly add 400 μL of preheated serum-free DMEM medium.

[0262] (6) Add the mixture from (4) to each well and shake gently. Incubate at 37°C for 5-6 hours, then replace with preheated complete culture medium.

[0263] (7) Extract total RNA after culturing in an incubator for 30 hours.

[0264] Trizol method for extracting total RNA from tissues

[0265] (1) Cool the centrifuge to 4°C.

[0266] (2) Cells: Discard the cell culture medium (discard the supernatant after centrifuging the suspended cells), add 500 μL of Trizol, incubate at room temperature for 10 min, pipette a few times, and transfer the Trizol to a 1.5 mL RNase-free centrifuge tube. Tissue: After taking the tissue, place it in a 1.5 mL RNase-free centrifuge tube, weigh it, add 1 mL of Trizol for every 100 mg of tissue, add grinding beads, and grind using a tissue homogenizer.

[0267] (3) Add one-fifth of the volume of Trizol in chloroform, shake vigorously for 15 seconds, and let stand at room temperature for 2 minutes.

[0268] (4) Place the solution in a centrifuge at 12,000 rpm for 15 minutes.

[0269] (5) After removing the centrifuge, transfer the clear liquid in the upper layer to a brand new 1.5ml centrifuge tube using a pipette, discard the precipitate, and record the volume of the supernatant.

[0270] (6) Add the same volume of isopropanol as the supernatant to the centrifuge tube, shake slowly for 15 seconds, and let stand in a 4°C refrigerator for 15 minutes.

[0271] (7) After standing, take it out and put it into a 4℃ low temperature high speed centrifuge, 12000rpm, 10min.

[0272] (8) Discard the supernatant and keep the precipitated RNA.

[0273] (9) Add 1 ml of 75% ethanol prepared with DEPC water to each tube and rinse the precipitated RNA.

[0274] (10) Put it back into the 4℃ low temperature high speed centrifuge and centrifuge at 12000 rpm for 5 min.

[0275] (11) Discard the supernatant and air-dry the RNA precipitate until it becomes transparent.

[0276] (12) Add an appropriate amount of DEPC water depending on the size of the RNA precipitate, shake slowly to mix, and use Nano Drop to detect the RNA concentration and purity. The OD260 / OD280 should be between 1.8 and 2.0. The RNA can then proceed to the next step.

[0277] Cy5-labeled RT-PCR

[0278] (1) Reverse transcription

[0279] Reverse transcription is assigned to the following groups:

[0280] The total volume is 10 μl. After preparing the mixture according to the above formula, gently shake to mix and perform reverse transcription on a PCR instrument. PCR instrument program: 50℃, 30 min; 85℃, 5 s; 4℃ incubation.

[0281] (2) RT-PCRs

[0282] The PCR reaction system is as follows:

[0283] The PCR reaction procedure is as follows:

[0284] Reaction primers:

[0285] Cy5-labeled PCR samples were added to a 6% non-denaturing PAGE gel and run at 220V for 35 min. Development was performed using a G:BOX system, and the band grayscale values ​​were analyzed using ImageJ software. The percentage of inclusion in human TRPV1 gene and mouse Trpv1 exon 7 (%incl) was calculated using the following formula:

[0286] Exon 7 inclusion percentage = Full-length transcript / (Full-length transcript + Δ7 transcript) × 100%

[0287] Pain Behavioral Experiment

[0288] (1) Hot plate experiment

[0289] Mice were placed on a hot metal plate and observed through an acrylic transparent box. Timing began when the mouse was placed in the hot plate. Pain was indicated when the mouse licked its hind paws, flipped its hind paws forward, swung its hind paws, and jumped off the hot plate; this time was recorded as the hot plate reaction time, indicating the mouse's thermal pain threshold. The hot plate temperature used in this experiment was 52℃. To avoid damage to the plantar tissue, the maximum testing time was set to 25 seconds. Mice were removed immediately after the test, and baseline values ​​were determined before drug administration. Before the formal test, mice were placed on the same metal plate at room temperature for 30 seconds to acclimatize. Each mouse was tested three times, with intervals of 15 minutes or more between each test. The average of the three tests for each mouse was taken.

[0290] (2) Tail-drift test

[0291] First, restrain the mouse with a clean towel to maintain a comfortable and natural posture. Then, immerse the tip of the mouse's tail 2-3 cm above the water in pre-set hot water. The mouse will wag its tail in response to the stimulation. Record the time from immersion to tail wag; this time can be used as an evaluation standard for the mouse's thermal pain threshold at that temperature. In this experiment, the hot water temperature was set at 48℃. To avoid damage to the tail tip tissue, the maximum test time was set to 10 seconds. The mouse was removed immediately after the test, and baseline values ​​were determined before drug administration. Mice needed to acclimatize before the formal test. On the first day, wrap the mouse in a towel and immerse the tail tip in room temperature water until it stopped struggling. Remove the mouse after 10 seconds of stillness in the water. Each mouse was tested three times, with intervals of 15 minutes or more between each test. The average of the three tests for each mouse was taken.

[0292] (3) Mechanical pain threshold test (von Frey)

[0293] Von Frey cilia are a series of fibers with varying lengths and diameters. When the tips of the cilia contact the sole of a mouse's hind foot, they generate pressure. When the pressure increases to a certain level, the mouse will lick its hind foot, flip its hind foot forward, and shake its hind foot. In this experiment, the pressures corresponding to the von Frey cilia were 0.07g, 0.16g, 0.4g, 0.6g, 1.0g, 1.4g, and 2.0g. We applied stimulation to the same hind foot of the mouse in ascending order of the pressure. Each stimulation was given 10 times. When there was no response after the 6th stimulation, a larger pressure was applied. When 5 positive responses were observed, the pressure at that point was recorded as the mouse's baseline mechanical pain threshold. Each stimulation maintained the same degree of cilia curvature for 5 seconds, with at least a 3-minute interval between each stimulation.

[0294] Statistics and Analysis

[0295] All data are expressed as mean ± standard deviation (mean ± SD) and statistical analysis was performed using GraphPad Prism 8.0 software. One-way ANOVA and two-way ANOVA were used to analyze the differences between groups for each parameter. P < 0.05 was considered statistically significant.

[0296] Example 1: ASO walking method for primary and fine screening

[0297] In this embodiment, 13 20nt-long alpha-splicing sites (ASOs) were designed targeting exon 7 and flanking introns of TRPV1. The target sites covered a 200nt sequence: the 10nt portion of the 3' end of intron 6, the entire 180nt exon 7, and the 10nt portion of the 5' end of intron 7. Adjacent ASOs moved one position every 15nt, with a 5nt overlap. The ASOs were named SEQ ID NO. 001 to SEQ ID NO. 013 according to their target position, moving from the 5' end to the 3' end. Using Vazyme's ExFect Transfection Reagent at a concentration of 25nM, the ASOs were transfected into cultured 293T cells to observe their effect on the splicing of endogenous TRPV1 exon 7. RNA samples collected from cells were analyzed using fluorescent semi-quantitative RT-PCR to analyze changes in the abundance of full-length mRNA and mRNA lacking exon 7, and the inclusion percentage of exon 7 was calculated. A buffer (without ASO added during transfection) and SEQ ID NO. 000 (unrelated ASO) were used as two negative controls. The results are shown in Figure 2. SEQ ID NO. 005 and SEQ ID NO. 008 significantly reduced the inclusion percentage of exon 7. SEQ ID NO. 005 targets nucleotides 50 to 71 of exon 7, while SEQ ID NO. 008 targets nucleotides 96 to 115 of exon 7. This suggests that these two regions may contain splicing enhancer sequences, and ASOs may inhibit splicing by affecting the binding of specific splicing activators to these splicing regulatory sequences through a locating effect. Subsequently, based on the initial screening results described above, this embodiment designed a fine screening experiment for two regions. The fine screening target coverage areas were: nucleotides 41 to 80 of exon 7 and nucleotides 86 to 125 of exon 7. Adjacent ASOs were moved one position. ASOs were named SEQ ID NO. 014 to SEQ ID NO. 053 according to their position of action, from the 5' end to the 3' end. The results are shown in Figure 3. The inclusion percentage of SEQ ID NO. 020 to SEQ ID NO. 033 decreased significantly, and the inclusion percentage of the region near SEQ ID NO. 005, SEQ ID NO. 023 to SEQ ID NO. 024, was relatively low. Similarly, the inclusion percentage of SEQ ID NO. 035 to SEQ ID NO. 053 also decreased significantly, and the inclusion percentages of the regions SEQ ID NO. 036-037, SEQ ID NO. 043-044, SEQ ID NO. 046-047, and SEQ ID NO. 049-052 also decreased significantly.FL: Full-length transcript; Δ7: Exon 7 skipped transcript; %incl (percentage of exon 7 included) = FL / (Δ7+FL) × 100%. *, P<0.05; **, P<0.01; ***, P<0.001. Significantly different from the buffer control group (n=3).

[0298] Example 2: ASO Length Optimization

[0299] Based on the initial and fine screening results of ASOs, this embodiment designed a length optimization experiment in the region near the target site. Because SEQ ID NO.023 and SEQ ID NO.024 had low inclusion percentages, and the target site of SEQ ID NO.005 was located between them, 13 ASOs with lengths of 22nt, 21nt, 19nt, and 18nt were designed in the region of nucleotides 50 to 71 of exon 7, with adjacent ASOs shifted one nucleotide position. In the results of Example 1, the inclusion percentage of SEQ ID NO.035 to SEQ ID NO.053 showed a significant decrease, and several ASOs with lengths close to the average of SEQ ID NO.008 existed. Therefore, 80 ASOs with lengths of 30nt, 29nt, 28nt, 27nt, 26nt, 25nt, 24nt, 23nt, 22nt, 21nt, 19nt, and 18nt were further designed in the region of nucleotides 95 to 124 of exon 7, with adjacent ASOs shifted one nucleotide position. The ASOs were named SEQ ID NO.54 to SEQ ID NO.145. They were transfected into 293T cells at a concentration of 25 nM. A buffer and SEQ ID NO.000 were used as negative controls, and SEQ ID NO.005 and SEQ ID NO.008 were used as positive controls. The effect of ASOs on the splicing of exon 7 of the endogenous TRPV1 gene was observed. Approximately 30 hours after transfection, cells were collected, RNA was extracted, and the abundance changes of full-length mRNA and mRNA lacking exon 7 were analyzed using semi-quantitative fluorescent RT-PCR to calculate the inclusion percentage of exon 7. The results are shown in Figure 3. The ASOs optimized for the length of SEQ ID NO.005 did not show a significant improvement compared to the positive controls. However, among the length-optimized ASOs designed for SEQ ID NO.008, SEQ ID NO.095, SEQ ID NO.081, SEQ ID NO.085, and SEQ ID NO.080 showed stronger splicing inhibition effects. The results are shown in Figure 4, where FL: full-length transcript; Δ7: transcript skipped by exon 7; %incl (percentage of exon 7 included) = FL / (Δ7+FL) × 100%. *, P<0.05; **, P<0.01; ***, P<0.001. The differences were statistically significant compared with SEQ ID NO.005 and SEQ ID NO.008 (n=3).

[0300] Further dose-dependent response analysis was performed on SEQ ID NO.095, SEQ ID NO.081, SEQ ID NO.085, and SEQ ID NO.080. 0 nM, 5 nM, 10 nM, and 25 nM ASOs were transfected into 293T cells, and the inhibitory effect of different ASO concentrations on TRPV1 exon 7 splicing was detected by semi-quantitative fluorescent RT-PCR. The results are shown in Figure 5. At a dose of 5 nM, the full-length transcripts of SEQ ID NO.095, SEQ ID NO.081, and SEQ ID NO.085 were all lower than those of SEQ ID NO.080, and there was no significant difference among them. Therefore, these three ASOs were selected for further optimization analysis. Wherein, FL: full-length transcript; Δ7: transcript skipped by exon 7; %incl (percentage of exon 7 inclusion) = FL / (Δ7+FL)×100%. *, P<0.05; **, P<0.01, significantly different from SEQ ID NO.080 (n=3).

[0301] Example 3: Improving the splice suppression effect of ASO

[0302] This embodiment will further enhance the efficacy of the screened ASOs. Introns 6 and 7 flanking exon 7 of the TRPV1 gene are classic GT / AG type introns. The 5D-ASO technology involves adding a near-perfect 5' splice site sequence to one side of an ASO sequence of a target RNA, constructing a bifunctional ASO. This 5' splice site sequence is typically 6-11 nt and perfectly pairs with part or all of the sequence in the 5' end of the U1 snRNA single-stranded free sequence, acting as a 5' splice site decoy (5D), disrupting the U1 snRNP's recognition of the true splice site, thereby inhibiting splicing. Based on SEQ ID NO.095, SEQ ID NO.081, and SEQ ID NO.085 screened out in Example 2, we first added one of three 9-nt decoy sequences to the 5' and 3' ends of these three ASOs, respectively. These three decoy sequences were named SEQ ID NO.178 (CAGGTAAGT), SEQ ID NO.179 (AGGTAAGTA), and SEQ ID NO.180 (GGTAAGTAT). The bifunctional ASO sequences designed for SEQ ID NO.095 were named SEQ ID NO.146 to SEQ ID NO.151, the bifunctional ASO sequences designed for SEQ ID NO.081 were named SEQ ID NO.152 to SEQ ID NO.157, and the bifunctional ASO sequences designed for SEQ ID NO.085 were named SEQ ID NO.158 to SEQ ID NO.163. ASO was transfected into 293T cells at a concentration of 25 nM. SEQ ID NO. 000 and buffer were used as negative controls, while SEQ ID NO. 095, SEQ ID NO. 081, and SEQ ID NO. 085 were used as positive controls. The effect of ASO on exon 7 splicing of the endogenous TRPV1 gene was observed. Approximately 30 hours after transfection, cells were collected, RNA was extracted, and the abundance changes of full-length mRNA and mRNA lacking exon 7 were analyzed using semi-quantitative fluorescent RT-PCR. The inclusion percentage of exon 7 was calculated. The results are shown in Figure 6. Compared with its positive control SEQ ID NO. 081, SEQ ID NO. 154 showed a significantly lower inclusion percentage of exon 7, indicating a significant inhibition of normal splicing. Wherein, FL: full-length transcript; Δ7: transcript skipping exon 7; %incl (expon 7 inclusion percentage) = FL / (Δ7+FL)×100%.*, P<0.05; **, P<0.01; ***, P<0.001, significantly different from SEQ ID NO.095 (n=3); #, P<0.05; ##, P<0.01; ###, P<0.001, significantly different from SEQ ID NO.081 (n=3); &, P<0.05; &&, P<0.01; &&&, P<0.001, significantly different from SEQ ID NO.085 (n=3).

[0303] Subsequently, SEQ ID NO. 154 was further optimized. SEQ ID NO. 154 is composed of SEQ ID NO. 083 and its 5' end SEQ ID NO. 180. The optimization strategy designed 8nt (SEQ ID NO. 181 to SEQ ID NO. 182), 7nt (SEQ ID NO. 183 to SEQ ID NO. 185), and 6nt decoy sequences based on SEQ ID NO. 180 and added them to the 5' end of SEQ ID NO. 081. The bifunctional ASO with added 8-nt decoy was named SEQ ID NO. 164 to SEQ ID NO. 165, the bifunctional ASO with added 7-nt decoy was named SEQ ID NO. 166 to SEQ ID NO. 168, and the bifunctional ASO with added 6-nt decoy was named SEQ ID NO. 169 to SEQ ID NO. 172. Transfected into 293T cells at a concentration of 10 nM, with buffer and SEQ ID NO. 000 at 25 nM as negative controls, and SEQ ID NO. 154 at 25 nM and 10 nM as positive controls. The results are shown in Figure 6. Under the 10 nM condition, SEQ ID NO. 164 and SEQ ID NO. 166 showed stronger splicing repression than SEQ ID NO. 154, with a significantly reduced exon 7 inclusion percentage. Wherein, FL: full-length transcript; Δ7: transcript skipping exon 7; %incl (exon 7 inclusion percentage) = FL / (Δ7+FL)×100%. *, P<0.05; **, P<0.01; ***, P<0.001. Significant difference compared to SEQ ID NO. 154 group (n=3).

[0304] Example 4: Comparison of ASO effects between human and rodent sources

[0305] This embodiment confirms that human-derived target sequence ASOs can also be applied to mice. By comparing the target region of SEQ ID NO. 081 on exon 7 of the human TRPV1 gene and the mouse Trpv1 gene, specifically nucleotides 95 to 119, three different bases were found in this sequence between humans and mice: position 99 (G in humans, C in mice), position 102 (G in humans, T in mice), and position 105 (C in humans, T in mice). Based on SEQ ID NO. 164 and SEQ ID NO. 166, two additional ASOs matching the mouse-derived gene were designed and named SEQ ID NO. 173 and SEQ ID NO. 174. Furthermore, neonatal C57BL / 6N mice were administered the drugs via intraventricular injection (ICV), with the saline group serving as a negative control. 15 μg of SEQ ID NO.164, SEQ ID NO.173, SEQ ID NO.166, and SEQ ID NO.174 were administered to the lateral ventricles of newborn C57BL / 6N brains once daily (twice daily) at P1 and P3. Samples were collected at P7 and RNA was extracted. The abundance changes of full-length mRNA and mRNA lacking exon 7 were analyzed using semi-quantitative fluorescent RT-PCR, and the inclusion percentage of exon 7 was calculated. The results are shown in Figure 7. SEQ ID NO.164, SEQ ID NO.173, SEQ ID NO.166, and SEQ ID NO.174 significantly reduced the inclusion percentage of exon 7 in the TRPV1 gene in the brain and spinal cord, but had no significant effect in the dorsal root ganglion (DRG). SEQ ID NO.164, compared to SEQ ID NO.166 and SEQ ID NO.173, exhibited stronger splicing inhibition in the brain, with no significant difference from SEQ ID NO.173, suggesting the possibility of using SEQ ID NO.164 as a final candidate drug. FL: full-length transcript; Δ7: transcript skipping exon 7; %incl (percentage of exon 7 inclusion) = FL / (Δ7+FL)×100%. *, P<0.05; **, P<0.01; ***, P<0.001, significantly different from saline (n=3); #, P<0.05; ##, P<0.01; ###, P<0.001, significantly different from SEQ ID NO.164 (n=3). In this example, ASO used in animals was diluted to a concentration of 7.5 μg / μL with physiological saline.

[0306] Example 5: Verification of the analgesic effect of ASO

[0307] In this embodiment, SEQ ID NO.164, screened in Example 4, was used to inject adult C57BL / 6N mice into the lateral ventricle to observe changes in pain behavior. Eight-week-old adult C57BL / 6N mice were divided into three groups: a saline group, a SEQ ID NO.175 group (based on the SEQ ID NO.164 sequence, with one base changed every two nucleotide positions, serving as a negative control), and a SEQ ID NO.164 group. On day 0, using a stereotaxic instrument, saline, 300 μg of SEQ ID NO.175, and 300 μg of SEQ ID NO.164 were administered via lateral ventricle injection. Samples were collected from the saline and SEQ ID NO.175 groups on day 42 post-injection, and from the SEQ ID NO.164 group on days 28, 35, and 42 post-injection in three batches. This embodiment tested the analgesic effect of ASO through mouse pain behavior experiments. The thermal pain behavior experiments included the hot plate test and the tail flick test, while the mechanical pain behavior experiments included the von Frey test. All mice underwent baseline behavioral testing two days prior to injection, and behavioral changes were continuously observed at days 7, 14, 21, 28, 35, and 42 post-injection. Mice were weighed before each observation. The results are shown in Figure 8. In the tail flick test, mice injected with SEQ ID NO. 164 showed increased tolerance to hot water starting on day 7, with significant improvements observed from days 14 to 42. The same phenomenon was observed in the hot plate test, suggesting that SEQ ID NO. 164 enhances thermal pain tolerance in mice. In the von Frey experiment, mice injected with SEQ ID NO.164 showed no significant changes on days 7 and 14, but exhibited increased paw pressure tolerance on days 21, 28, 35, and 42, indicating that SEQ ID NO.164 significantly increased the mechanical pain threshold in mice. However, this example also revealed a decrease in body weight in mice in both the SEQ ID NO.175 and SEQ ID NO.164 groups, with the weight recovery in the SEQ ID NO.164 group being worse than the negative control SEQ ID NO.175 group. FL: Full-length transcript; Δ7: Transcript skipping exon 7; %incl (percentage of exon 7 inclusion) = FL / (Δ7+FL) × 100%. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Significant difference compared to saline (n = 8). In this example, all animals using ASO were diluted to a concentration of 30 μg / μL with physiological saline.

[0308] This embodiment used fluorescent RT-PCR to detect the inclusion of exon 7 of the Trpv1 gene in the brain, spinal cord, and DRG in the saline group, SEQ ID NO.175 group, and SEQ ID NO.164 group (sampled at days 28, 35, and 42). The results are shown in Figure 9. Significant splicing repression was observed in the brain and spinal cord at days 28, 35, and 42, while only slight changes were observed in the DRG. L: Full-length transcript; Δ7: Transcript skipping exon 7; %incl (percentage of exon 7 inclusion) = FL / (Δ7+FL) × 100%. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Significant difference compared to saline (n = 3).

[0309] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. 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 of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An antisense oligonucleotide that promotes exon 7 skipping in the TRPV1 gene, characterized by: The antisense oligonucleotide targets exon 7 of the TRPV1 gene, or the junction of intron 6 and exon 7, or the junction of exon 7 and intron 7.

2. The antisense oligonucleotide as described in claim 1, characterized in that: The target site of the antisense oligonucleotide is the sequence of nucleotides 95 to 123 of exon 7 of the TRPV1 gene.

3. The antisense oligonucleotide as described in claim 2, characterized in that: The antisense oligonucleotide sequences are shown in SEQ ID NO.081, SEQ ID NO.085 and SEQ ID NO.

095.

4. The antisense oligonucleotide as described in claim 2, characterized in that: The antisense oligonucleotide was obtained by appending the 8-base sequence of SEQ ID NO.181 to the 5' end of the sequence SEQ NO.081 using the 5' splice site decoy technique, resulting in the sequence shown in SEQ ID NO.

164.

5. The antisense oligonucleotide as described in claim 2, characterized in that: In the sequence of the antisense oligonucleotide, one or more Ts can be replaced by Us.

6. The antisense oligonucleotide as described in claim 2, characterized in that: The antisense oligonucleotide is a single-stranded oligonucleotide in which the monomeric glycosyl group is chemically modified with 2′-O-methoxyethyl or 2′-O-methyl, and one non-bridging oxygen atom in the phosphodiester bond that links the monomer is replaced by a sulfur atom in part or all of them.

7. The antisense oligonucleotide as described in claim 6, characterized in that: The chemical modifications include, but are not limited to, morpholine modification.

8. The antisense oligonucleotide that promotes exon 7 skipping of the TRPV1 gene as described in claim 3, characterized in that: The antisense oligonucleotide targets the precursor mRNA of TRPV1 gene expression, promoting exon 7 skipping at the splicing level, resulting in mature mRNA that does not contain exon 7.

9. The use of the antisense oligonucleotide that promotes exon 7 skipping of the TRPV1 gene as described in any one of claims 1-8 in the preparation of a drug for treating chronic pain.

Images