Antisense oligonucleotide for reducing XDH gene expression in targeted manner and application of antisense oligonucleotide

Abstract

The invention discloses antisense oligonucleotides capable of reducing XDH gene expression in a targeted mode and application of the antisense oligonucleotides, and belongs to the technical field of biological medicines.The antisense oligonucleotides mediate target mRNA degradation through an RNase H (RNase H) dependent cleavage mechanism, and the mRNA expression of XDH genes is horizontally knocked down after transcription; or pre-mRNA splicing is regulated and controlled through the steric hindrance effect, and inhibition of XDH gene expression or selective generation of variable splicing products is achieved at the transcriptional level. The antisense oligonucleotide molecule has a specific nucleic acid sequence, is combined with chemical modification design, can specifically bind XDH mRNA molecules in a targeting manner, promote XDH mRNA degradation and inhibit XDH protein synthesis, so that the expression level of XDH in cells is reduced, uric acid synthesis is reduced, the purpose of reducing uric acid is finally achieved, and cell experiments and animal model tests show that the antisense oligonucleotide molecule has a good application prospect. The antisense oligonucleotide molecule disclosed by the invention has a good uric acid reducing effect, and is long in drug effect duration and high in drug safety.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to antisense oligonucleotides (ASOs) for targeted reduction of XDH gene mRNA and protein expression and their use in the preparation of drugs for treating hyperuricemia. Background Art

[0002] There are over 170 million people in my country with hyperuricemia, and the number is increasing annually. Hyperuricemia (HUA) is a metabolic disorder characterized by abnormally elevated uric acid levels in the blood. Long-term, uncontrolled elevated uric acid levels can lead to gout, kidney damage, and increase the risk of cardiovascular and cerebrovascular diseases and metabolic syndrome. It has become the fourth most common metabolic disorder after the "three highs" (high blood pressure, high blood sugar, and high blood lipids).

[0003] Purine metabolism in the liver is the primary pathway for uric acid production. Xanthine oxidoreductase (XOR) in hepatocytes is encoded by the XDH gene and exists as either xanthine dehydrogenase (XDH) or xanthine oxidase (XO). XDH and XO catalyze the conversion of purine to uric acid using different substrates. As a key regulatory enzyme for uric acid synthesis in the body, XOR is also an important drug target for clinical uric acid-lowering therapies. Currently, first-line treatments for hyperuricemia, such as febuxostat and allopurinol, work by inhibiting the catalytic activity of XOR, thereby reducing uric acid synthesis. However, these small molecule drugs have adverse reactions and limitations in their application. Common adverse reactions include hepatotoxicity and renal toxicity. Limitations include a short duration of efficacy and a high risk of relapse after discontinuation of treatment. Summary of the Invention

[0004] In view of the deficiencies of the above-mentioned prior art methods, the present invention provides antisense oligonucleotide molecules that target and reduce the expression of the XDH gene and their use in the preparation of drugs for improving hyperuricemia.

[0005] To achieve the above object, the present invention adopts the following technical solutions: The antisense oligonucleotides are targeted to reduce the expression of XDH gene, and the sequences of the antisense oligonucleotides are shown in SEQ ID NOs: 1-107.

[0006] Furthermore, the antisense oligonucleotide described in the present invention comprises a gap region consisting of 10 2'-deoxynucleotides, and the gap region is flanked by wing regions consisting of 5 nucleotides at the 5' and 3' ends, respectively. The nucleotides in the 5' wing region and the 3' wing region are modified with 2'-methoxyethoxy (2'-MOE) sugar, the nucleotides in the gap region are modified with 2'-deoxyribose, all internucleotide bonds in the gapmer are phosphorothioate (PS) bonds, and all cytosine residues in all gapmers contain 5-methylation modification.

[0007] Furthermore, the antisense oligonucleotide of the present invention comprises at least one N-acetylgalactosamine (GalNAc) targeting ligand for specific binding to the asialoglycoprotein receptor (ASGPR), wherein the targeting ligand is located at the 5' or 3' end of the antisense oligonucleotide and is covalently coupled to the oligonucleotide via a chemical linker, wherein the linker is selected from a monovalent, bivalent or trivalent branched structure, and the GalNAc ligand is positioned at the 5' or 3' end of the antisense oligonucleotide.

[0008] The present invention also provides a composition capable of reducing the expression of XDH gene, comprising the antisense oligonucleotide and a pharmaceutically acceptable carrier.

[0009] The present invention also provides the use of the antisense oligonucleotide in preparing a drug for reducing XDH gene expression.

[0010] The present invention also provides use of the antisense oligonucleotide or the composition in preparing a drug for treating hyperuricemia.

[0011] The present invention provides antisense oligonucleotide (ASO) molecules. Their mechanism of action is that these ASO molecules use the above-mentioned specific nucleic acid sequences and are combined with chemical modification design to target and specifically bind to XDH mRNA molecules, promote the degradation of XDH mRNA, inhibit XDH protein synthesis, thereby reducing the expression level of XDH in cells, and ultimately reduce uric acid synthesis, thereby achieving the effect of lowering uric acid.

[0012] The antisense oligonucleotides designed in the present invention (1) employ a 5-10-5 gapmer structure, with each ASO sequence being 20 bases long; (2) having a GC content of 45% to 65% in each ASO sequence; (3) having a binding energy ∆G°37 of ≥-8 kcal / mol for each ASO with XDH mRNA; (4) predicting the number of potential off-target genes for each ASO to be ≤3 based on gene sequence alignment; and (5) excluding any sequence containing a motif that triggers immune stimulation. The present invention introduces multiple parameters (1)-(5) above based on the classic "sliding window" design strategy, thereby improving the efficiency of ASO sequence screening.

[0013] The sequences of the ASOs targeting the XDH gene provided by the present invention are shown in the table below: Number Sequence Number Sequence XDH ASO-1 CCACAAGGTGTCAGTATATG XDH ASO-55 AGGCAAAGGATACACGATCT XDH ASO-2 CACAAGGTGTCAGTATATGT XDH ASO-56 GGATACACGATCTTGTTCTG XDH ASO-3 CTTTTGGCAATTCTCTCCTA XDH ASO-57 GGCAAAGGATACACGATCTT XDH ASO-4 ACTTGAAGAAGAAGCTGAGG XDH ASO-58 GCAAAGGATACACGATCTTG XDH ASO-5 GAACTTGAAGAAGAAGCTGA XDH ASO-59 CAAAGGATACACGATCTTGT XDH ASO-6 AACTTGAAGAAGAAGCTGAG XDH ASO-60 AGGATACACGATCTTGTTCT XDH ASO-7 AGAACTTGAAGAAGAAGCTG XDH ASO-61 AAGGATACACGATCTTGTTC XDH ASO-8 TGAAGAAGAAGCTGAGGGTG XDH ASO-62 GATGGCATCTTTGATGGCAA XDH ASO-9 CTTGAAGAAGAAGCTGAGGG XDH ASO-63 CGGATGGCATCTTTGATGGC XDH ASO-10 TTGAAGAAGAAGCTGAGGGT XDH ASO-64 GGATGGCATCTTTGATGGCA XDH ASO-11 GGATGTCTGCCACCAGTTAT XDH ASO-65 TGGCATCTTTGATGGCAAAG XDH ASO-12 CAGCAGGCAAAGGATACACG XDH ASO-66 ATGGCATCTTTGATGGCAAA ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ GCCCAACACAAGTAACCTAG XDH ASO-18 TCAGGTCCCCTTCTTTGTAC XDH ASO-72 TGCCCAACACAAGTAACCTA XDH ASO-19 TGCCACAAGGTGTCAGTATA XDH ASO-73 ATGTCCTCCTCAGACTGACC XDH ASO-20 TTGCCACAAGGTGTCAGTAT XDH ASO-74 CCCACCATGTCCTCCTCAGA XDH ASO-21 GTCAGGATACACTTTCTTCT XDH ASO-75 GTCCTCCTCAGACTGACCCT XDH ASO-22 AGCATTCTCATTAAGGAGGT XDH ASO-76 TGTCCTCCTCAGACTGACCC XDH ASO-23 TTCTTTATAGCATCCTGAGG XDH ASO-77 CCACCATGTCCTCCTCAGAC XDH ASO-24 AGTCAGGATACACTTTCTTC XDH ASO-78 CTCAGACTGACCCTTGGGCA XDH ASO-25 GTTCTTTATAGCATCCTGAG XDH ASO-79 AGACTGACCCTTGGGCACCT XDH ASO-26 TGCATTTTTCTCCACCACCT XDH ASO-80 TCAGACTGACCCTTGGGCAC XDH ASO-27 TCTGCATTTTTCTCCACCAC XDH ASO-81 CACCATGTCCTCCTCAGACT XDH ASO-28 TAGCATTCTCATTAAGGAGG XDH ASO-82 CCATGTCCTCCTCAGACTGA XDH ASO-29 GCATTTTTCTCCACCACCTA XDH ASO-83 CATGTCCTCCTCAGACTGAC XDH ASO-30 ACTGTCAGGTAGAACTTGAA XDH ASO-84 ACCATGTCCTCCTCAGACTG XDH ASO-31 CAGGTAGAACTTGAAGAAGA XDH ASO-85 TCCTCAGACTGACCCTTGGG XDH ASO-32 GGTAGAACTTGAAGAAGAAG XDH ASO-86 TCCTCCTCAGACTGACCCTT XDH ASO-33 GTAGAACTTGAAGAAGAAGC XDH ASO-87 CTCCTCAGACTGACCCTTGG XDH ASO-34 GAAGAAGAAGCTGAGGGTGA XDH ASO-88 CCTCCTCAGACTGACCCTTG XDH ASO-35 AAGAAGAAGCTGAGGTGAG XDH ASO-89 GAGTGGTCTTGAGGGCTGAG XDH ASO-36 CTGTCAGGTAGAACTTGAAG XDH ASO-90 AGTGGTCTTGAGGCTGAGA XDH ASO-37 TCAGGTAGAACTTGAAAG XDH ASO-91 CGGAGCAGTGTGTACATACT XDH ASO-38 GTCAGGTAGAACTTGAAAGAA XDH ASO-92 GGAGCAGTGTGTACATACTC XDH ASO-39 TGTCAGGTAGAACTTGAAGA XDH ASO-93 GAGCAGTGTGTACATACTCA XDH ASO-40 AGAAGAAGCTGAGGGTGAGG XDH ASO-94 AGCAGTGTGTACATACTCAT XDH ASO-41 AAGAAGCTGAGGTGAGGGGT XDH ASO-95 GCAGTGTGTACATACTCATG XDH ASO-42 AGAAGCTGAGGTGAGGGTG XDH ASO-96 CAGTGTGACATACTCATGA XDH ASO-43 GAAGAAGCTGAGGTGAGGG XDH ASO-97 AGTGTGTACATACTCATGAC XDH ASO-44 GGGATGTCTGCCACCAGTTA XDH ASO-98 TGTCTTTCAGCCTCTGGGAA XDH ASO-45 CAGGAAGGGATGTCTGCCAC XDH ASO-99 CACAAACTGTCTGGAGATCT XDH ASO-46 CCAGGAAGGGATGTCTGCCA XDH ASO-100 TCACAAACTGTCTGGAGATC XDH ASO-47 AAGGGATGTCTGCCACCAGT XDH ASO-101 GTTCACAAACTGTCTGGAGA XDH ASO-48 AGGGATGTCTGCCACCAGTT XDH ASO-102 TTCACAAACTGTCTGGAGAT XDH ASO-49 GGAAGGGATGTCTGCCACCA XDH ASO-103 GTGTCTTTCAGCCTCTGGGGA XDH ASO-50 GAAGGGATGTCTGCCACCAG XDH ASO-104 GGTTCACAAACTGTCTGGAG XDH ASO-51 AGGAAGGGATGTCTGCCACC XDH ASO-105 AGTGTCTTTCAGCCTCTGGG XDH ASO-52 GTCCCAGTCTTCATGAAGCC XDH ASO-106 GAGTGTCTTTCAGCCTCTGG XDH ASO-53 AAACCAGCGCAGCTGCTCCA XDH ASO-107 GGAGTGTCTTTCAGCCTCTG XDH ASO-54 AGCAAACCAGCGCAGCTGCT The present invention has the following beneficial effects: (1) The antisense oligonucleotides designed in the present invention mediate the degradation of target mRNA through an RNase H (RNase H)-dependent cleavage mechanism, achieving knockdown of XDH gene mRNA expression at the post-transcriptional level, and further leading to a reduction in XDH protein products at the translational level, thereby achieving targeted regulation of XDH gene expression; (2) The antisense oligonucleotides designed in the present invention regulate pre-mRNA splicing through steric hindrance, thereby inhibiting XDH gene expression or selectively generating alternative splicing products at the transcriptional level; (3) The targeting ligand contained in the antisense oligonucleotide designed by the present invention has a delivery function and is used to specifically bind to the asialoglycoprotein receptor (ASGPR) to enhance the hepatocyte targeting of the oligonucleotide. The number of potential off-target genes predicted by the ASO is ≤3.

[0014] (4) The antisense oligonucleotides of the present invention are used to prepare drugs for treating diseases with XDH gene products as direct targets or pathological conditions associated with abnormal expression or dysfunction of the XDH gene, providing a way to treat hyperuricemia; (5) The antisense oligonucleotides provided by the present invention have been tested in cell experiments and animal models. The ASO molecules can not only effectively reduce the expression of the target XDH gene (as shown in Example 1), but also have a good uric acid-lowering effect in vivo (as shown in Example 2). Moreover, compared with allopurinol, the duration of drug effect (as shown in Example 3) and drug safety (as shown in Example 4) are significantly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a diagram showing the effects of ASOs numbered XDH ASO-1 to XDH ASO-15 targeting XDH in Example 1 of the present invention on reducing the expression of the target gene XDH in a mouse cell line; Figure 2 This is a graph showing the serum uric acid level measurement in the hyperuricemia mouse model according to Example 2 of the present invention; Figure 3 This is a diagram showing the short-term uric acid-lowering effect of the ASO numbered XDH ASO-12 in Example 2 of the present invention in a hyperuricemia mouse model; Figure 4 This is a diagram showing the long-term uric acid-lowering effect of the ASO numbered XDH ASO-12 in Example 3 of the present invention in a hyperuricemia mouse model; Figure 5 The anatomical diagram of the liver and kidney of mice after treatment with different doses of XDH ASO-12, febuxostat, and allopurinol in Example 4 of the present invention; Figure 6 This is a graph showing changes in body weight of mice treated with different doses of XDH ASO-12, febuxostat, and allopurinol according to Example 4 of the present invention; Figure 7 This is a graph showing the kidney and liver function index detection results of mice after being treated with different doses of XDH ASO-12, febuxostat, and allopurinol according to Example 4 of the present invention, wherein: A is the kidney function index CRE2 detection result, and B is the liver function index AST detection result. DETAILED DESCRIPTION

[0016] Example 1 Experimental verification of the ability of ASOs numbered XDH ASO-1 to XDH ASO-15 to reduce the expression of the target gene XDH in cells cultured in vitro

[0017] Step 1: Cell culture and ASO transfection Cell culture: AML12 cells (a normal mouse liver cell line) were seeded into 12-well plates. 1 mL of complete medium (containing 10% fetal bovine serum) was added to each well. The cells were cultured at 37°C and 5% CO2 in a CO2 incubator until the cell density reached 70%-80%.

[0018] ASO transfection: Lipofectamine 2000 transfection reagent was used in the cell line to mix the ASO targeting XDH (50 / 100 / 150 nM) with the liposome transfection reagent according to the ratio in the instructions. A negative control group (transfection of ASO that does not target any gene sequence), a blank control group (no transfection of ASO) and a positive siRNA control group (siRNA sequence that has been reported in the literature to have the effect of knocking down XDH expression) were set up. The transfection operation was as follows: (1) 50 μL of opti-MEM serum-free medium was added to a 1.5 mL sterile centrifuge tube, and 2 μL of ExFect was added, vortexed to mix, and allowed to stand for 3 minutes; (2) 50 μL of Opti-MEM serum-free medium was added to a 1.5 mL sterile centrifuge tube. (1) Add the appropriate amount of ASO (final transfection concentration: 50 / 100 / 150 nM) to the culture medium, vortex to mix, and let it stand for 3 minutes. (2) Add the ExFect-opti-MEM dropwise to the ASO-opti-MEM, vortex to mix, and let it stand at room temperature for 15 minutes before transfection. (3) Add the ExFect / ASO complex mixture dropwise to the culture medium and gently shake the culture dish to evenly disperse the ExFect / ASO. (4) Incubate overnight for 24 hours. (5) Harvest the cells, extract RNA, and proceed to subsequent experiments.

[0019] Step 2: Use fluorescence quantitative PCR technology to detect the knockdown effect of target gene XDH expression at the mRNA level Detection of target mRNA expression by qPCR: Total RNA was extracted from cells (using TRIzol reagent), reverse transcribed into cDNA (using a reverse transcription kit), and the expression level of XDH mRNA was detected by qPCR. The primer sequences are as follows: XDH (target gene) detection primers: forward 5′- TAGAAGAAAGTTGGGGCTGTGCG -3′, reverse 5′-CAGATGGGGGTCAAGCAGGCA -3′; GAPDH (internal reference gene) detection primers: forward 5'-CATCACTGCCACCCAGAAGACTG -3', reverse 5'-ATGCCAGTGAGCTTCCCGTTCAG -3'.

[0020] Reaction conditions: pre-denaturation at 95°C for 30 s, denaturation at 95°C for 10 s, annealing at 60°C for 30 s, instrument default settings, for a total of 40 cycles; The results are as follows Figure 1As shown, the X-axis of the bar graph indicates the experimental groups, namely: (1) Blank, blank experimental group, cells were cultured normally without additional treatment; (2) NC, negative control group, cells were treated with three ASO molecules with different final concentrations (50nM, 100nM, 150nM) that did not target any gene sequence; (3) 1-15, ASO experimental group, cells were treated with three ASO molecules with different final concentrations (50nM, 100nM, 150nM) 1-15 respectively; (4) siRNA, positive control group, cells were treated with siRNA that was published in the literature and was clearly able to effectively reduce XDH expression. The Y-axis of the bar graph indicates the relative expression level of XDH mRNA.

[0021] Real-time quantitative PCR assays revealed that, compared to a blank control (untreated cells) or a negative control (NC group, in which cells were treated with an ASO sequence that did not target any gene), treatment with XDH-targeted ASOs, numbered XDH ASO-1 through XDH ASO-15, reduced XDH mRNA expression. This finding provides strong experimental evidence for the use of XDH-targeted ASOs in the treatment of hyperuricemia. The experiments demonstrated that the ASOs of the present invention specifically bind to XDH mRNA, effectively blocking its translation process and thereby reducing XDH protein synthesis.

[0022] Example 2 Verification of the short-term uric acid-lowering function of ASO numbered XDH ASO-12 in animals based on a hyperuricemia mouse model Step 1: Establishment of hyperuricemia animal model and administration of ASO numbered XDH ASO-12 (1) Animal adaptive feeding: Experimental mice (strain: C57BL / 6, male, 8 weeks old) were adaptively fed under standard feeding conditions for 1 week to ensure stable health status; (2) Drug preparation: a. Potassium oxonate: 200 mg / mL, dissolved in 0.5% sodium carboxymethylcellulose solution; b. Hypoxanthine: 100 mg / mL, dissolved in 0.5% sodium carboxymethylcellulose solution; (3) Model construction and grouping: c. Blank control group: 0.5% sodium carboxymethylcellulose solution (150 μL) was administered orally for the first time, and the same solution (150 μL) was administered orally again 2 hours later; d. Modeling group: The animals were first gavaged with hypoxanthine solution (100 mg / mL, 150 μL) and then 2 hours later with potassium oxonate solution (200 mg / mL, 150 μL). Modeling was continued until the end of treatment.

[0023] (4) Model validation Nine days after modeling, orbital blood was collected from the mice to test the serum uric acid level. After confirming that the serum uric acid level in the modeling group was significantly higher than that in the blank control group, the next step of drug administration was carried out.

[0024] (5) Dosage in different groups e. Experimental group: ASO-12 targeting XDH was administered at doses of 15, 10, and 5 mg / kg (finally dissolved in 100 μL of saline) and injected into the mice via the tail vein 9 days after modeling.

[0025] f. Negative control group: normal saline, 100 μL / mouse, injected into mice via the tail vein 9 days after modeling.

[0026] g. Positive control group: Allopurinol, dose of 25 mg / kg, 9 days after modeling, allopurinol was administered orally once 7 hours after modeling every day for 5 days.

[0027] h. Blank control group: a group without hyperuric acid model treatment.

[0028] Step 2: Serum uric acid level test: Five days after administration, orbital blood was collected from the mice, serum was separated, and uric acid levels were measured using an automatic biochemical analyzer to simulate the uric acid-lowering effect of short-term administration.

[0029] like Figure 2 As shown in the figure, the X-axis of the bar graph indicates the experimental groups, namely the non-modeling group and the modeling group; the Y-axis indicates the blood uric acid level of the mice (unit: μmol / L). *** indicates that there is a statistically significant difference in the blood uric acid values ​​between the modeling group and the non-modeling group, p < 0.001; after the modeling of steps (1), (2), (3), and (4) in step 1, the serum uric acid value of the modeling group mice was significantly higher than that of the non-modeling group (P < 0.001), indicating that the hyperuricemia mouse model was successfully established.

[0030] like Figure 3As shown, the X-axis of the bar graph indicates the experimental groups, namely: (1) blank control group without modeling, mice were fed normally without drug stimulation; (2) negative control group, mice were gavaged with normal saline after modeling; (3) ASO experimental group, mice were treated with three doses (5 / 10 / 15 mg / kg) of ASO-12 targeting XDH after modeling; (4) positive control group, mice were treated with allopurinol after modeling. ** and *** respectively indicate that there are statistically significant differences in blood uric acid values ​​between the two comparison groups, among which **P<0.01; ***P<0.001. ASO numbered XDH ASO-12 at different doses (5 / 10 / 15 mg / kg) can effectively reduce the uric acid level of hyperuricemic mice and restore them to normal. The results showed that in the short term (5 days after administration), different concentrations (5, 10, 15 mg / kg) of ASO targeting XDH can significantly reduce the serum uric acid level of hyperuricemic mice. This result indicates that ASO targeting XDH can effectively intervene in the uric acid metabolic pathway and reduce the production of uric acid, thereby playing an important role in the treatment of hyperuricemia.

[0031] Example 3 ASO No. XDH ASO-12 can maintain a long-term uric acid-lowering effect in hyperuricemic mice Since small molecule chemical drugs such as allopurinol have a short efficacy period and uric acid rebounds easily after drug discontinuation, this example uses a hyperuricemia mouse model to detect the change in uric acid level of ASO numbered ASO-12 compared with allopurinol over a period of time after drug discontinuation.

[0032] The specific experimental procedures are as follows: Step 1: Establishment of a hyperuricemia animal model and administration of ASO, as in Step 1 of Example 2. Step 2: After the administration procedure is completed, the drug (ASO or allopurinol) is discontinued. All model mice remain under the potassium oxonate / hypoxanthine stimulation for 28 days, simulating the scenario of a patient taking medication for a period of time and then stopping it for a long time. Step 3: Serum uric acid level measurement, as in Step 2 of Example 2.

[0033] like Figure 4 As shown in the figure, the X-axis of the bar graph indicates the experimental groups, namely: (1) blank control group, mice were fed normally without drug stimulation to establish the model; (2) negative control group, mice were gavaged with normal saline after model establishment; (3) ASO experimental group, mice were treated with ASO-12 targeting XDH after model establishment, and the test was performed 28 days after administration; (4) positive control group, mice were treated with allopurinol after model establishment, and the test was performed 28 days after administration. * and *** respectively indicate that there is a statistically significant difference in blood uric acid values ​​between the two comparison groups, among which *P<0.05; ***P<0.001.

[0034] In a hyperuricemia mouse model, blood uric acid levels rebounded to elevated levels 28 days after allopurinol was discontinued. In contrast, blood uric acid levels remained normal 28 days after discontinuation of ASO-12. This result suggests that the XDH-targeting ASO, ASO-12, significantly outperforms allopurinol in maintaining its effects.

[0035] Example 4 ASO numbered XDH ASO-12 has good safety In this example, a gradient dose administration experiment was conducted to evaluate the safety of ASO numbered ASO-12 in mice to verify its clinical application as a potential therapeutic drug.

[0036] The specific steps are as follows: Step 1: Experimental grouping and drug treatment 1. Animal Adaptation Feeding: Upon arrival, experimental mice (C57BL / 6, male, 8 weeks old) were adaptively fed under standard housing conditions for 1 week to ensure stable health.

[0037] 2. Experimental groups and drug treatments: a. Experimental group: XDH-targeting ASO, doses of 1, 5, and 10 mg / kg / mouse (dissolved in 100 μL saline), injected once into the tail vein.

[0038] b. Negative control group: 100 μL of normal saline per mouse, injected once into the tail vein.

[0039] c. Positive control group: Allopurinol at doses of 25, 125, and 250 mg / kg / animal (dissolved in 100 μL 3dH2O); febuxostat at doses of 20, 100, and 200 mg / kg / animal (dissolved in 200 μL 0.5% sodium carboxymethylcellulose solution). Both drugs were administered orally once daily.

[0040] Step 2: Dynamic monitoring of physiological indicators: 1. Body weight changes: The mice were weighed at a fixed time every other day (11:00 AM).

[0041] 2. Liver and kidney function assessment: On days 10 and 14 after administration, eye blood was collected from mice and serum was separated. AST (a marker of liver injury) and CRE (a key marker of kidney injury) were measured using a biochemical analyzer.

[0042] like Figure 5-7 As shown, healthy mice were treated with normal saline or low, medium and high doses of ASO-12, allopurinol and febuxostat, and the liver and kidney organs (such as Figure 5 ), weight (such as Figure 6 ), renal injury marker CRE2 (such as Figure 7 As shown in A), liver injury marker AST (as shown in Figure 7 (shown in B) indicator.

[0043] Commonly used uric acid-lowering drugs (allopurinol, febuxostat) exhibit varying degrees of weight loss or renal toxicity (mainly manifested as acute kidney injury, abnormalities in tissue anatomy or the kidney injury marker CRE) under medium- to high-dose treatment under experimental conditions. This result is generally consistent with the instructions for use of the relevant drugs, which also indicates that such drugs should not be taken long-term. The ASO numbered ASO-12 targeting XDH of the present invention precisely regulates target expression, maintains the uric acid metabolism intervention effect, does not cause significant metabolic disorders or organ damage, and is safe, providing key experimental basis for its clinical translation.

[0044] The present invention provides ASO and a prepared drug for inhibiting hyperuricemia. In in vitro cultured cell lines and hyperuricemia mouse models, ASO targeting XDH can effectively reduce the gene expression level of XDH, thereby intervening in the uric acid metabolic pathway and reducing uric acid production. Therefore, this invention is expected to provide a new approach for the treatment of hyperuricemia, play an important role in the treatment of hyperuricemia, and promote the application of antisense oligonucleotide technology in the treatment of metabolic diseases. The drug for inhibiting hyperuricemia of the present invention is used for the adjuvant treatment of hyperuricemia and has broad application prospects.

Claims

1. An antisense oligonucleotide targeting and reducing XDH gene expression, characterized in that: The sequences of the antisense oligonucleotides are shown in SEQ ID NOs: 1-107.

2. The antisense oligonucleotide targeting and reducing XDH gene expression according to claim 1, characterized in that: The antisense oligonucleotide comprises a gap region consisting of 10 2'-deoxynucleotides, wherein the gap region is flanked by wing regions consisting of 5 nucleotides at the 5' and 3' ends, respectively. The nucleotides in the 5' and 3' wing regions are modified with 2'-methoxyethoxy (2'-MOE) sugars, the nucleotides in the gap region are modified with 2'-deoxyribose, all internucleotide bonds in the gapmer are phosphorothioate (PS) bonds, and all cytosine residues in all gapmers contain 5-methylation modifications.

3. The antisense oligonucleotide targeting and reducing XDH gene expression according to claim 1, characterized in that: The antisense oligonucleotide contains at least one N-acetylgalactosamine (GalNAc) targeting ligand for specific binding to the asialoglycoprotein receptor (ASGPR). The targeting ligand is located at the 5' or 3' end of the antisense oligonucleotide and is covalently coupled to the oligonucleotide via a chemical linker. The linker is selected from a monovalent, bivalent or trivalent branched structure. The GalNAc ligand is positioned at the 5' or 3' end of the antisense oligonucleotide.

4. A composition capable of reducing XDH gene expression, comprising the antisense oligonucleotide according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier.

5. Use of the antisense oligonucleotide according to any one of claims 1 to 3 in the preparation of a drug for reducing XDH gene expression.

6. Use of the antisense oligonucleotide according to any one of claims 1 to 3 or the composition according to claim 4 in the preparation of a medicament for treating hyperuricemia.

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