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

Abstract

The invention discloses antisense oligonucleotides for reducing XDH gene expression in a targeted manner and application of the antisense oligonucleotides, and belongs to the technical field of biological medicines. The antisense oligonucleotide mediates the degradation of the target mRNA through an RNase H (RNase H) dependent cleavage mechanism, and the mRNA expression of the XDH gene is horizontally knocked down after transcription; 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

[0001] This application is a divisional application filed on July 27, 2025, application number: 2025110363692, invention title: Antisense oligonucleotides targeting and reducing XDH gene expression and their applications. Technical Field

[0002] This invention belongs to the field of biomedical technology, specifically relating to antisense oligonucleotides (ASO) for targeting and reducing the expression of XDH gene mRNA and protein, and their application in the treatment of hyperuricemia. Background Technology

[0003] Hyperuricemia (HUA) is a metabolic disorder characterized by an abnormally high concentration of uric acid in the blood. Long-term high uric acid levels, if not controlled in time, can lead to gout, kidney damage, and increase the risk of cardiovascular and cerebrovascular diseases as well as metabolic syndrome. It has now become the fourth major category of metabolic disorders after the "three highs" (hypertension, hyperglycemia, and hyperlipidemia).

[0004] Purine metabolism in the liver is the main pathway for uric acid production. Xanthine oxidoreductase (XOR) in hepatocytes is encoded by the XDH gene and exists in two forms: xanthine dehydrogenase (XDH) or xanthine oxidase (XO). XDH and XO catalyze the conversion of purines to uric acid using different substrates. As a key regulatory enzyme in uric acid synthesis in the body, XOR is also an important drug target for clinical uric acid-lowering treatment. Currently, first-line drugs for treating hyperuricemia, such as febuxostat and allopurinol, work by inhibiting the catalytic activity of XOR, thereby reducing uric acid synthesis levels. However, these small-molecule drugs have adverse reactions and limitations in application. Common adverse reactions include hepatotoxicity and nephrotoxicity, and limitations include short duration of action and a high relapse rate after discontinuation. Summary of the Invention

[0005] To address the shortcomings of the existing technologies, this invention provides an antisense oligonucleotide molecule that targets and reduces XDH gene expression and its application in the preparation of drugs to improve hyperuricemia.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: Antisense oligonucleotides that target and reduce XDH gene expression mediate the degradation of target mRNA through an RNase H-dependent cleavage mechanism, thereby knocking down XDH gene mRNA expression at the posttranscriptional level.

[0007] Furthermore, the antisense oligonucleotide comprises a gap region consisting of 10 2'-deoxynucleotides, with 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, and the nucleotides in the gap region are modified with 2'-deoxyribose. All nucleotides in the gapmer are bonded by phosphate thioester (PS) bonds, and all cytosine residues in all gapmers are 5-methylated.

[0008] The antisense oligonucleotide of the present invention comprises at least one N-acetylgalactosamine (GalNAc) as a targeting ligand for specifically binding to the desialylate glycoprotein 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 monovalent, divalent, or trivalent branched structures, and the GalNAc ligand is located at the 5' or 3' end of the antisense oligonucleotide.

[0009] The antisense oligonucleotides designed in this invention (1) adopt a 5-10-5 gapmer structure, with each ASO sequence being 20 bases in length; (2) each ASO sequence contains 45% to 65% GC; (3) each ASO has a binding energy ∆G°37 ≥ -8 kcal / mol to XDH mRNA; (4) based on gene sequence information alignment, each ASO predicts ≤3 potential off-target genes; and (5) each ASO excludes any motif sequences that trigger immune stimulation. This invention introduces multiple parameters (1)-(4) above on the basis of the classic "sliding window" design strategy, thereby improving the efficiency of ASO sequence screening.

[0010] The sequence of an ASO targeting the XDH gene provided by this invention is 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 XDH ASO-13 CAGGCAAAGGATACACGATC XDH ASO-67 GGCATCTTTGATGGCAAAGA XDH ASO-14 AGCAGGCAAAGGATACACGA XDH ASO-68 CATCTTTGATGGCAAAGAAG XDH ASO-15 GCAGGCAAAGGATACACGAT XDH ASO-69 GCATCTTTGATGGCAAAGAA XDH ASO-1                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            & ​ ​ ​ ​ ​ ​ 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 ASO molecule described in this invention contains all of the above-mentioned sequences and chemical modification features.

[0011] The present invention also provides the application of the antisense oligonucleotide in the treatment of hyperuricemia.

[0012] The present invention has the following beneficial effects: (1) The antisense oligonucleotides designed in this invention mediate the degradation of target mRNA through RNase H-dependent cleavage mechanism, thereby knocking down the mRNA expression of the XDH gene at the posttranscriptional level and further reducing the XDH protein product at the translational level, thus achieving targeted regulation of XDH gene expression. (2) The antisense oligonucleotides designed in this invention contain a targeting ligand with a delivery function, which is used to specifically bind to the desialyl glycoprotein receptor (ASGPR), enhance the hepatocyte targeting of the oligonucleotides, and the number of potential off-target genes predicted by ASO is ≤3.

[0013] (3) The antisense oligonucleotides of the present invention are used to prepare drugs for treating diseases that directly target XDH gene products or pathological conditions related to abnormal or dysfunctional expression of XDH gene, providing a pathway for the treatment of hyperuricemia. (4) This invention provides antisense oligonucleotide molecules. Their mechanism of action is that these ASO molecules, using the specific nucleic acid sequences described above and combined with chemical modifications, can target and specifically bind to XDH mRNA molecules, promoting XDH mRNA degradation, inhibiting XDH protein synthesis, thereby reducing XDH expression levels in cells, ultimately decreasing uric acid synthesis and achieving the effect of lowering uric acid. Cell experiments and animal model tests have shown that the ASO molecules of this invention can not only efficiently 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), and compared with allopurinol, the duration of efficacy (as shown in Example 3) and drug safety (as shown in Example 4) are significantly improved. Attached Figure Description

[0014] Figure 1 This is a diagram showing the effect of the ASOs targeting XDH, numbered XDH ASO-1 to XDH ASO-15, on reducing the expression of the target gene XDH in mouse cell lines in Example 1 of the present invention. Figure 2 This is a graph showing the serum uric acid level measurement in the hyperuricemia mouse model of Example 2 of the present invention; Figure 3 This is a graph showing the short-term uric acid-lowering effect of ASO XDH ASO-12 in a mouse model of hyperuricemia, as described in Example 2 of this invention. Figure 4 This is a graph showing the long-term uric acid-lowering effect of ASO XDH ASO-12 in a mouse model of hyperuricemia, as described in Example 3 of this invention. Figure 5 Anatomical diagrams of the liver and kidneys of mice treated with different doses of XDH ASO-12, febuxostat, and allopurinol in Example 4 of this invention. Figure 6 This is a schematic diagram showing the changes in body weight of mice treated with different doses of XDH ASO-12, febuxostat, and allopurinol in Example 4 of the present invention. Figure 7The graphs show the detection results of kidney and liver function indicators in mice treated with different doses of XDH ASO-12, febuxostat, and allopurinol in Example 4 of the present invention. In the graphs, A represents the detection results of the kidney function indicator CRE2, and B represents the detection results of the liver function indicator AST. Figure 8 This is a graph showing the effect of qPCR detection in Example 5 of the present invention on the reduction of target gene XDH expression in mouse cell lines by 31 ASOs (data are expressed as mean ± standard error). Figure 9 In Example 6 of this invention, the inhibitory effect of 31 ASOs targeting XDH on XDH protein expression in the AML12 cell line was verified by Western blotting, with β-Tubulin used as an internal control protein for sample loading correction. Figure 10 This is a graph showing the short-term uric acid-lowering effect of 31 ASOs in a hyperuricemic mouse model in Example 7 of the present invention. Figure 11 The graph shows the detection results of kidney and liver function indicators in mice treated with 31 ASO and allopurinol at 10 times the effective therapeutic dose in Example 9 of the present invention. In the graph: A is the detection result of the kidney function marker CRE, B is the detection result of the liver function AST, and C is the detection result of the liver function ALT. Detailed Implementation

[0015] The molecules with XDH numbers XDH ASO-1 to XDH ASO-15 used in Examples 1-4 were synthesized strictly according to their sequence and chemical modification characteristics.

[0016] Example 1: Validation of the ability of cell lines XDH ASO-1 to XDH ASO-15 to ASO to reduce the expression of the target gene XDH in in vitro cultured cell lines.

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

[0018] ASO transfection: Lipofectamine 2000 transfection reagent was used in the cell line to mix XDH-targeting ASO (50 / 100 / 150 nM) with liposome transfection reagent according to the instructions. A negative control group (transfected with ASO that does not target any gene sequence), a blank control group (no ASO transfection) and a positive siRNA control group (siRNA sequence that has been reported in the literature to have a knockdown effect on XDH expression) were set up. The transfection operation was as follows: (1) Add 50 μL of opti-MEM serum-free medium to a 1.5 mL sterile centrifuge tube and add 2 μL of ExFect, vortex to mix, and let stand for 3 minutes; (2) Add 50 μL of siRNA to a 1.5 mL sterile centrifuge tube. Add an appropriate amount of ASO to opti-MEM (final transfection concentration is 50 / 100 / 150 nM), vortex to mix, and let stand for 3 minutes; (3) Add ExFect-opti-MEM to ASO-opti-MEM, vortex to mix, and let stand at room temperature for 15 minutes before transfection; (4) Add the ExFect / ASO complex mixture to the culture medium, and gently shake the culture dish to disperse ExFect / ASO evenly; (5) Incubate overnight for 24 h; (6) Collect cells, extract RNA, and conduct subsequent experiments.

[0019] Step 2: Use quantitative real-time PCR to detect the knockdown effect of the target gene XDH at the mRNA level. qPCR detection of target mRNA expression: Total RNA was extracted from cells (using TRIzol reagent), and cDNA was synthesized by reverse transcription (using a reverse transcription kit). The expression level of XDH mRNA was detected using qPCR. 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: 95°C pre-denaturation for 30 seconds, 95°C denaturation for 10 seconds, 60°C annealing for 30 seconds, instrument default settings, for a total of 40 cycles; The results are as follows Figure 1 As shown, columnar Figure XThe axes indicate 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 different final concentrations (50nM, 100nM, 150nM) of ASO molecules that did not target any gene sequence; (3) 1-15, ASO experimental group, cells were treated with three different final concentrations (50nM, 100nM, 150nM) of ASO molecules 1-15 respectively; (4) siRNA, positive control group, cells were treated with siRNA that was published in the literature and clearly could effectively reduce XDH expression. The Y-axis of the bar chart indicates the relative expression level of XDH mRNA.

[0021] Real-time quantitative PCR analysis showed that, compared with the blank control (cells without any treatment) or the negative control group (NC group, i.e., cells treated with ASO sequences that do not target any gene), the expression level of XDH mRNA was reduced after treatment with XDH-targeted ASOs numbered XDH ASO-1 to XDH ASO-15. This finding provides strong experimental evidence for the application of XDH-targeted ASOs in the treatment of hyperuricemia. Experiments demonstrate that the ASOs of this invention effectively block the translation process of XDH by specifically binding to XDH mRNA, thereby reducing XDH protein synthesis.

[0022] Example 2: Validation of the short-term uric acid-lowering function of ASO (XDH ASO-12) in a hyperuricemic mouse model.

[0023] Step 1: Establishment of an animal model of hyperuricemia and administration of ASO (code XDH ASO-12) (1) Adaptive feeding of animals: The experimental mice (strain: C57BL / 6, male, 8 weeks old) were adapted to feeding under standard feeding conditions for 1 week to ensure stable health status; (2) Drug preparation: a. Potassium oxazine: 200 mg / mL, dissolved in 0.5% sodium carboxymethyl cellulose solution; b. Hypoxanthine: 100 mg / mL, dissolved in 0.5% sodium carboxymethyl cellulose solution; (3) Model building and grouping: c. Blank control group: 0.5% sodium carboxymethyl cellulose solution (150 μL) was administered by gavage for the first time, and the same solution (150 μL) was administered by gavage for the second time 2 hours later. d. Modeling group: Initially administered hypoxanthine solution (100 mg / mL, 150 μL) by gavage, followed by potassium oxonate solution (200 mg / mL, 150 μL) by gavage 2 hours later. Modeling continued until the end of treatment.

[0024] (4) Model validation Nine days after modeling, orbital blood was collected from mice to detect serum uric acid levels. After confirming that the serum uric acid level in the model group was significantly higher than that in the blank control group, the next step of drug administration was carried out.

[0025] (5) Different groups of drugs were administered. e. Experimental group: ASO-12 targeting XDH was administered at doses of 15, 10, and 5 mg / kg (finally dissolved in 100 μL of physiological saline) and injected into mice via the tail vein 9 days after modeling.

[0026] f. Negative control group: physiological saline, dose 100 μL / mouse, injected into mice via tail vein 9 days after modeling.

[0027] g. Positive control group: Allopurinol, dose of 25 mg / kg, administered by gavage once daily for 5 days after modeling, 7 hours after modeling.

[0028] h. Blank control group: The group that did not undergo the high uric acid model treatment.

[0029] Step 2: Serum uric acid level test: Five days after administration, orbital blood was collected from mice, serum was separated, and uric acid levels were measured using a fully automated biochemical analyzer to simulate the effect of short-term administration in lowering uric acid.

[0030] like Figure 2 As shown, columnar Figure X The Y-axis indicates the experimental groups, namely the non-modeling group and the modeling group; the Y-axis indicates the serum uric acid level of mice (unit: μmol / L). *** indicates that there is a statistically significant difference in serum uric acid values ​​between the modeling group and the non-modeling group, p < 0.001; after modeling by processing in steps (1), (2), (3), and (4) of 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.

[0031] like Figure 3 As shown, columnar Figure XThe axes indicate the experimental groups, namely: (1) blank control group without modeling, mice were fed normally without drug stimulation to induce modeling; (2) negative control group, mice were gavaged with physiological 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 *** indicate that there are statistically significant differences in serum uric acid values ​​between the pairwise comparison groups, where **P<0.01; ***P<0.001. Different doses (5 / 10 / 15 mg / kg) of ASO designated as XDH ASO-12 can effectively reduce the uric acid level of hyperuricemic mice and restore it to normal. The results show 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 metabolism pathway and reduce uric acid production, thus playing an important role in the treatment of hyperuricemia.

[0032] Example 3: ASO product XDH ASO-12 was able to maintain a long-lasting uric acid-lowering effect in hyperuricemic mice. Because small molecule chemical drugs such as allopurinol have a short duration of action and uric acid levels tend to rebound after drug withdrawal, this embodiment uses a hyperuricemic mouse model to detect the changes in uric acid levels of ASO-12 compared to allopurinol over a period of time after drug withdrawal.

[0033] The specific experimental procedures are as follows: Step 1: Establishment of the hyperuricemia animal model and administration of ASO, same as Step 1 in Example 2. Step 2: After completing the administration procedure, stop administration (ASO or allopurinol). All model mice continue to be stimulated with potassium oxonate / hypoxanthine for 28 days to simulate the scenario of long-term drug withdrawal after a period of medication in patients. Step 3: Detection of serum uric acid levels, same as Step 2 in Example 2.

[0034] like Figure 4 As shown, columnar Figure X The axes indicate the experimental groups, namely: (1) blank control group without modeling, mice were fed normally without drug stimulation to induce modeling; (2) negative control group, mice were gavaged with physiological saline after modeling; (3) ASO experimental group, mice were treated with ASO-12 targeting XDH after modeling, and the results were measured 28 days after administration; (4) positive control group, mice were treated with allopurinol after modeling, and the results were measured 28 days after administration. * and *** indicate that there are statistically significant differences in serum uric acid values ​​between the pairwise comparison groups, where *P<0.05; ***P<0.001.

[0035] In a mouse model of hyperuricemia, serum uric acid levels rebounded to hyperuricemic levels 28 days after administration of allopurinol and subsequent withdrawal. In contrast, serum uric acid levels remained normal 28 days after administration of ASO-12 (a drug targeting XDH). This result indicates that ASO-12, targeting XDH, significantly outperformed allopurinol in maintaining the therapeutic effect.

[0036] Example 4: The ASO designated XDH ASO-12 has good security. This embodiment uses a gradient dose administration experiment to evaluate the safety of ASO-12 in mice, thereby verifying its clinical application as a potential therapeutic agent.

[0037] The specific steps are as follows: Step 1: Experimental grouping and drug treatment 1. Animal acclimatization feeding: After arrival, the experimental mice (C57BL / 6, male, 8 weeks old) were acclimatized for 1 week under standard feeding conditions to ensure stable health.

[0038] 2. Experimental grouping and drug treatment: a. Experimental group: ASO targeting XDH, at doses of 1, 5, and 10 mg / kg / animal (dissolved in 100 μL of physiological saline), administered once via tail vein injection.

[0039] b. Negative control group: 100 μL / animal saline, injected once via tail vein.

[0040] c. Positive control group: Allopurinol, at doses of 25, 125, and 250 mg / kg / animal (dissolved in 100 μL of 3dH2O); Febuxostat, at doses of 20, 100, and 200 mg / kg / animal (dissolved in 200 μL of 0.5% sodium carboxymethyl cellulose solution), both drugs were administered by gavage once daily.

[0041] Step 2: Dynamic monitoring of physiological indicators: 1. Weight changes: The mice were weighed at a fixed time every other day (11:00 AM).

[0042] 2. Liver and kidney function indicators: Blood was collected from the eyeballs of mice on days 10 and 14 after drug administration, and serum was separated. AST (a marker of liver injury) and CRE (a key marker of kidney injury) were detected using a biochemical analyzer.

[0043] like Figure 5-7 As shown, healthy mice were treated with physiological saline and low, medium, and high doses of ASO-12, allopurinol, and febuxostat, respectively. Liver and kidney organ function was then assessed. Figure 5 ),weight( Figure 6 ), kidney injury marker CRE2 ( Figure 7 As shown in A), liver injury marker AST ( Figure 7 (As shown in B) Indicator.

[0044] Commonly used uric acid-lowering drugs (allopurinol, febuxostat) all showed varying degrees of weight loss or nephrotoxicity (mainly manifested as acute kidney injury, abnormal tissue anatomy, or abnormal CRE markers of kidney injury) under high-dose treatment in experimental conditions. This result is basically consistent with the instructions for use of the relevant drugs, which also indicates that such drugs are not suitable for long-term use. However, the ASO-12 targeting XDH of this invention, through precise regulation of target expression, maintains the intervention effect of uric acid metabolism without causing significant metabolic disorders or organ damage, and has a safe profile, providing key experimental evidence for its clinical translation.

[0045] The 31 ASO sequences in Examples 5-9 below are numbered ASO-3, ASO-5, ASO-12, ASO-13, ASO-15-21, ASO-23, ASO-25-27, ASO-29, ASO-31, ASO-36, ASO-44, ASO-46, ASO-49, ASO-51, ASO-52, ASO-62, ASO-63, ASO-65, ASO-67, ASO-92, ASO-93, ASO-100, and ASO-103, with ASO-12 being a positive control sequence.

[0046] All 31 ASO molecules mentioned above were synthesized according to the specified sequences and chemical modifications.

[0047] Example 5: Validation of the ability of the above 31 ASOs to reduce the expression of the target gene XDH in in vitro cultured cell lines using real-time quantitative PCR. This embodiment aims to verify, at the mRNA level, the inhibitory activity of 31 ASOs targeting XDH on XDH gene expression in AML12 cells using quantitative real-time PCR technology.

[0048] Step 1: Cell Culture and Transfection Cell culture: AML12 cells (normal mouse hepatocyte line) were used at a density of 1 × 10⁶ cells per well. 5 Cells were seeded at a density of 10% in 12-well plates, and 1 mL of DMEM / F12 complete medium containing 10% fetal bovine serum and 1% penicillin / streptomycin was added to each well. The cells were then cultured in a 37°C, 5% CO2 incubator until they reached 70%-80% confluence before transfection.

[0049] ASO transfection: Using Lipofectamine 2000 transfection reagent, ASO targeting XDH was transfected into cells at a final concentration of 100 nM. Specific steps included: mixing the transfection reagent and ASO separately with Opti-MEM serum-free medium, allowing them to stand at room temperature, then mixing the two solutions to form a transfection complex; removing the original cell culture medium, adding the transfection complex, culturing for 6 hours, then replacing with complete culture medium, and continuing culturing until the designated time point for cell collection.

[0050] Step 2: Real-time quantitative PCR detection RNA extraction and reverse transcription: Total RNA was extracted from cells using the TRIzol method. After testing its purity and concentration, 1 μg of RNA was used to synthesize cDNA using a reverse transcription kit.

[0051] qPCR reaction: Amplification was performed using the SYBR Green kit on a real-time quantitative PCR instrument. The primer sequences are as follows (all specificity was verified by BLAST): XDH (target gene): Forward primer sequence: 5'-TAGAAGAAAGTTGGGGCTGTGCG-3', reverse primer sequence: 5'-CAGATGGGGGTCAAGCAGGCA-3', the amplification product of this primer pair has a fragment length of 187 bp; GAPDH (internal reference gene): forward primer sequence 5'-CATCACTGCCACCCAGAAGACTG-3', reverse primer sequence 5'-ATGCCAGTGAGCTTCCCGTTCAG-3', the amplification product of this primer pair has a fragment length of 154 bp.

[0052] The reaction volume was 20 μL. The reaction conditions included pre-denaturation at 95 °C for 30 seconds, followed by 40 cycles of denaturation, annealing / extension, and finally melting curve analysis. The relative expression level of XDH mRNA was calculated using the 2^(-ΔΔCq) method, and normalized using GAPDH as an internal reference gene.

[0053] qPCR experimental results are as follows Figure 8 As shown: columnar Figure X The Y-axis represents the experimental group (NC group: negative control group, cells transfected with scramble ASO; experimental group: cells transfected with 100 nM ASO molecules of 31 sequences respectively); the Y-axis represents the relative expression level of XDH mRNA (NC group as 1-fold reference).

[0054] Real-time quantitative PCR analysis showed that, compared with the negative control, the positive sequence ASO-12 and 30 other ASOs significantly reduced the expression level of the target gene XDH mRNA, with inhibition rates ranging from 50% to 80%, confirming that the 31 ASO molecules have highly efficient gene expression inhibition activity at the mRNA level.

[0055] Example 6: Validation of the inhibitory effect of 31 ASOs targeting XDH on XDH protein expression in the AML12 cell line by Western blotting. In this embodiment, Western blotting was used to verify the inhibitory effect of ASO on XDH expression at the protein level, further clarifying its regulatory function.

[0056] Step 1: Cell Culture and ASO Transfection The cell culture process, ASO transfection steps, and experimental grouping were the same as in Example 5. Cells were collected 48 hours after transfection for total protein extraction.

[0057] Step 2: Total protein extraction and Western blot analysis Total protein extraction: Collect cells, wash twice with pre-cooled PBS, add strong RIPA lysis buffer containing protease inhibitors, stand on ice for 10 minutes, then sonicate to disrupt the cells, and continue lysis on ice for 20 minutes; after low-temperature high-speed centrifugation for 15 minutes, aspirate the supernatant to obtain the total protein extract.

[0058] Western blotting of proteins: 50 μg of protein sample was subjected to SDS-PAGE electrophoresis, followed by transfer to a PVDF membrane. After blocking with 5% skim milk, the sample was sequentially incubated with XDH primary antibody and HRP-labeled secondary antibody, and finally developed using ECL chemiluminescence reagent. β-Tubulin was used as an internal control protein to verify changes in XDH protein expression.

[0059] The results are as follows Figure 9 As shown: Figure 9 The A in the figure indicates that, compared with the negative control group, the content of XDH protein in cells was significantly reduced after ASO treatment with reagents 3 / 5 / 12 / 13 / 15. Figure 9 B indicates that, compared with the negative control group, the content of XDH protein in cells was significantly reduced after treatment with ASOs 16 / 17 / 18 / 19 / 20 / 21 / 23 / 25 / 26 / 27. Figure 9 The C value indicates that, compared with the negative control group, the XDH protein content in cells was significantly reduced after treatment with ASOs 29 / 31 / 36 / 44 / 46 / 49 / 51 / 52. Figure 9The D in the figure indicates that (compared to the negative control group, treatment with ASOs 62 / 63 / 65 / 67 / 92 / 93 / 100 / 103 significantly reduced the XDH protein content in cells). In the Western blot pattern, the top row shows the XDH protein bands, and the bottom row shows the β-Tubulin internal control bands; the intensity of the band color indicates the expression level of the corresponding protein. The results confirm that the positive sequence ASO-12 and 30 other ASO molecules can effectively reduce the expression level of XDH protein, and the inhibitory effect is consistent with that at the mRNA level.

[0060] Example 7: Validation of the short-term uric acid-lowering function of 31 ASOs in animals using a hyperuricemic mouse model. 1. Preparation of experimental animals and induction of hyperuricemia model (1) Adaptation feeding of experimental animals: Healthy male mice of C57BL / 6 strain (8 weeks old) were selected and acclimatized for 7-10 days in a standard environment (temperature 22±2℃, humidity 50%±10%, 12-hour light-dark cycle) to ensure that the animals’ physiological state was stable before they could be used for experiments.

[0061] (2) Preparation of modeling reagents: a. Potassium oxazine solution: Weigh an appropriate amount of potassium oxazine, dissolve it in 0.5% sodium carboxymethyl cellulose aqueous solution, and prepare a suspension with a final concentration of 200 mg / mL. b. Hypoxanthine solution: Weigh hypoxanthine and dissolve it in 0.5% sodium carboxymethyl cellulose solution using the same method to prepare a suspension with a concentration of 100 mg / mL.

[0062] (3) Model building and experimental grouping: Mice were randomly divided into the following four groups: Blank control group: No hyperuricemia mouse model was constructed. The mice were treated with 0.5% sodium carboxymethyl cellulose solution. 150 μL was administered by gavage for the first time, and the same volume of solvent was administered by gavage for the second time after an interval of 2 hours. Negative model treatment group: hypoxanthine solution (150 μL, equivalent to 100 mg / kg) was administered by gavage, followed by potassium oxonate solution (150 μL, equivalent to 200 mg / kg) by gavage 2 hours later, once daily; after the model was established, an equal volume of physiological saline (100 μL / animal) was injected via tail vein.

[0063] Model ASO treatment group: On the 9th day after model establishment and confirmation of successful modeling, a low dose (15 mg / kg) of XDH ASO preparation (dissolved in 100 μL of physiological saline) was injected via the tail vein. Model positive drug treatment group: The positive control group was treated with allopurinol, a common clinical uric acid-lowering drug, as a positive drug to treat the hyperuricemia mouse model. Starting from the 9th day of modeling, allopurinol (25 mg / kg) was administered by gavage 7 hours after modeling, and the treatment continued for 5 days. (4) Model validity verification: Nine days after the initial modeling, blood was collected from the orbital cavity and centrifuged to prepare serum samples. Serum uric acid levels were then measured using a fully automated biochemical analyzer.

[0064] 2. Drug efficacy index detection and data analysis Five days after administration of the aforementioned 31 ASOs and positive control drugs, serum samples were collected again via orbital blood collection. Serum uric acid concentration was quantified using a fully automated biochemical analyzer. The statistical differences in uric acid levels between the treatment groups, the model group, and the positive control group were compared to evaluate the uric acid-lowering effect of the 31 different XDH ASO molecules at low doses. Figure 10 As shown, a single injection of a low dose (15 mg / kg) including the positive sequence ASO-12 and 30 other ASOs significantly reduced uric acid levels in a mouse model of hyperuricemia, with all ASO treatment groups recovering to near-normal uric acid levels. This result indicates that the 30 ASOs targeting XDH can effectively intervene in the uric acid metabolism pathway, reducing uric acid production and thus playing an important role in the treatment of hyperuricemia.

[0065] 3. The long-term uric acid-lowering function of 31 ASOs has been verified in animals based on a mouse model of hyperuricemia.

[0066] Example 8: 31 ASOs showed good safety in mice. 1. Experimental design and dosing regimen (1) Experimental animals and feeding conditions: Healthy male mice of the C57BL / 6 strain (8 weeks old) were selected and acclimatized for 7-10 days in a standard environment (temperature 22±2℃, relative humidity 50±10%, 12 h / 12 ​​h light and dark cycle) to confirm that their physiological state was stable before they were used for experiments.

[0067] (2) Grouping and drug administration: Mice were randomly divided into the following four groups for intervention: Wild-type mice: C57BL / 6 strain male mice raised under normal feeding conditions without any intervention; Blank control group: A single injection of an equal volume of normal saline (100 μL / animal) via the tail vein. ASO treatment experimental group: 31 different XDH ASO molecules (dissolved in 100 μL of physiological saline) were injected once via tail vein at a dose 10 times the effective therapeutic dose (150 mg / kg). Positive drug allopurinol group: Administered daily by gavage at 10 times the effective therapeutic dose (250 mg / kg) (dissolved in 100 μL deionized water) for 5 consecutive days, then discontinued. 2. Safety Indicator Observation and Analysis Methods Since the most significant side effect of conventional uric acid-lowering drugs is liver and kidney toxicity, this embodiment uses liver and kidney function biochemical tests as the core indicator for safety evaluation. On day 10 after drug administration, orbital blood was collected from mice. After serum separation, the following indicators were measured in each serum sample using a fully automated biochemical analysis system: Liver injury markers: aspartate aminotransferase (AST) and alanine aminotransferase (ALT); Kidney injury marker: Creatinine (CRE).

[0068] 3. Results and Safety Conclusions like Figure 11 As shown, Figure 11 The A in the figure indicates that, compared with the wild-type control group and the saline-treated negative control group, the renal function index serum creatinine CRE of mice in the ASO treatment group of 31 sequences was normal, and the index of this index was also normal in mice in the febuxostat-treated group. Figure 11 B indicates that, compared with the wild-type control group and the saline-treated negative control group, the liver function index aspartate aminotransferase (AST) in the ASO treatment group of 31 sequences was normal, while the index in the febuxostat positive drug treatment group was slightly elevated. Figure 11 The C in the figure indicates that, compared with the wild-type control group and the saline-treated negative control group, the liver function index alanine aminotransferase (ALT) was normal in the ASO treatment group of mice with the 31 sequences, while this index was slightly elevated in the mice treated with the positive drug febuxostat. During the experimental observation period, no significant abnormalities were observed in the renal function index CRE and the liver function indexes AST and ALT in the wild-type mouse group, the negative control group, and the ASO treatment group with the 31 sequences; however, the positive control drug febuxostat induced a slight increase in liver function indexes, suggesting a certain risk of organ damage, which is consistent with the known clinical safety characteristics of the aforementioned drugs. The XDH-targeting ASO sequences provided by this invention did not induce significant liver and kidney toxicity or weight suppression at high doses, indicating that they have an excellent tolerability window. Combined with its previously verified uric acid-lowering efficacy, the ASO molecules provided by this invention show great potential for clinical translation in lowering uric acid.

[0069] This invention provides an antisense oligonucleotide (ASO) and a prepared drug for inhibiting hyperuricemia. In in vitro cultured cell lines and hyperuricemia mouse models, the ASO targeting XDH effectively reduces XDH gene expression levels, thereby intervening in the uric acid metabolism pathway and reducing uric acid production. Therefore, this invention is expected to provide a new approach for the treatment of hyperuricemia, playing an important role in the treatment of hyperuricemia and promoting the application of antisense oligonucleotide technology in the treatment of metabolic diseases. The hyperuricemia-inhibiting drug of this invention has broad application prospects for adjunctive treatment of hyperuricemia.

Claims

1. An antisense oligonucleotide targeted to reduce expression of an XDH gene, characterized in that: The antisense oligonucleotide comprises the sequence defined by the following number corresponding and the sequence synthesized in the form of chemical modification: 。 2. A composition capable of reducing the expression of XDH gene, comprising the antisense oligonucleotide of claim 1 and a pharmaceutically acceptable carrier.

3. Use of the antisense oligonucleotide of claim 1 in the preparation of a drug for reducing the expression of XDH gene.

4. Use of the antisense oligonucleotide of claim 1 or the composition of claim 2 in the preparation of a drug for treating hyperuricemia.

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