XDH nucleic acid-targeted oligonucleotide and use thereof

Abstract

The present invention relates to the field of biomedicine, and specifically relates to an XDH nucleic acid-targeted oligonucleotide and a use thereof. The oligonucleotide contains consecutive sequences that are at least 80%, 90%, 95%, 99%, or 100% identical to at least 12 consecutive nucleotides in a sequence GCCCACCATGTCC TCCTCAGACTGACCCTT, TGCCCAACACAAGTAACCTAG, or TTGCCACAAGGTGTCAGTATATGT. The oligonucleotide or the modified oligonucleotide can be used for treating XDH-related diseases or symptoms.

Description

Oligonucleotides targeting XDH nucleic acids and their applications Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to oligonucleotides targeting XDH nucleic acid and their applications. Background Technology

[0002] Hyperuricemia (HUA) is caused by purine metabolism disorders leading to excessive production of uric acid (SUA) or decreased excretion of uric acid (UA). Globally, there are approximately 1 billion patients. In my country, the prevalence of gout and hyperuricemia has been rising rapidly in recent years, becoming the second leading metabolic disease after diabetes. Meta-analysis shows that the overall prevalence of hyperuricemia in China is 13.3% (1.1% for gout), estimating approximately 177 million hyperuricemia patients and 14.66 million gout patients. Hyperuricemia is not only a major cause of gout but also a contributing factor to kidney disease, metabolic syndrome, insulin resistance, type 2 diabetes, and non-alcoholic fatty liver disease, exhibiting a linear dose-response relationship.

[0003] Uric acid in the human body mainly originates from purine metabolism. Xanthine oxidoreductase (XOR) is the key and rate-limiting enzyme in uric acid production. XOR exists in two forms: xanthine oxidase (XO) and xanthine dehydrogenase (XDH), which catalyze the oxidation of hypoxanthine to xanthine and xanthine to uric acid, respectively. Most XOR in the liver exists in the form of XDH. Xanthine dehydrogenase is crucial for treating hyperuricemia and gout. Allopurinol and febuxostat, used clinically to treat gout, are inhibitors of XDH, but they have serious adverse reactions.

[0004] Antisense oligonucleotide (ASO) therapeutics typically consist of 15-30 chemically modified nucleotides linked primarily by phosphate thioester bonds. Once inside the cell, ASO drugs bind to complementary target mRNAs via base pairing and are then degraded by ribonuclease H1, thereby inhibiting target gene expression. In addition, the mechanisms of action of ASO drugs include translation inhibition, splicing regulation, and increased protein translation. Summary of the Invention

[0005] The inventors designed a series of antisense oligonucleotide sequences that can effectively inhibit the expression of XDH at the transcriptional level, thereby inhibiting the production of uric acid.

[0006] On one hand, the present invention provides an oligonucleotide containing at least 80%, 90%, 95%, 99%, or 100% identical continuous sequences to at least 12 consecutive nucleotides in the sequences GCCCACCATGTCCTCCTCAGACTGACCCTT, TGCCCAACACAAGTAACCTAG, TTGCCACAAGGTGTCAGTATATGT, TAGAACTTGAAGAAGAAGCT, AGAACTTGAAGAAGAAGCTG, AACTTGAAGAAGAAGCTGAG, GGTTCACAAACTGTCTGGAG, GTTCACAAACTGTCTGGAGA, CTGAAGTAGTGGAAGGGGTT, TGAGTAGTGGAAGGGGTTC, CACAAACTGTCTGGAGATCT, TCACAAACTGTCTGGAGATC, CAGGCAAAGGATACACGATC, or TTTTGGCAATTCTCTCCTAA.

[0007] Preferably, the oligonucleotide consists of 12 to 27 linked nucleosides; more preferably, the oligonucleotide consists of 14 to 20 linked nucleosides.

[0008] Preferably, the modified oligonucleotide is a modified oligonucleotide.

[0009] Preferably, at least one nucleoside in the modified oligonucleotide contains a modified nucleic acid base. Preferably, the modified nucleic acid base is 5-methylcytosine.

[0010] Preferably, the modified oligonucleotide is a gamper, which includes a gap composed of a linked deoxyribonucleotide and 5' and 3' wings composed of linked ribonucleotides, wherein the gamper is located between the 5' and 3' wings.

[0011] Preferably, at least one nucleotide of the wing segment of the spacer comprises a modified sugar. Preferably, the modified sugar is a 2'-modified sugar. Preferably, the 2'-modified sugar is 2'-O-methoxyethyl modified. Preferably, the modified sugar is a bicyclic sugar. Preferably, the modification is 4'-CH(CH3)-O-2' (referred to as "restricted ethyl" or "cEt") modification.

[0012] Preferably, the modified oligonucleotide comprises: a nick consisting of linked deoxynucleosides and 5' and 3' wings consisting of linked nucleosides, wherein the spacer is located between the 5' and 3' wings, and wherein each nucleoside in each wing comprises a modified sugar. Preferably, the nick consists of 3-16 linked nucleosides. Preferably, the modified oligonucleotide comprises: a nick consisting of 8-12 linked deoxynucleosides and 5' and 3' wings consisting of 2-6 linked nucleosides, wherein the nick is located between the 5' and 3' wings, and wherein each nucleoside in each wing comprises a modified sugar. Preferably, the modified oligonucleotide comprises: a nick consisting of 10 linked deoxynucleosides and 5' and 3' wings consisting of 5 linked nucleosides, wherein the nick is located between the 5' and 3' wings, and wherein each nucleoside in each wing comprises a modified sugar; wherein each nucleoside in each wing comprises a 2'-O-methoxyethyl sugar or a restrictive ethyl sugar. Preferably, the modified oligonucleotide comprises: a nick consisting of 10 linked deoxynucleotides and 5' and 3' wings consisting of 3 linked nucleotides, wherein the nick is located between the 5' and 3' wings and wherein each nucleotide of each wing comprises a modified sugar; wherein each nucleotide of each wing comprises a 2'-O-methoxyethyl sugar or a restricted ethyl sugar.

[0013] Preferably, at least one internucleotide bond in the modified oligonucleotide is a modified internucleotide bond. Preferably, all internucleotide bonds in the modified oligonucleotide are modified internucleotide bonds. Preferably, the modified internucleotide bonds are phosphate thioester internucleotide bonds.

[0014] In a preferred embodiment, the single-stranded modified oligonucleotide comprises a notched segment consisting of 10 linked deoxynucleosides and 5' and 3' wings consisting of 5 linked nucleosides, respectively, wherein the notched segment is located between the 5' and 3' wings, and each nucleoside in each wing is a 2'-O-methoxyethyl modified nucleoside, the internucleotide bonds in the entire modified oligonucleotide are phosphate thioester bonds, and all cytosines in the entire modified oligonucleotide are 5-methylcytosine. In another preferred embodiment, the single-stranded modified oligonucleotide comprises a notched segment consisting of 10 linked deoxynucleosides and 5' and 3' wings consisting of 3 linked nucleosides, respectively, wherein the notched segment is located between the 5' and 3' wings, and each nucleoside in each wing is a restricted ethyl sugar modified nucleoside, the internucleotide bonds in the entire modified oligonucleotide are phosphate thioester bonds, and all cytosines in the entire modified oligonucleotide are 5-methylcytosine.

[0015] Preferably, the nucleotide sequence of the 10 linked deoxyribonucleotide nicks is selected from: CATGTCCTC, CATGTCCTCC, ATGTCCTCCT, CTTGAAGAAG, TTGAAGAAGA, GAAGAAGAA, TGAGAAGAA, GAAGAAGAAG, AAGAAGAAGC, ACAAACTGTC, CAAACTGTCT, GGTGTCAGT, GGTGTCAGTA, AAACTGTCTG, GTAGTGGAAG, ACTGTCTGGA, AACTGTCTGG, ACACAAGTAA, GGCAAAGGAT, GGCAAAGGAT, GCAAAGGATA, CAAAGGATAC, AAAGGATACA, GAAGAAGCTG, GGCAATTCTC, GCAATTCTCT;

[0016] Accordingly, the 5' wing segment is selected from GCCCA, CCCAC, CCACC, TAGAA, AGAAC, GAACT, AACTT, ACTTG, GGTTC, GTTCA, CCACAA, CACAA, TTCAC, CTGAA, TGAAG, CACAA, TCACA, TCACA, GCCCA, CAGCA, AGCAG, GCAGG, CAGGC, TTGAA, CTTTT, TTTTTG;

[0017] Accordingly, the 3' wing segment is selected from CTCAG, TCAGA, CAGAC, AAGCT, AGCTG, GCTGA, CTGAGG, TGGAG, GGAGA, AATTG, TATGT, GAGAT, GGGTT, GGTTC, GATCT, AGATC, CCTAG, AACCG, CACGA, ACGAT, CGATC, AGGGT, TCCTA, and CCTAA.

[0018] Alternatively, the nucleotide sequence of the 10 linked deoxyribonucleotides forming the nick is selected from: CACCATGTCC, ACCATGTCCT, CCATGTCCTC, CATGTCCTCC, ATGTCCTCCT, TGTCCTCCTC, GTCCTCCTCA, TCCTCCCTCAG, CCTCCTCAGA, CTCCTCAGAC, TCCTCAGACT, CCTCAGACTG, CTCAGACTGA, TCAGACTGAC, CAGACTGACC, CCAACACAAG, CAACACAAGT, AACCAAGTA, ACACAAGTAA, CACAAGTAAC, ACAAGTAACC, CCACAAGGTG, CACAAGGTGT, ACAAGGTGTC, CAAGGTGTCA, AAGGTGTCAG, AGGTGTCAGT, GGTGTCAGTA, GTGTCAGTAT, TGTCAGTATA;

[0019] Accordingly, the 5' wing segment is selected from GCC, CCC, CCA, CAC, ACC, CCA, CAT, ATG, TGT, GTC, TCC, CCT, CTC, TCC, CCT, TGC, GCC, CCC, CCA, CAA, AAC, TTG, TGC, GCC, CCA, CAC, ACA, CAA, AAG, AGG;

[0020] Accordingly, the 3' wing segment is selected from TCC, CCT, CTC, TCA, CAG, AGA, GAC, ACT, CTG, TGA, GAC, ACC, CCC, CCT, CTT, TAA, AAC, ACC, CCT, CTA, TAG, TCA, CAG, AGT, GTA, TAT, ATA, TAT, ATG, and TGT.

[0021] On the other hand, the present invention provides a liver-targeting oligonucleotide, the structure of which comprises the above-mentioned oligonucleotide and one or more ligands of N-acetylgalactosamine (GalNAc) derivatives. Preferably, the ligands of the N-acetylgalactosamine (GalNAc) derivatives are attached to the oligonucleotide via a linker. Preferably, the linker is a monovalent, divalent, or trivalent branched linker.

[0022] More preferably, the structure of the liver-targeting oligonucleotide is as follows:

[0023] N-acetylgalactosamine is attached to the 5' end of the oligonucleotide.

[0024] On the other hand, the present invention provides a pharmaceutical composition comprising the modified oligonucleotide or a salt thereof, or a liver-targeting oligonucleotide, and a pharmaceutically acceptable carrier. Preferably, the composition is an injectable pharmaceutical composition, such as a subcutaneous or intravenous injection pharmaceutical composition.

[0025] On the other hand, the present invention provides the use of the aforementioned oligonucleotide or its salt, or liver-targeting oligonucleotide, for the preparation of medicaments for the prevention or treatment of XDH-related diseases or symptoms. Preferably, the disease or symptom is hyperuricemia or gout. Attached Figure Description

[0026] Figure 1 shows the effect of MOE-modified antisense oligonucleotides on reducing XDH gene expression in primary hepatocytes of suckling mice.

[0027] Figure 2 shows the effect of cEt-modified antisense oligonucleotides on reducing XDH gene expression in primary hepatocytes of suckling mice.

[0028] Figure 3 shows the inhibitory effect of different concentrations of antisense oligonucleotides on XDH mRNA. Detailed Implementation

[0029] definition

[0030] "Antisense oligonucleotide" or "ASO" refers to an oligonucleotide having a nucleobase sequence complementary to that of a target nucleic acid or a region or segment thereof. Antisense oligonucleotides can specifically hybridize with a target nucleic acid or a region or segment thereof, and this hybridization leads to RNase H-mediated cleavage of the target nucleic acid.

[0031] "XDH nucleic acid" refers to any nucleic acid that encodes XDH. For example, in some embodiments, XDH nucleic acid includes, but is not limited to, DNA sequences encoding XDH and any RNA sequences transcribed from DNA, including any mRNA sequences encoding XDH proteins, such as pre-mRNA or spliced ​​mRNA sequences.

[0032] Throughout this application, the term "about" is used to indicate values ​​that include inherent variations in the error of the method / apparatus used to determine the value, or variations that exist between study subjects. Typically, the term "about" means encompassing a variation of approximately or less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, depending on the specific circumstances.

[0033] The term “or” is used in the claims to mean “and / or” unless explicitly indicated to refer only to alternatives or that the alternatives are mutually exclusive, although this disclosure supports the definition of alternatives and “and / or” only.

[0034] "2′-O-methoxyethyl" (also known as 2′-MOE) refers to 2′-O(CH2)2-OCH3 replacing the 2′-OH group on the ribosyl ring. Sugars modified with 2′-O-methoxyethyl are modified sugars.

[0035] "2′-MOE nucleoside" (2′-O-methoxyethyl nucleoside) refers to a nucleoside containing a sugar moiety modified with 2′-MOE.

[0036] "2′-substituted nucleoside" or "2′-modified nucleoside" means a nucleoside containing a 2′-substituted or 2′-modified sugar moiety. As used herein, "2′-substituted" or "2′-modified" with respect to the sugar moiety means a sugar moiety containing at least one 2′-substituent group other than H or OH.

[0037] "Bicyclic sugar" refers to a furanose ring modified by two atoms bridging it. Bicyclic sugars are modified sugars. "Bicyclic nucleic acid" or "BNA" refers to a nucleoside or nucleotide in which the furanose portion includes a bridging link between two carbon atoms on the furanose ring, thus forming a bicyclic system.

[0038] "cEt" or "restricted ethyl" refers to a bicyclic nucleotide having a bicyclic sugar moiety comprising a bridge connecting a 4'-carbon and a 2'-carbon, wherein the bridge has the formula: 4'-CH(CH3)-O-2'. "cEt-modified nucleotide" refers to a bicyclic nucleotide having a bicyclic sugar moiety comprising a bridge connecting a 4'-carbon and a 2'-carbon, wherein the bridge has the formula: 4'-CH(CH3)-O-2'. Its structural formula is as follows:

[0039] Where Bx represents any nucleobase.

[0040] A "spacer" or "gapmer" refers to an antisense oligonucleotide containing an inner region of multiple nucleotides that support RNase H cleavage between an outer region containing one or more nucleotides, wherein the nucleotides containing the inner region are chemically different from the one or more nucleotides containing the outer region. The inner region may be referred to as a "gap" or "segment," and the outer region may be referred to as a "wing." In some embodiments, the antisense oligonucleotide is a spacer.

[0041] The term "complementary" is used to describe the relationship between nucleotide bases and / or polynucleotides that are capable of hybridizing with each other. For example, when two nucleotide sequences are aligned in opposite directions, such a polynucleotide or one or more regions thereof matches the nucleotide sequence of another polynucleotide or one or more regions thereof. As described herein, nucleobase matching or complementary nucleobases include the following pairs: adenine (A) with thymine (T), adenine (A) with uracil (U), cytosine (C) with guanine (G), and 5-methylcytosine (…). m C) with guanine (G). Complementary polynucleotides and / or nucleic acids do not need to have nucleobase complementarity at every nucleoside and may include one or more nucleobase mismatches. Therefore, this disclosure also includes isolated polynucleotides complementary to sequences as disclosed or used herein, as well as those substantially similar nucleic acid sequences.

[0042] Nucleobases can be naturally occurring or synthetic. Nucleobases and sugar bases can be modified or unmodified independently. "Modified nucleosides" refers to nucleosides that contain modified nucleobases and / or modified sugar moieties. Modified nucleosides can include abase-free nucleosides lacking nucleobases.

[0043] 5-methylcytosine ( m C) refers to cytosine with a methyl group attached to the 5-position. 5-Methylcytosine is a modified nucleobase.

[0044] In the context of oligonucleotides, "continuous" refers to adjacent nucleosides, nucleobases, sugar moieties, or links between nucleosides. For example, "continuous nucleobases" means nucleobases that are adjacent to each other in the sequence.

[0045] "Connected nucleosides" refers to adjacent nucleosides that are linked together through inter-nucleosides.

[0046] "Nucleoside linkage" is a covalent connection between adjacent nucleosides in a polynucleotide. As used in this article, "modified nucleoside linkage" refers to any nucleoside linkage other than phosphodiester nucleoside linkage.

[0047] "Thiophosphate linkage" refers to a modified phosphate linkage in which one of the non-bridging oxygen atoms is replaced by a sulfur atom.

[0048] "Polynucleotide" refers to a polymer of linked nucleosides, each of which may be independently modified or unmodified. Unless otherwise stated, a polynucleotide consists of 8-80 linked nucleosides. "Modified polynucleotide" refers to a polynucleotide in which at least one sugar, nucleobase, or inter-nucleoside linkage is modified. "Unmodified polynucleotide" refers to a polynucleotide that does not contain any sugar, nucleobase, or inter-nucleoside modifications.

[0049] Example 1: General method for preparing MOE-Gapmer antisense oligonucleotides using solid-phase technology

[0050] Unless otherwise stated, all reagents and solutions used in the synthesis of oligomers were purchased from commercial sources. Standard phosphoramide structural units and solid supports were used to incorporate nucleoside residues, including, for example, T, A, G, and... m C residues. All monomers used (β-D-2'-deoxyribonucleoside and β-D-2'-(MOE)ribonucleoside) were phosphoramidite solutions of 0.06 M in anhydrous acetonitrile.

[0051] A 500 nmol synthesis column made of Universal CPG solid support was packed into an LK-48E synthesizer, and the specified sequence was synthesized using a phosphoramide coupling method. For the coupling step, the phosphoramide monomer was delivered in an amount more than four times the loading on the solid support and phosphoramide condensation was carried out for 10 min. All other steps were performed according to the manufacturer's standard protocol. Dimethoxytriphenylmethyl (DMT) was removed from the 5'-hydroxyl group of the nucleotide using a solution of 3% trichloroacetic acid in dichloromethane. BTT (0.35 M, containing 0.5% NMI) in anhydrous acetonitrile was used as an activator in the coupling step. Phosphothiophosphate bonding was introduced by a contact time of 3 min with a 0.2 M solution of diphenylacetyl disulfide in 1:1 pyridine / acetonitrile.

[0052] After the specified sequence was synthesized, the specified sequence bound to the solid support was suspended in ammonia (25wt%-30wt%) and heated at 85°C for 2 h. The solid support was then filtered off and the ammonia was removed under reduced pressure. The residue was purified by high-performance liquid chromatography to prepare the MOE-Gapmer antisense oligonucleotides shown in Table 1.

[0053] The MOE-Gapmer antisense oligonucleotides in Table 1 are 20 nucleotides in length and are designed as 5-10-5 spacers. The nick contains 10 2'-deoxynucleotides and is flanked by wings containing 5 nucleotides each (in the 5' and 3' directions). All nucleotides in the 5' and 3' wings are modified with 2'-MOE sugars, and all nucleotides in the nick are modified with 2'-deoxy sugars. All internucleotide bonds in the spacer are phosphate thioester (P=S) bonds, and all cytosine residues in the spacer are 5-methylcytosine.

[0054] Table 1. Effects of MOE-modified antisense oligonucleotides on reducing XDH gene expression in primary hepatocytes of suckling rats.

[0055] Example 2: Real-time quantitative PCR detection of the effect of MOE-modified antisense oligonucleotides on reducing XDH gene expression in primary hepatocytes of neonatal rats.

[0056] Liver fragments were collected from 5-day-old suckling mice. After washing 2-3 times with PBS, the fragments were minced as finely as possible with scissors. Approximately 5 ml of Hanks buffer was added, and the fragments were pipetted and passed through a cell strainer. The fragments were transferred to a new 10 cm dish, 3 ml of Hanks buffer was added, along with 100 μl each of collagenase I and IV. The mixture was incubated at 37°C, and the cells were pipetted every 15 minutes. After two pipetting cycles, PBS was added, and the cells were passed through a cell strainer again. The cells were centrifuged at 1000 rpm for 3 minutes, and the cells were collected at the bottom of the centrifuge tube. The cells were resuspended in DMEM + 10% FBS + 1% P / S medium and transferred to 15 cm dishes. The cells were cultured at 37°C. After approximately 7-10 days of confluence, the cells were digested and plated for ASO knockdown efficiency detection. 1.5 × 10⁶ cells per well. 5 Cells were transfected with different oligonucleotide sequences into the corresponding wells using Lipofectamine RNAiMax (ThermoFisher) transfection reagent after 12 hours. The final concentration of the oligonucleotides was 134 nM. The blank control group was transfected with DEPC water. The results are shown in Figure 1 and Table 1. The real-time quantitative PCR results showed that all 25 antisense oligonucleotides in this example could significantly inhibit XDH mRNA expression, with knockdown efficiencies ranging from 25% to 83%.

[0057] Example 3: Detection of the efficiency of MOE-modified antisense oligonucleotides in knocking down XDH mRNA in mice.

[0058] This experiment used 4-6 week old C57 / B6 mice, weighing approximately 18-20 grams. Mice were intraperitoneally injected with 100 mg / kg of ASO drug. Two to three days later, liver and kidney tissues were collected, and RNA was extracted to detect the XDH knockdown efficiency. The results are shown in Table 2. Compared with the negative control group, antisense oligonucleotides SG25-1, SG25-2, SG25-3, SG25-4, SG25-5, SG25-7, SG25-9, SG25-10, SG25-11, SG25-12, SG25-14, SG25-15, SG25-16, SG25-17, SG25-18, SG25-22, and SG25-25 significantly inhibited the expression of XDH mRNA in mouse liver.

[0059] Table 2. Detection of the knockdown efficiency of MOE-modified antisense oligonucleotides on XDH mRNA in mice.

[0060] Example 4: General method for preparing cEt-Gapmer antisense oligonucleotides using solid-phase technology

[0061] Unless otherwise stated, all reagents and solutions used in the synthesis of oligomers were commercially available. Standard phosphoramide structural units and solid supports were used to incorporate nucleoside residues, including, for example, T, A, G, and mC residues. The phosphoramide solution used for the β-D-2'-deoxyribonucleoside monomer (DNA) was 0.06 M in anhydrous acetonitrile, and the phosphoramide solution used for the 4'-CH(CH3)-O-2' (referred to as "cEt" when in the S configuration) ribonucleoside was 0.1 M in anhydrous acetonitrile.

[0062] A 500 nmol synthesis column made of Universal CPG solid support was packed into an LK-48E synthesizer, and the specified sequence was synthesized using a phosphoramide coupling method. For the coupling step, the DNA phosphoramide monomer was delivered in an amount more than four times the loading on the solid support and phosphoramide condensation was carried out for 10 min; the cEt phosphoramide monomer was delivered in an amount more than four times the loading on the solid support and phosphoramide condensation was carried out for 20 min. All other steps were performed according to the standard protocol supplied by the manufacturer. Dimethoxytriphenylmethyl (DMT) was removed from the 5'-hydroxyl group of the nucleotide using a solution of 3% trichloroacetic acid in dichloromethane. BTT (0.35 M, containing 0.5% NMI) in anhydrous acetonitrile was used as an activator in the coupling step. Phosphothiophosphate bonding was introduced by a contact time of 3 min with a 0.2 M solution of diphenylacetyl disulfide in 1:1 pyridine / acetonitrile.

[0063] After the specified sequence was synthesized, the specified sequence bound to the solid support was suspended in ammonia (25wt%-30wt%) and heated at 85°C for 2 h. The solid support was then filtered off and the ammonia was removed under reduced pressure. The residue was purified by high-performance liquid chromatography to prepare cEt-Gapmer antisense oligonucleotides as shown in Table 3.

[0064] The cEt-Gapmer antisense oligonucleotides in Table 3 are 16 nucleotides in length and are designed as a 3-10-3 spacer. The nick contains 10 2'-deoxynucleotides and is flanked by wings containing 3 nucleotides each (in the 5' and 3' directions). All nucleotides in the 5' and 3' wings are modified with cEt sugars, and all nucleotides in the nick are modified with 2'-deoxy sugars. All internucleotide bonds in the spacer are phosphate thioester (P=S) bonds, and all cytosine residues in the spacer are 5-methylcytosine.

[0065] Table 3. Effects of cEt-modified antisense oligonucleotides on reducing XDH gene expression in primary hepatocytes of neonatal rats.

[0066] Example 5: Real-time quantitative PCR detection of the effect of cEt-modified antisense oligonucleotides on reducing XDH gene expression in primary hepatocytes of neonatal rats.

[0067] Primary liver cells from suckling rats were sorted according to the method in Example 3. After the cells reached confluence, they were enzymatically digested and plated for ASO knockdown efficiency detection. 1.5 × 10⁶ cells were placed in each well. 5 Cells were transfected with different oligonucleotide sequences into the corresponding wells using Lipofectamine RNAiMax (ThermoFisher) transfection reagent after 12 hours. The final concentration of the oligonucleotides was 30 nM. The blank control group was transfected with DEPC water. The remaining methods were the same as in Example 3. The results are shown in Figure 2 and Table 3. The real-time quantitative PCR results showed that all 30 cEt-modified antisense oligonucleotides tested in this example could significantly inhibit XDH mRNA expression, with knockdown efficiencies ranging from 34% to 84%.

[0068] Example 6: XDH mRNA expression levels were detected after different concentrations of antisense oligonucleotides were freely uptaken into primary hepatocytes of neonatal rats.

[0069] The cells used in this experiment were primary neonatal rat hepatocytes, isolated using the same method as in Example 3. These cells were seeded into 24-well plates at a density of 1.5 × 10⁶ cells per well. 5 Cells were sampled, and after 12 hours, oligonucleotides of different concentrations were added and mixed to achieve final concentrations of 0M, 1M, and 5M. RNA was extracted and detected after 24 hours. The results are shown in Figure 3 and Table 4. Compared with the negative control group (0M, 100%), ASO at concentrations of 1M and 5M could inhibit XDH mRNA expression to varying degrees.

[0070] Table 4. Inhibitory effect of different concentrations of antisense oligonucleotides on XDH mRNA (free diffusion)

[0071] Example 7: Detection of the efficiency of cEt-modified antisense oligonucleotides in knocking down XDH mRNA in mice.

[0072] This experiment used 4-6 week old C57 / B6 mice, weighing approximately 18-20 grams. Mice were intraperitoneally injected with 30 mg / kg of ASO. Two to three days later, liver, kidney, and intestinal tissues were collected, and RNA was extracted to detect the XDH knockdown efficiency. The results, shown in Table 5, indicate that compared to the negative control group, the tested ASO significantly inhibited the expression of XDH mRNA in mouse liver.

[0073] Table 5. Detection of the knockdown efficiency of cEt-modified antisense oligonucleotides on XDH mRNA in mice.

Claims

1. An oligonucleotide, characterized in that, It contains at least 12 consecutive nucleotides that are at least 80%, 90%, 95%, 99%, or 100% identical to the sequence GCCCACCATGTCCTCCTCAGACTGACCCTT, TGCCCAACACAAGTAACCTAG, TTGCCACAAGGTGTCAGTATATGT, TAGAACTTGAAGAAGAAGCT, AGAACTTGAAGAAGAAGCTG, AACTTGAAGAAGAAGCTGAG, GGTTCACAAACTGTCTGGAG, GTTCACAAACTGTCTGGAGA, CTGAAGTAGTGGAAGGGGTT, TGAGTAGTGGAAGGGGTTC, CACAAACTGTCTGGAGATCT, TCACAAACTGTCTGGAGATC, CAGGCAAAGGATACACGATC, or TTTTGGCAATTCTCTCCTAA.

2. The oligonucleotide of claim 1, wherein, It consists of 12 to 27 linked nucleosides; more preferably, the oligonucleotide consists of 14 to 20 linked nucleosides, for example, specifically 14, 16, 18 or 20 linked nucleosides.

3. The oligonucleotide of claim 2, wherein, The oligonucleotide is a modified oligonucleotide; preferably, at least one nucleoside in the modified oligonucleotide contains a modified nucleic acid base; more preferably, the modified nucleic acid base is 5-methylcytosine.

4. The oligonucleotide of claim 2, wherein, The modified oligonucleotide is a gamper, which includes a gap composed of linked deoxynucleosides and 5' wings and 3' wings composed of linked nucleosides, wherein the gamper is located between the 5' wings and the 3' wings; Preferably, at least one nucleoside of the wing segment of the spacer contains a modified sugar; more preferably, the modified sugar is a 2'-modified sugar; even more preferably, the 2'-modified sugar is 2'-O-methoxyethyl modified. Preferably, the modified sugar is a bicyclic sugar; more preferably, the modification is a 4'-CH(CH3)-O-2' modification. Preferably, the modified oligonucleotide comprises: a nick consisting of linked deoxynucleotides and 5' wings and 3' wings consisting of linked nucleotides, wherein the spacer segment is located between the 5' wings and the 3' wings, and wherein each nucleotide of each wing comprises a modified sugar; preferably, the nick consists of 3-16 linked nucleotides; Preferably, the modified oligonucleotide comprises: a nick consisting of 8-12 linked deoxynucleotides and 5' and 3' wings consisting of 2-6 linked nucleotides, wherein the nick is located between the 5' and 3' wings and wherein each nucleotide of each wing comprises a modified sugar; Preferably, the modified oligonucleotide comprises: a nick consisting of 10 linked deoxynucleotides and 5' and 3' wings consisting of 5 linked nucleotides, wherein the nick is located between the 5' and 3' wings and wherein each nucleotide of each wing comprises a modified sugar; wherein each nucleotide of each wing comprises a 2'-O-methoxyethyl sugar or a restricted ethyl sugar; More preferably, the modified oligonucleotide comprises: a nick consisting of 10 linked deoxynucleotides and 5' and 3' wings consisting of 3 linked nucleotides, wherein the nick is located between the 5' and 3' wings and wherein each nucleotide of each wing comprises a modified sugar; wherein each nucleotide of each wing comprises a 2'-O-methoxyethyl sugar or a restricted ethyl sugar.

5. The oligonucleotide of claim 4, wherein, At least one internucleotide bond in the modified oligonucleotide is a modified internucleotide bond; preferably, all internucleotide bonds in the modified oligonucleotide are modified internucleotide bonds; preferably, the modified internucleotide bonds are phosphate thioester internucleotide bonds.

6. The oligonucleotide of claim 4, wherein, The single-stranded modified oligonucleotide comprises a nick consisting of 10 linked deoxynucleosides and 5' wings and 3' wings consisting of 5 linked nucleosides, respectively, wherein the nick is located between the 5' wings and the 3' wings, and each nucleoside in each wing is a 2'-O-methoxyethyl modified nucleoside, the internucleotide bonds in the entire modified oligonucleotide are phosphate thioester bonds, and all cytosines in the entire modified oligonucleotide are 5-methylcytosine; Alternatively, the single-stranded modified oligonucleotide comprises a nick consisting of 10 linked deoxynucleosides and 5' wings and 3' wings consisting of 3 linked nucleosides, respectively, wherein the nick is located between the 5' wings and the 3' wings, and each nucleoside in each wing is a restricted ethyl sugar modified nucleoside, the internucleotide bonds in the entire modified oligonucleotide are phosphate thioester bonds, and all cytosines in the entire modified oligonucleotide are 5-methylcytosine.

7. The oligonucleotide of claim 6, wherein, The nucleotide sequences of the 10 linked deoxyribonucleotide nicks are selected from: CCATGTCCTC, CATGTCCTCC, ATGTCCTCCT, CTTGAAGAAG, TTGAAGAAGA, GAAGAAGAA, TGAGAAGAA, GAAGAAGAAG, AAGAAGAAGC, ACAAACTGTC, CAAACTGTCT, GGTGTCAGT, GGTGTCAGTA, AAACTGTCTG, GTAGTGGAAG, ACTGTCTGGA, AACTGTCTGG, ACACAAGTAA, GGCAAAGGAT, GGCAAAGGAT, GCAAAGGATA, CAAAGGATAC, AAAGGATACA, GAAGAAGCTG, GGCAATTCTC, GCAATTCTCT; Accordingly, the 5' wing segment is selected from GCCCA, CCCAC, CCACC, TAGAA, AGAAC, GAACT, AACTT, ACTTG, GGTTC, GTTCA, CCACAA, CACAA, TTCAC, CTGAA, TGAAG, CACAA, TCACA, TCACA, GCCCA, CAGCA, AGCAG, GCAGG, CAGGC, TTGAA, CTTTT, TTTTTG; Accordingly, the 3' wing segment is selected from CTCAG, TCAGA, CAGAC, AAGCT, AGCTG, GCTGA, CTGAGG, TGGAG, GGAGA, AATTG, TATGT, GAGAT, GGGTT, GGTTC, GATCT, AGATC, CCTAG, AACCG, CACGA, ACGAT, CGATC, AGGGT, TCCTA, and CCTAA.

8. The oligonucleotide of claim 6, wherein, The nucleotide sequences of the 10 linked deoxyribonucleotide nicks are selected from: CACCATGTCC, ACCATGTCCT, CCATGTCCTC, CATGTCCTCC, ATGTCCTCCT, TGTCCTCCTC, GTCCTCCTCA, TCCTCCCTCAG, CCTCCTCAGA, CTCCTCAGAC, TCCTCAGACT, CCTCAGACTG, CTCAGACTGA, TCAGACTGAC, CAGACTGACC, CCAACACAAG, CAACACAAGT, AACCAAGTA, ACACAAGTAA, CACAAGTAAC, ACAAGTAACC, CCACAAGGTG, CACAAGGTGT, ACAAGGTGTC, CAAGGTGTCA, AAGGTGTCAG, AGGTGTCAGT, GGTGTCAGTA, GTGTCAGTAT, TGTCAGTATA; Accordingly, the 5' wing segment is selected from GCC, CCC, CCA, CAC, ACC, CCA, CAT, ATG, TGT, GTC, TCC, CCT, CTC, TCC, CCT, TGC, GCC, CCC, CCA, CAA, AAC, TTG, TGC, GCC, CCA, CAC, ACA, CAA, AAG, AGG; Accordingly, the 3' wing segment is selected from TCC, CCT, CTC, TCA, CAG, AGA, GAC, ACT, CTG, TGA, GAC, ACC, CCC, CCT, CTT, TAA, AAC, ACC, CCT, CTA, TAG, TCA, CAG, AGT, GTA, TAT, ATA, TAT, ATG, and TGT.

8. A liver-targeting oligonucleotide, characterized in that its structure comprises the oligonucleotide as described in any one of claims 1 to 8 and a ligand of one or more N-acetylgalactosamine GalNAc derivatives; Preferably, the ligand of the N-acetylgalactosamine GalNAc derivative is attached to the oligonucleotide via a linker.

9. The liver-targeted oligonucleotide of claim 8, wherein, The linker is a monovalent, divalent or trivalent branched linker; preferably, the liver-targeted oligonucleotide structure is: N-acetylgalactosamine is attached to the 5' end of the oligonucleotide.

10. A pharmaceutical composition comprising, in combination, a therapeutically effective amount of a compound of claim 1 and a pharmaceutically acceptable carrier. The oligonucleotide or a salt thereof as described in any one of claims 1 to 8, or the liver-targeting oligonucleotide as described in claim 8 or 9, and a pharmaceutically acceptable carrier; Preferably, the composition is an injectable pharmaceutical composition, such as a subcutaneous or intravenous injection pharmaceutical composition.

11. Use of the oligonucleotide or salt thereof as claimed in any one of claims 1 to 8, or the liver-targeting oligonucleotide as claimed in claim 8 or 9, in the preparation of a medicament for the prevention or treatment of XDH-related diseases or symptoms; preferably, the disease or symptom is hyperuricemia or gout.

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