WO2025139010A1 - Sirna for inhibiting xdh gene expression and modifier and use thereof - Google Patents

Abstract

Provided siRNA for inhibiting XDH gene expression and a modifier and use thereof. The siRNA comprises a sense strand and an antisense strand. The sense strand and/or the antisense strand have a length in the range of 19-25 nucleotides, and the antisense strand is reversely complementary to a segment on a target gene; and the sense strand has a nucleotide sequence as shown in SEQ ID NO: 1-14, and the antisense strand has a nucleotide sequence as shown in SEQ ID NO: 18-31. siRNA molecules and modified siRNA molecules have high stability and/or high inhibitory activity. Ligand-conjugated siRNA molecules have good liver targeting performance and the capability to promote cell endocytosis while maintaining high inhibitory activity and stability, can reduce the impact on other tissues or organs and reduce the amount of siRNA molecules used, and can achieve the purposes of reducing toxicity and reducing costs.

Description

siRNA for inhibiting XDH gene expression and its modified products and applications Technical Field

The invention belongs to the field of biomedicine, and specifically relates to siRNA for inhibiting XDH gene expression and its modified products and applications.

Background Art

Hyperuricemia refers to the level of uric acid in the blood exceeding the normal range, generally less than 420μmol/L for men and less than 360μmol/L for women. Increased uric acid levels are the biochemical basis for hyperuricemia and gout. Gout is a chronic metabolic disease caused by the deposition of urate crystals in joints and other tissues, manifested as recurrent acute arthritis, chronic joint deformities, tophi, kidney damage and urinary tract stones.

The occurrence of hyperuricemia and gout is related to genetic and environmental factors. Genetic factors mainly include gene mutations that affect the production and excretion of uric acid, such as xanthine oxidase gene and renal uric acid transporter gene. Environmental factors mainly include high-purine diet, alcoholism, obesity, hypertension, kidney disease, drugs, etc. The mechanism of hyperuricemia and gout involves the formation and deposition of urate crystals, activation of inflammatory response, increase of oxidative stress, damage to endothelial function, etc.

In recent years, with economic development and changes in lifestyle, the prevalence of hyperuricemia and gout has increased and is becoming younger. According to statistics, there are about 930 million patients with hyperuricemia and gout worldwide, and it is expected to reach 1.18 billion in 2025. The overall prevalence of hyperuricemia in my country is 13.3%, about 186 million people; the overall prevalence of gout is 1.1%, about 15 million people.

Hyperuricemia and gout not only affect the quality of life of patients, but are also closely related to the incidence of a variety of chronic non-communicable diseases, such as cardiovascular disease, metabolic syndrome, chronic kidney disease, etc. Therefore, timely diagnosis and treatment of hyperuricemia and gout have important clinical significance and public health value. Current treatment methods mainly include drug therapy and non-drug therapy. Drug therapy is mainly divided into two stages: acute phase and remission phase. The acute phase is mainly for anti-inflammatory and analgesic, and the remission phase is mainly for lowering uric acid levels. Non-drug treatment mainly includes improving lifestyle, controlling diet, losing weight, increasing exercise, etc. The comprehensive use of drug therapy and non-drug treatment can effectively control the development of hyperuricemia and gout and prevent the occurrence of complications. The main drugs for the clinical treatment of hyperuricemia and gout are currently the following:

At present, the main drugs for clinical treatment of hyperuricemia and gout include xanthine oxidase (XDH) inhibitors: by inhibiting xanthine oxidase, uric acid production is reduced, and blood uric acid levels are lowered. They are suitable for patients with increased uric acid production. Commonly used drugs include allopurinol and febuxostat. Allopurinol is a first-line uric acid-lowering drug with a starting dose of 50 to 100 mg/d. The maximum dose is 800 mg/d, and the dosage needs to be adjusted according to renal function. Although it is effective and inexpensive, special attention should be paid to its hypersensitivity reaction in the Chinese population (the incidence of hypersensitivity reaction in Taiwan is 2.7%). Once it occurs, the mortality rate is as high as 30%. It has been confirmed that the occurrence of allopurinol hypersensitivity reaction is significantly correlated with HLA-B*5801, and the frequency of carrying this genotype in the Han population is 10% to 20%. Febuxostat is a specific xanthine oxidase inhibitor with a starting dose of 20 mg/d and a maximum dose of 80 mg/d. It is suitable for patients with renal insufficiency, but attention should still be paid to its potential cardiovascular risks.

Summary of the invention

The present invention designs corresponding siRNA for XDH, and effectively delivers siRNA to the liver by coupling GalNAc, thereby interfering with XDH mRNA in the liver, effectively reducing the expression of XDH protein and the synthesis of uric acid, thereby playing a role in treating hyperuricemia.

The XDH gene targeted by the present invention is the gene shown by Genbank registration number NM-000379.4.

The first aspect of the present invention discloses a siRNA for inhibiting the expression of xanthine oxidase gene, comprising a sense strand and an antisense strand; the sense strand and/or the antisense strand has a length ranging from 19 to 25 nucleotides, and the antisense strand is reversely complementary to a segment on the target gene;

The sense strand and/or the antisense strand may have any length of 19, 20, 21, 22, 23, 24 or 25 nucleotides; the sense strand and the antisense strand can complement each other to form a double-stranded RNA.

The sense strand has a nucleotide sequence as shown in SEQ ID NO: 1-14, and the antisense strand has a nucleotide sequence as shown in SEQ ID NO: 18-31. The sequence of the siRNA for inhibiting the expression of the xanthine oxidase gene is specifically as follows:

Furthermore, the 3' ends of the sense strand and the antisense strand of the nucleotide sequence are connected with at most 2 additional nucleotides to constitute overhangs, and the overhangs are selected from A, C, G, U, T or modified nucleotides of A, C, G, U, T.

Furthermore, the siRNA is also modified, and the modified siRNA is selected from a modification of the sugar portion at the 2' position, or at least one phosphate group is a phosphate group containing a modified group, or one or more nucleotide analogs; the polynucleic acid molecule is chemically synthesized using naturally occurring nucleotides or various modified nucleotides, and the modified nucleotides are designed to increase the biological stability of the molecule or increase the physical stability of the duplex formed between the polynucleic acid molecule and the target nucleic acid.

The 2' modified nucleotides include 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-ODMAP), TO-dimethylaminoethoxyethyl (2'-O-DMAEOE) or 2'-ON-methyl. Acetamido (2'-O-NMA) modified nucleotides.

The phosphate group containing a modified group is specifically a phosphorothioate group formed by replacing at least one oxygen atom in the phosphodiester bond with a sulfur atom;

The nucleotide analogue is selected from an isonucleotide, LNA, ENA, cEt BNA, UNA or GNA.

The structure of the positive strand of the modified siRNA molecule is shown in SEQ ID NO: 35-46; the structure of the antisense strand of the modified siRNA molecule is shown in SEQ ID NO: 47-58, which are as follows:

Among them, dA represents deoxyribonucleotide A; mA, mU, mC and mG represent ribonucleotides A, U, C and G modified by 2'-O-methyl, respectively; fA, fU, fC and fG represent ribonucleotides A, U, C and G modified by 2'-fluoro, respectively; -s- represents that the two nucleotides before and after are connected by a thiophosphate backbone; GNA-U represents the modification of ribonucleotide U by GNA.

As a specific embodiment of the present invention, a conjugate is obtained by coupling a modified siRNA for inhibiting the expression of the XDH target gene with a ligand, which can help the siRNA be delivered to the target organ or tissue and enter the cell; the ligand is conjugated to the 3' end of the sense chain, and the ligand includes but is not limited to GalNAc, cholesterol, biotin, vitamins, galactose derivatives or analogs, lactose derivatives or analogs, N-acetylglucosamine derivatives or analogs; the ligand is preferably GalNAc.

In some embodiments, the positive chain structure of the siRNA molecule conjugated with the ligand is shown in SEQ ID NO: 59-61; the antisense chain structure of the modified siRNA molecule is shown in SEQ ID NO: 62-64:

L-96 is GalNac-L96, a G-rich oligonucleotide with a longer GalNAc linker.

The second aspect of the present invention discloses a biological material related to the above siRNA, which is any of the following:

1) A vector containing the above-mentioned siRNA molecule;

2) A reagent or a kit containing the above-mentioned siRNA or the vector described in 1);

3) A pharmaceutical composition, consisting of the above-mentioned siRNA molecules and other pharmaceutically acceptable components.

The pharmaceutically acceptable other components include, but are not limited to, water, saline, pH buffer, protective agent, osmotic pressure regulator, excipient, diluent, disintegrant, binder, lubricant, sweetener, preservative or a combination thereof. The protective agent may be at least one of inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose and glucose.

The above-mentioned carriers include, but are not limited to, magnetic nanoparticles (such as Fe ₂ O ₃ ), carbon nanotubes, mesoporous silica, calcium phosphate nanoparticles, polyethyleneimine, polyamidoamine dendrimers, polylysine, chitosan, poly D- or L-lactic acid/glycolic acid copolymers, poly(aminoethyl ethylene phosphate) and poly methacrylate-N,N-dimethylaminoethyl ester and one or more of their derivatives.

Furthermore, the dosage form of the pharmaceutical composition may be a liquid preparation (e.g., injection) or a lyophilized powder injection. The lyophilized powder injection is mixed with a liquid excipient during administration to prepare a liquid preparation. The liquid preparation may be, but is not limited to, administered subcutaneously, intramuscularly, or intravenously, and may also be, but is not limited to, administered to the lungs by spraying, or administered to other organs (e.g., liver) via the lungs by spraying.

The third aspect of the present invention discloses the use of the above-mentioned biomaterial in the treatment of hyperuricemia.

In some embodiments, the present invention provides an in vivo method comprising alleviating or treating a disease or symptom mediated by an XDH gene in a subject, wherein the disease or symptom comprises hyperuricemia or gout. The method may comprise administering to the subject an effective amount, such as a preventive effective amount or a therapeutic effective amount, of the above-mentioned siRNA molecule or pharmaceutical composition.

The present invention achieves the following beneficial technical effects:

1. siRNA molecules and modified siRNA molecules have high stability and/or high inhibitory activity.

2. While maintaining high inhibitory activity and stability, the ligand-conjugated siRNA molecules also have good liver targeting and the ability to promote cell endocytosis, which can reduce the impact on other tissues or organs and reduce the amount of siRNA molecules used, thereby achieving the purpose of reducing toxicity and reducing costs.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows that the naked siRNA sequence in Example 1 can reduce the expression of XDH mRNA in Hep-G2 cells.

Figure 2 shows that the siRNA modified sequence in Example 2 can reduce the expression of XDH mRNA in Hep-G2 cells.

Figure 3 shows that the GalNAc-coupled siRNA modified sequence in Example 3 can reduce the expression of XDH mRNA in Hep-G2 cells.

4A-D show the IC50 of the siRNA modified sequence coupled with GalNAc in Example 4 in reducing the expression of XDH mRNA in Hep-G2 cells.

Figure 5 shows that the GalNAc-coupled siRNA modified sequence in Example 5 can reduce the expression of XDH mRNA in the liver of SD rats.

FIG. 6 is a graph showing the efficacy of the GalNAc coupling sequence in Example 6 in a mouse hyperuricemia model.

FIG. 7 is a graph showing the results of the drug efficacy persistence of the GalNAc coupling sequence in Example 7 in a mouse hyperuricemia model.

FIG8 is a graph showing the results of the drug efficacy persistence of the GalNAc coupling sequence in Example 8 in the cynomolgus monkey hyperuricemia model.

DETAILED DESCRIPTION

It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

Example 1: Screening of naked sequence knockdown effects

1) Human liver cancer cells Hep-G2 (purchased from the cell bank of Kunming Institute of Chinese Academy of Sciences) in the logarithmic growth phase were digested with trypsin, and then digestion was terminated with complete medium supplemented with 10% FBS. The cells were collected by centrifugation, resuspended with medium supplemented with 10% FBS, counted with a hemocytometer, and then 50,000 cells were added to each well of a 24-well plate for culture.

2) Preparation of a mixture of LipoRNAiMAX (Lipofectamine RNAiMAX) (purchased from Invitrogen) and siRNA, specifically using the following sequences: wherein overhangs are generated in some sequences, and the overhangs are in brackets in each sequence in the table below.

10 nM/well siRNA and 1.5 μL LipoRNAiMAX (Invitrogen) were diluted in 25 μL serum-free culture medium (Opti-MEM, purchased from Gibco), and then the siRNA solution and LipoRNAiMAX (Invitrogen) solution were mixed and allowed to stand at room temperature for 5 minutes.

3) Add 50 μL of the corresponding group of siRNA and LipoRNAiMAX (invitrogen) mixed solution into each well.

4) After 48 hours of culture, discard the culture medium, wash twice with enzyme-free PBS, add lysis solution (BioFlux) to lyse the cells, add chloroform (MREDA) for extraction, shake and mix, let stand at room temperature for 2-3 minutes, centrifuge, transfer the layered supernatant to the well plate and combine with the binding solution, use a nucleic acid extractor, according to the kit instructions (MagaBio plus total RNA purification kit II, manufacturer Bori, batch number BSC69L1E) to arrange the well plate in order and use the BSC69 program to extract RNA.

5) Prepare the qPCR system on ice, add 1 μL One Step SYBR Green Mix (Novozyme), 10 μL 2*One Step SYBR Green Mix (Novozyme), 0.4 μL hYJH-012 3P F, 0.4 μL hYJH-012 3P R to each well, dilute 100 ng RNA in 8.2 μL RNase ddH ₂ O (Novozyme) and add to the well, mix well and place in qPCR instrument for reaction.

PCR reaction conditions: 50°C, 15 min pre-denaturation, 95°C, 1 min, 95°C annealing for 15 s, 60°C extension for 1 min, for 39 cycles.

PCR primers: hYJH-012 3P F (SEQ ID NO: 65): NF05_300300, hYJH-012 3P R (SEQ ID NO: 66): NF05_300301,

Novozyme test kit: batch number 7E750A3

The results showed that after LipoRNAiMAX transfection, the YJH-012-1609 sequence had an inhibition rate of 43%, the YJH-012-3043 sequence had an inhibition rate of 21%, the YJH-012-3047 sequence had an inhibition rate of 53%, and the YJH-012-ARH sequence (positive control sequence) had an inhibition rate of 50%, and there were significant differences, as shown in Figure 1.

Example 2: Screening of knockdown effects of modified sequences

1) Human hepatoma cell Hep-G2 (purchased from the cell bank of Kunming Institute of Chinese Academy of Sciences) in the logarithmic growth phase was digested with trypsin, and then digestion was terminated with complete medium supplemented with 10% FBS. The cells were collected by centrifugation and cultured with medium supplemented with 10% FBS. Resuspend the cells, count the cells using a hemocytometer, and then add 50,000 cells to each well of a 24-well plate for culture.

2) The preparation of the mixture of LipoRNAiMAX (invitrogen) and siRNA was carried out by specifically selecting the following sequences: wherein overhangs were generated in some sequences, and the overhangs are indicated in brackets in each sequence in the table below.

10 nM/well siRNA and 1.5 μL LipoRNAiMAX (purchased from Invitrogen) were diluted in 25 μL serum-free culture medium (Opti-MEM, purchased from Gibco), and then the above siRNA solution and LipoRNAiMAX (Invitrogen) solution were mixed and allowed to stand at room temperature for 5 minutes.

3) Add 50 μL of the corresponding group of siRNA and LipoRNAiMAX (invitrogen) mixed solution into each well.

4) After 48 hours of culture, discard the culture medium, wash twice with enzyme-free PBS, add lysis solution (BioFlux) to lyse the cells, add chloroform (MREDA) for extraction, shake and mix, let stand at room temperature for 2-3 minutes, centrifuge, transfer the layered supernatant to the well plate and combine with the binding solution, use a nucleic acid extractor, according to the kit instructions (MagaBio plus total RNA purification kit II, manufacturer Bori, batch number BSC69M1E) to arrange the well plate in order and use the BSC69 program to extract RNA.

5) Prepare qPCR system on ice, add 1μL One Step SYBR Green Mix (Novozymes), 10μL 2*One Step SYBR Green Mix (Novozymes), 0.4μL hYJH-012 4P F, 0.4μL hYJH-012 4P R to each well, dilute 100ng RNA in 8.2μL RNase ddH2O (Novozymes) and add to the wells, mix well and put into qPCR instrument for reaction.

PCR reaction conditions: 50°C, 15 min pre-denaturation, 95°C, 1 min, 95°C annealing for 15 s, 60°C extension for 1 min, for 39 cycles.

PCR primers: hYJH-012 4P F (SEQ ID NO: 67): NF07_182005, hYJH-012 4P R (SEQ ID NO: 68): NF07_182006,

Novozyme test kit: batch number 7E750A3

The results showed that after LipoRNAiMAX transfection, YJH-012-1609 and modified sequences had inhibitory effects, and the YJH-012-1609mE1 sequence had the best inhibition rate, which could reach 70%; YJH-012-3047 and modified sequences had inhibitory effects, and the YJH-012-3047mE+2 sequence had the best inhibition rate, which could reach 61%, and there was a significant difference; the inhibition rates of the positive control modified sequences YJH-012-ALN m1 and YJH-012-ARH m1 were 58% and 36%, respectively. As shown in Figure 2.

Example 3: Knockdown effect of GalNAc coupling sequence in Hep-G2 cells

1. The GalNAc conjugates are as follows:

2. Knockdown experiment:

1) Human liver cancer cells Hep-G2 (purchased from the cell bank of Kunming Institute of Chinese Academy of Sciences) in the logarithmic growth phase were digested with trypsin, and then digestion was terminated with complete medium supplemented with 10% FBS. The cells were collected by centrifugation, resuspended with medium supplemented with 10% FBS, counted with a hemocytometer, and then 50,000 cells were added to each well of a 24-well plate for culture.

2) Preparation of LipoRNAiMAX (invitrogen) and siRNA mixture, specifically using the following sequence: Overhangs were generated in some sequences, and the overhangs are shown in brackets in the following table.

10 nM/well siRNA and 1.5 μL LipoRNAiMAX (Invitrogen) were diluted in 25 μL serum-free culture medium (Opti-MEM, purchased from Gibco), and then the siRNA solution and LipoRNAiMAX (Invitrogen) solution were mixed and allowed to stand at room temperature for 5 minutes.

3) Add 50 μL of the corresponding group of siRNA mixture to each well.

4) After 48 hours of culture, discard the culture medium, wash twice with enzyme-free PBS, add lysis solution (BioFlux) to lyse the cells, add chloroform (MREDA) for extraction, shake and mix, let stand at room temperature for 2-3 minutes, centrifuge, transfer the layered supernatant to the well plate and combine with the binding solution, use a nucleic acid extractor, according to the kit instructions (MagaBio plus total RNA purification kit II, manufacturer Bori, batch number BSC69M1E) to arrange the well plate in order and use the BSC69 program to extract RNA.

5) Prepare the qPCR system on ice. Add 1 μL One Step SYBR Green Mix (Novozyme), 10 μL 2*One Step SYBR Green Mix (Novozyme), 0.4 μL hYJH-012 4P F, and 0.4 μL hYJH-012 4P R to each well. Dilute 100 ng RNA in 8.2 μL RNase ddH ₂ O (Novozyme) and add to the well. Mix well and place in the qPCR instrument for PCR. Line reaction.

PCR reaction conditions: 50°C, 15 min pre-denaturation, 95°C, 1 min, 95°C annealing for 15 s, 60°C extension for 1 min, for 39 cycles.

PCR primers: hYJH-012 4P F (SEQ ID NO: 67): NF07_182005, hYJH-012 4P R (SEQ ID NO: 68): NF07_182006,

Novozyme test kit: batch number 7E750A3

The results are shown in Figure 3. The inhibition rates of sequences YJH-012-1609mS2-L96, YJH-012-1609mE1-L96 and YJH-012-3047mE+2-L96 on XDH mRNA were 20.33%, 60.33% and 47.67%, respectively.

Example 4: IC50 of GalNAc coupling sequence in Hep-G2 cells

1) Human liver cancer cells Hep-G2 (purchased from the cell bank of Kunming Institute of Chinese Academy of Sciences) in the logarithmic growth phase were digested with trypsin, and then digestion was terminated with complete medium supplemented with 10% FBS. The cells were collected by centrifugation, resuspended with medium supplemented with 10% FBS, counted with a hemocytometer, and then 50,000 cells were added to each well of a 24-well plate for culture.

2) Preparation of a mixture of LipoRNAiMAX (invitrogen) and siRNA using the same sequence as in Example 3;

50nM/well NC siRNA and 1.5μL LipoRNAiMAX (invitrogen) were diluted in 25μL serum-free culture medium (Opti-MEM, purchased from gibco), and then the above siRNA solution was mixed with LipoRNAiMAX (invitrogen) solution to prepare a stock solution, which was allowed to stand at room temperature for 5 minutes. At the same time, three siRNAs, YJH-012-1609mS2-L96, YJH-012-1609mE1-L96, and YJH-012-3047mE+2-L96, were selected, and 50nM/well siRNA was directly diluted in 50μL serum-free culture medium (Opti-MEM, purchased from gibco), and a stock solution was prepared, which was allowed to stand at room temperature for 5 minutes. Then the stock solutions of the four siRNAs were diluted in turn to siRNA dilution solutions with siRNA concentrations of 10nM, 2nM, and 0.4nM.

3) Add 50 μL of the corresponding group of siRNA mixed solution to each well.

4) After 48 hours of culture, the culture medium was discarded, the cells were washed twice with enzyme-free PBS, and then lysed with lysis solution (BioFlux), and chloroform (MREDA) was added for extraction. After oscillation and mixing, the cells were allowed to stand at room temperature for 2-3 minutes, centrifuged, and the separated cells were separated. The supernatant was transferred to the well plate and combined with the binding solution. A nucleic acid extractor was used to extract RNA using the BSC69 program after arranging the well plate in order according to the kit instructions (MagaBio plus total RNA purification kit II, manufacturer Bioer, batch number BSC69M1E).

5) Prepare qPCR system on ice, add 1μL One Step SYBR Green Mix (Novozymes), 10μL 2*One Step SYBR Green Mix (Novozymes), 0.4μL hYJH-012 4P F, 0.4μL hYJH-012 4P R to each well, dilute 100ng RNA in 8.2μL RNase ddH2O (Novozymes) and add to the wells, mix well and put into qPCR instrument for reaction.

PCR reaction conditions: 50°C, 15 min pre-denaturation, 95°C, 1 min, 95°C annealing for 15 s, 60°C extension for 1 min, for 39 cycles.

PCR primers: hYJH-012 4P F (SEQ ID NO: 67): NF07_182005, hYJH-012 4P R (SEQ ID NO: 68): NF07_182006,

Novozyme test kit: batch number 7E750A3

The results showed that the IC50 values plotted according to the inhibition rate of the GalNAc coupling sequence in Hep-G2 cells were shown in Figures 4A-D. The IC50 values of YJH-012-1609mS2-L96, YJH-012-1609mE1-L96 and YJH-012-3047mE+2-L96 were 4.517 nM, 4.017 nM and 11.89 nM, respectively.

Example 5: Knockdown effect of GalNAc coupling sequence in rat liver

The siRNAs of interest identified from the in vitro studies were evaluated in vivo. The pharmacodynamic activity of the following GalNAc-conjugated siRNAs targeting XDH was analyzed in rats after subcutaneous injection of siRNA: wherein overhangs were generated in some sequences, the overhangs are in brackets in each sequence in the table below.

Two dose concentrations (1 mg/kg and 4 mg/kg) were set for each GalNAc-coupled siRNA, and the drug was administered subcutaneously once a week for multiple times. Rat liver samples were collected on the 21st day after the start of administration, and the XDH mRNA levels in all samples were analyzed by RT-qPCR. Rat liver tissue was lysed using a lysis buffer (BioFlux), and chloroform (MREDA) was added for extraction. After oscillation and mixing, it was allowed to stand at room temperature for 2-3 minutes, centrifuged, and the supernatant after stratification was transferred to a well plate and combined with the binding solution. A nucleic acid extractor was used to arrange the well plate in order according to the kit instructions (MagaBio Plus Total RNA Purification Kit II, manufacturer Biori, batch number C692307003) and the total RNA was extracted using the BSC69 program.

2) Prepare qPCR system on ice, add 1μL One SteP SYBR Green Mix (Novozymes), 10μL 2*One SteP SYBR Green Mix (Novozymes), 0.4μL rYJH-012-2P F (rXDH-2P F), 0.4μL rYJH-012-2P R (rXDH-2P R) to each well, dilute total RNA of liver tissue in 8.2μL RNase deionized water (Novozymes) and add to the wells, mix well and put into qPCR instrument for reaction.

qPCR reaction conditions: 50°C, 15 min pre-denaturation; 95°C, 1 min, 95°C annealing for 15 s, 60°C extension for 1 min, for 39 cycles.

PCR primers: rYJH-012-2P F (SEQ ID NO:69):NF09-130069, rYJH-012-2P R (SEQ ID NO:70):NF09-130070.

Novozyme test kit: batch number 7E711D3

As shown in Figure 5, injection of 4 mg/kg of the above sequences (YJH-012-1609mS2-L96, YJH-012-1609mE1-L96, and YJH-012-3047mE+2-L96) had 42.08%, 49.00%, and 54.90% inhibitory effects on rat liver XDH mRNA, respectively. The results showed that injection of GalNAc-coupled siRNA could downregulate XDH mRNA expression.

Example 6: Efficacy of GalNAc coupling sequence in mouse hyperuricemia model

The pharmacodynamic activity of the following GalNAc-conjugated siRNA targeting XDH was analyzed in mice after subcutaneous injection of siRNA: Overhangs were generated in some sequences, and the overhangs are in brackets in each sequence in the table below.

1) Each GalNAc-coupled siRNA was set at one dose concentration (8 mg/kg) and administered subcutaneously once on the first day of the experiment. Modeling was performed 30 minutes after administration, and modeling mice were intraperitoneally injected with 600 mg/kg potassium oxonate every day.

2) On the 14th day after the start of the experiment, blood was collected 2 hours after modeling and serum was separated to measure the uric acid level.

As shown in Figure 6, 8 mg/kg of the above sequences (YJH-012-1609mS2-L96, YJH-012-1609mE1-L96 and YJH-012-3047mE+2-L96) were injected, and the uric acid-lowering effects on hyperuricemia model mice were 20.15%, 40.82% and 40.36% on the 14th day after administration. The results show that the injection of GalNAc-coupled siRNA can Downregulates the blood uric acid level in hyperuricemia model mice.

Example 7: Durability of efficacy of GalNAc conjugated sequence in mouse hyperuricemia model

The pharmacodynamic durability of siRNA was evaluated in a mouse hyperuricemia model. The pharmacodynamic activity of the following GalNAc-conjugated siRNA targeting XDH was analyzed in mice after subcutaneous injection of siRNA: Overhangs were generated in some sequences, and the overhangs are in brackets in each sequence in the table below.

1) GalNAc-coupled siRNA was set at three dose concentrations (1 mg/kg, 4 mg/kg and 8 mg/kg) and a single subcutaneous administration was performed on the first day of the experiment. Modeling was performed 30 minutes after administration, and modeling mice were intraperitoneally injected with 600 mg/kg potassium oxonate every day.

2) On days -1, 4, 7, 10, 14, 21, and 28 after the start of the experiment, blood was collected 2 hours after modeling and serum was separated to measure uric acid levels.

As shown in Figure 7, a single administration of 4 mg/kg or 8 mg/kg of YJH-012-1609mE1-L96 can reduce the blood uric acid level of hyperuricemia model mice for 28 days. However, the 1 mg/kg dose group showed significant uric acid-lowering effect only on the 4th day after administration.

Example 8: Durability of efficacy of GalNAc conjugated sequences in the cynomolgus monkey hyperuricemia model

The pharmacodynamic durability of siRNA was evaluated in the cynomolgus monkey hyperuricemia model. The pharmacodynamic activity of the following GalNAc-conjugated siRNA targeting XDH was analyzed in cynomolgus monkeys after subcutaneous injection of siRNA: wherein overhangs were generated in some sequences, and the overhangs are in brackets in each sequence in the table below.

1) GalNAc-coupled siRNA was set at one dose concentration (10 mg/kg) and administered subcutaneously once on the first day of the experiment. Modeling was performed 30 minutes after administration, and the modeling cynomolgus monkeys were intraperitoneally injected with 600 mg/kg potassium oxonate every day.

2) On days -1, 1, 4, 7, 14, 21, 30, 45, 60, and 90 after the start of the experiment, blood was collected 2 hours after modeling and serum was separated to measure uric acid levels.

As shown in FIG8 , a single administration of 10 mg/kg of YJH-012-1609mE1-L96 can have a long-term effect of reducing the blood uric acid level in the hyperuricemia model cynomolgus monkey for 90 days.

The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims (13)

A siRNA for inhibiting the expression of xanthine oxidase gene, characterized in that it comprises a sense strand and an antisense strand; the antisense strand is reversely complementary to a segment on the target gene; The sense chain has a nucleotide sequence as shown in SEQ ID NO: 1-14, and the antisense chain has a nucleotide sequence as shown in SEQ ID NO: 18-31. The siRNA according to claim 1, characterized in that the 3' ends of the sense strand and the antisense strand of the nucleotide sequence are connected to at most 2 additional nucleotides to form overhangs. The siRNA according to claim 2, characterized in that the nucleotides at the protruding ends are selected from A, C, G, U, T or modified nucleotides of A, C, G, U, T. The siRNA according to claim 1 or 3, characterized in that at least one nucleotide in the siRNA is modified; the modified nucleotide is selected from a phosphate group containing a modified group, or one or more nucleotide analogs. The siRNA according to claim 4, characterized in that the 2' modified nucleotides include 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-ODMAP), T-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) or 2'-O-N-methylacetamido (2'-O-NMA) modified nucleotides. The siRNA according to claim 4, wherein the phosphate group containing the modified group is specifically a phosphorothioate group formed by replacing at least one oxygen atom in the phosphodiester bond with a sulfur atom. The siRNA as described in claim 4 is characterized in that the nucleotide analogue is selected from one of isonucleotides, LNA, ENA, cEt BNA, UNA or GNA. The siRNA as described in any one of claims 4 to 7 is characterized in that the positive chain structure of the modified siRNA molecule is shown in SEQ ID NO: 35-46; the antisense chain structure of the modified siRNA molecule is shown in SEQ ID NO: 47-58. The siRNA according to claim 8, characterized in that the 3' end of the sense strand is conjugated to a ligand; the ligand comprises GalNAc, cholesterol, biotin, vitamins, galactose derivatives or analogs, lactose derivatives or analogs, N-acetylglucosamine derivatives or analogs. The siRNA as described in claim 9 is characterized in that the positive chain structure of the siRNA molecule conjugated with the ligand is shown in SEQ ID NO: 59-61; the antisense chain structure of the modified siRNA molecule is shown in SEQ ID NO: 62-64. The biological material related to the siRNA according to any one of claims 1 to 10 is any one of the following: 1) A vector containing the siRNA according to any one of claims 1 to 10; 2) A reagent or a kit comprising the siRNA according to any one of claims 1 to 10 or the vector according to 1); 3) A pharmaceutical composition, consisting of the siRNA molecule according to any one of claims 1 to 10 and other pharmaceutically acceptable components. The biomaterial according to claim 11, characterized in that the dosage form of the pharmaceutical composition can be a liquid preparation or a lyophilized powder injection. Use of the siRNA according to any one of claims 1 to 10 or the biomaterial according to claim 11 in the treatment of hyperuricemia.