Sirna for inhibiting XDH gene expression and modifier and use thereof

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

[0001] The present 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

[0002] Hyperuricemia refers to blood uric acid levels exceeding the normal range, generally less than 420 μmol / L in men and less than 360 μmol / L in women. Elevated uric acid levels are the biochemical basis for hyperuricemia and gout, a chronic metabolic disease caused by the deposition of urate crystals in joints and other tissues. Symptoms include recurrent acute arthritis, chronic joint deformities, tophi, kidney damage, and urinary stones.

[0003] The development of hyperuricemia and gout is linked to both genetic and environmental factors. Genetic factors primarily include mutations in genes that affect uric acid production and excretion, such as those in the xanthine oxidase gene and the renal urate transporter gene. Environmental factors primarily include a high-purine diet, alcoholism, obesity, hypertension, kidney disease, and medications. The pathogenesis of hyperuricemia and gout involves the formation and deposition of urate crystals, activation of inflammatory responses, increased oxidative stress, and impaired endothelial function.

[0004] In recent years, with economic development and changes in lifestyle, the prevalence of hyperuricemia and gout has increased, with a younger population. According to statistics, approximately 930 million people worldwide suffer from hyperuricemia and gout, a number projected to reach 1.18 billion by 2025. In my country, the overall prevalence of hyperuricemia is 13.3%, or approximately 186 million people; the overall prevalence of gout is 1.1%, or approximately 15 million people.

[0005] 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 anti-inflammatory and analgesic, and the remission phase is mainly to reduce 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 the following:

[0006] Currently, the mainstay of clinical treatment for hyperuricemia and gout is xanthine oxidase (XDH) inhibitors. These inhibitors inhibit XDH, reducing uric acid production and lowering serum uric acid levels. They are suitable for patients with hyperuricemia. Commonly used drugs include allopurinol and febuxostat. Allopurinol is a first-line uric acid-lowering drug, with a starting dose of 50–100 mg / day and a maximum dose of 800 mg / day, requiring dosage adjustment based on renal function. Although highly effective and inexpensive, its use in the Chinese population warrants particular attention due to its potential for hypersensitivity reactions (the incidence in Taiwan is 2.7%), which can be fatal in up to 30%. A significant correlation has been established between allopurinol hypersensitivity reactions and HLA-B*5801, with the prevalence of this genotype in the Han Chinese population being 10–20%. Febuxostat is a specific XDH inhibitor with a starting dose of 20 mg / day and a maximum dose of 80 mg / day. It is suitable for patients with renal insufficiency, but its potential cardiovascular risks should be considered.

[0007] Summary of the Invention

[0008] The present invention designs corresponding siRNA for XDH, and effectively delivers siRNA to the liver by coupling with 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.

[0009] The XDH gene targeted by the present invention is the gene shown by Genbank registration number NM-000379.4.

[0010] The first aspect of the present invention discloses an siRNA for inhibiting the expression of the 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 reverse complementary to a segment on the target gene;

[0011] 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.

[0012] The sense strand has a nucleotide sequence as shown in SEQ ID NOs: 1 to 14, and the antisense strand has a nucleotide sequence as shown in SEQ ID NOs: 18 to 31. The sequence of the siRNA that inhibits the expression of the xanthine oxidase gene is specifically as follows:

[0013] Furthermore, the 3' ends of the sense strand and the antisense strand of the nucleotide sequence are connected with at most two additional nucleotides to form overhangs, and the overhangs are selected from A, C, G, U, T or modified nucleotides of A, C, G, U, T.

[0014] Furthermore, the siRNA is also modified, and the modified siRNA is selected from one or more of a modified sugar moiety at the 2' position, or at least one phosphate group is a phosphate group containing a modified group, or a nucleotide analog; 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.

[0015] 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.

[0016] 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;

[0017] The nucleotide analogue is selected from an isonucleotide, LNA, ENA, cEt BNA, UNA or GNA.

[0018] The structures of the sense strands of the modified siRNA molecules are shown in SEQ ID NOs: 35-46; the structures of the antisense strands of the modified siRNA molecules are shown in SEQ ID NOs: 47-58, as follows:

[0019] Among them, dA represents deoxyribonucleotide A; mA, mU, mC and mG represent 2'-O-methyl modified ribonucleotides A, U, C and G, respectively; fA, fU, fC and fG represent 2'-fluoro modified ribonucleotides A, U, C and G, respectively; -s- represents that the two nucleotides are connected by a thiophosphate backbone; GNA-U represents the modification of ribonucleotide U with GNA.

[0020] 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.

[0021] In some embodiments, the positive chain structure of the siRNA molecule conjugated to the ligand is shown in SEQ ID NOs: 59-61; the antisense chain structure of the modified siRNA molecule is shown in SEQ ID NOs: 62-64:

[0022] L-96 is GalNac-L96, a G-rich oligonucleotide with a longer GalNAc linker.

[0023] The second aspect of the present invention discloses a biological material related to the above-mentioned siRNA, which is any of the following:

[0024] 1) A vector containing the above-mentioned siRNA molecule;

[0025] 2) a reagent or kit containing the above-mentioned siRNA or the vector described in 1);

[0026] 3) Pharmaceutical composition, consisting of the above-mentioned siRNA molecules and other pharmaceutically acceptable components.

[0027] 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.

[0028] The above-mentioned carriers include, but are not limited to, magnetic nanoparticles (such as Fe2O3), 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.

[0029] Furthermore, the pharmaceutical composition may be in the form of a liquid preparation (e.g., an injection) or a lyophilized powder injection. The lyophilized powder injection is mixed with a liquid excipient during administration to form a liquid preparation. The liquid preparation may be administered subcutaneously, intramuscularly, or intravenously, or may be administered via spray to the lungs or to other organs (e.g., the liver) via spray administration through the lungs.

[0030] The third aspect of the present invention discloses the use of the above-mentioned biomaterial in treating hyperuricemia.

[0031] 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 prophylactically effective amount or a therapeutically effective amount, of the above-described siRNA molecule or pharmaceutical composition.

[0032] The present invention achieves the following beneficial technical effects:

[0033] 1. siRNA molecules and modified siRNA molecules have high stability and / or high inhibitory activity.

[0034] 2. While maintaining high inhibitory activity and stability, ligand-conjugated siRNA molecules also have good liver targeting and the ability to promote cellular 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

[0035] FIG1 is a graph showing that the naked siRNA sequence in Example 1 can reduce the expression of XDH mRNA in Hep-G2 cells.

[0036] FIG2 is a diagram showing that the siRNA modified sequence in Example 2 can reduce the expression of XDH mRNA in Hep-G2 cells.

[0037] FIG3 is a diagram showing that the modified sequence of siRNA coupled with GalNAc in Example 3 can reduce the expression of XDH mRNA in Hep-G2 cells.

[0038] 4A-D show the IC50 of the modified siRNA sequences coupled with GalNAc in Example 4 in reducing the expression of XDH mRNA in Hep-G2 cells.

[0039] FIG5 is a graph showing that the modified sequence of siRNA coupled with GalNAc in Example 5 can reduce the expression of XDH mRNA in the liver of SD rats.

[0040] FIG6 is a graph showing the efficacy of the GalNAc coupling sequence in Example 6 in a mouse hyperuricemia model.

[0041] FIG7 is a graph showing the results of the drug efficacy persistence of the GalNAc coupling sequence in Example 7 in a mouse hyperuricemia model.

[0042] 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

[0043] It is particularly important to note 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. It is obvious that relevant persons can modify or appropriately change and combine the methods and applications described herein to implement and apply the technology of the present invention without departing from the content, spirit, and scope of the present invention.

[0044] Example 1: Naked sequence knockdown effect screening

[0045] 1) Human hepatocellular carcinoma cells Hep-G2 (purchased from the cell bank of the Kunming Institute of the Chinese Academy of Sciences) in the logarithmic growth phase were trypsinized and then terminated with complete medium supplemented with 10% FBS. The cells were collected by centrifugation and resuspended in medium supplemented with 10% FBS. The cells were counted using a hemocytometer, and 50,000 cells were added to each well of a 24-well plate for culture.

[0046] 2) Preparation of a mixture of LipoRNAiMAX (Lipofectamine RNAiMAX) (purchased from Invitrogen) and siRNA, specifically using the following sequences: wherein overhangs were generated in some sequences, and the overhangs are indicated in brackets in each sequence in the table below.

[0047] 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.

[0048] 3) Add 50 μL of the corresponding group of siRNA and LipoRNAiMAX (Invitrogen) mixed solution to each well.

[0049] 4) After 48 hours of culture, the culture medium was discarded, and the cells were washed twice with enzyme-free PBS and lysed with lysis buffer (BioFlux). Chloroform (MREDA) was added for extraction, and the cells were shaken and allowed to stand at room temperature for 2-3 minutes. The supernatant was transferred to a well plate and combined with the binding buffer. RNA was extracted using a nucleic acid extraction instrument (MagaBio plus Total RNA Purification Kit II, BioRich, batch number BSC69L1E) according to the kit instructions. The well plates were arranged in order and RNA was extracted using the BSC69 protocol.

[0050] 5) Prepare the qPCR system on ice. Add 1 μL One Step SYBR Green Mix (Novagen), 10 μL 2*One Step SYBR Green Mix (Novagen), 0.4 μL hYJH-012 3P F, and 0.4 μL hYJH-012 3P R to each well. Dilute 100 ng of RNA in 8.2 μL RNase ddH2O (Novagen) and add to the wells. Mix well and place in a qPCR instrument for reaction.

[0051] 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.

[0052] PCR primers: hYJH-012 3P F (SEQ ID NO: 65): NF05_300300, hYJH-012 3P R (SEQ ID NO: 66): NF05_300301,

[0053] Novozymes test kit: batch number 7E750A3

[0054] 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.

[0055] Example 2: Screening of knockdown effects of modified sequences

[0056] 1) Human hepatocellular carcinoma cells Hep-G2 (purchased from the cell bank of the Kunming Institute of the Chinese Academy of Sciences) in the logarithmic growth phase were trypsinized and then terminated with complete medium supplemented with 10% FBS. The cells were collected by centrifugation and resuspended in medium supplemented with 10% FBS. The cells were counted using a hemocytometer, and 50,000 cells were added to each well of a 24-well plate for culture.

[0057] 2) The mixture of LipoRNAiMAX (Invitrogen) and siRNA was prepared using the following sequences: Overhangs were generated in some sequences, and the overhangs are indicated in brackets in the sequences in the table below.

[0058] 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.

[0059] 3) Add 50 μL of the corresponding group of siRNA and LipoRNAiMAX (Invitrogen) mixed solution to each well.

[0060] 4) After 48 hours of culture, the culture medium was discarded, and the cells were washed twice with enzyme-free PBS and lysed with lysis buffer (BioFlux). Chloroform (MREDA) was added for extraction, and the cells were shaken to mix and allowed to stand at room temperature for 2-3 minutes. The supernatant was transferred to a well plate and combined with the binding buffer. RNA was extracted using a nucleic acid extraction instrument (MagaBio plus Total RNA Purification Kit II, BioRich, batch number BSC69M1E) according to the kit instructions. The well plates were arranged in order and RNA was extracted using the BSC69 procedure.

[0061] 5) Prepare the qPCR system on ice. Add 1 μL One Step SYBR Green Mix (Novagen), 10 μL 2*One Step SYBR Green Mix (Novagen), 0.4 μL hYJH-012 4P F, and 0.4 μL hYJH-012 4P R to each well. Dilute 100 ng of RNA in 8.2 μL RNase ddH2O (Novagen) and add to the wells. Mix well and place in a qPCR instrument for reaction.

[0062] 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.

[0063] PCR primers: hYJH-012 4P F (SEQ ID NO: 67): NF07_182005, hYJH-012 4P R (SEQ ID NO: 68): NF07_182006,

[0064] Novozymes test kit: batch number 7E750A3

[0065] The results showed that after LipoRNAiMAX transfection, YJH-012-1609 and its modified sequences all had inhibitory effects, with the YJH-012-1609mE1 sequence having the highest inhibition rate, reaching 70%. YJH-012-3047 and its modified sequences all had inhibitory effects, with the YJH-012-3047mE+2 sequence having the highest inhibition rate, reaching 61%, with significant differences. The positive control modified sequences YJH-012-ALN m1 and YJH-012-ARH m1 had inhibition rates of 58% and 36%, respectively, as shown in Figure 2.

[0066] Example 3: Knockdown effect of GalNAc coupling sequence in Hep-G2 cells

[0067] 1. The GalNAc conjugates are as follows:

[0068] 2. Knockdown experiment:

[0069] 1) Human hepatocellular carcinoma cells Hep-G2 (purchased from the cell bank of the Kunming Institute of the Chinese Academy of Sciences) in the logarithmic growth phase were trypsinized and then terminated with complete medium supplemented with 10% FBS. The cells were collected by centrifugation and resuspended in medium supplemented with 10% FBS. The cells were counted using a hemocytometer, and 50,000 cells were added to each well of a 24-well plate for culture.

[0070] 2) The mixture of LipoRNAiMAX (Invitrogen) and siRNA was prepared using the following sequences: Overhangs were generated in some sequences, and the overhangs are indicated in brackets in the sequences in the table below.

[0071] 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.

[0072] 3) Add 50 μL of the corresponding group of siRNA mixture to each well.

[0073] 4) After 48 hours of culture, the culture medium was discarded, and the cells were washed twice with enzyme-free PBS and lysed with lysis buffer (BioFlux). Chloroform (MREDA) was added for extraction, and the cells were shaken to mix and allowed to stand at room temperature for 2-3 minutes. The supernatant was transferred to a well plate and combined with the binding buffer. RNA was extracted using a nucleic acid extraction instrument (MagaBio plus Total RNA Purification Kit II, BioRich, batch number BSC69M1E) according to the kit instructions. The well plates were arranged in order and RNA was extracted using the BSC69 procedure.

[0074] 5) Prepare the qPCR system on ice. Add 1 μL One Step SYBR Green Mix (Novagen), 10 μL 2*One Step SYBR Green Mix (Novagen), 0.4 μL hYJH-012 4P F, and 0.4 μL hYJH-012 4P R to each well. Dilute 100 ng of RNA in 8.2 μL RNase ddH2O (Novagen) and add to the wells. Mix well and place in a qPCR instrument for reaction.

[0075] 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.

[0076] PCR primers: hYJH-012 4P F (SEQ ID NO: 67): NF07_182005, hYJH-012 4P R (SEQ ID NO: 68): NF07_182006,

[0077] Novozymes test kit: batch number 7E750A3

[0078] The results are shown in FIG3 . 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.

[0079] Example 4: IC50 of GalNAc coupling sequence in Hep-G2 cells

[0080] 1) Human hepatocellular carcinoma cells Hep-G2 (purchased from the cell bank of the Kunming Institute of the Chinese Academy of Sciences) in the logarithmic growth phase were trypsinized and then terminated with complete medium supplemented with 10% FBS. The cells were collected by centrifugation and resuspended in medium supplemented with 10% FBS. The cells were counted using a hemocytometer, and 50,000 cells were added to each well of a 24-well plate for culture.

[0081] 2) Preparation of a mixture of LipoRNAiMAX (Invitrogen) and siRNA using the same sequence as in Example 3;

[0082] NC siRNA at 50 nM / well and 1.5 μL of LipoRNAiMAX (Invitrogen) were diluted separately in 25 μL of serum-free culture medium (Opti-MEM, purchased from Gibco). The siRNA solutions were then mixed with the LipoRNAiMAX (Invitrogen) solution to prepare stock solutions, which were then allowed to stand at room temperature for 5 minutes. Three siRNAs, YJH-012-1609mS2-L96, YJH-012-1609mE1-L96, and YJH-012-3047mE+2-L96, were also selected. 50 nM / well of these siRNAs were directly diluted in 50 μL of serum-free culture medium (Opti-MEM, purchased from Gibco) to prepare stock solutions, which were allowed to stand at room temperature for 5 minutes. The four siRNA stock solutions were then serially diluted to yield siRNA dilutions with concentrations of 10 nM, 2 nM, and 0.4 nM.

[0083] 3) Add 50 μL of the corresponding group of siRNA mixed solution to each well.

[0084] 4) After 48 hours of culture, the culture medium was discarded, and the cells were washed twice with enzyme-free PBS and lysed with lysis buffer (BioFlux). Chloroform (MREDA) was added for extraction, and the cells were shaken to mix and allowed to stand at room temperature for 2-3 minutes. The supernatant was transferred to a well plate and combined with the binding buffer. RNA was extracted using a nucleic acid extraction instrument (MagaBio plus Total RNA Purification Kit II, BioRich, batch number BSC69M1E) according to the kit instructions. The well plates were arranged in order and RNA was extracted using the BSC69 procedure.

[0085] 5) Prepare the qPCR system on ice. Add 1 μL One Step SYBR Green Mix (Novagen), 10 μL 2*One Step SYBR Green Mix (Novagen), 0.4 μL hYJH-012 4P F, and 0.4 μL hYJH-012 4P R to each well. Dilute 100 ng of RNA in 8.2 μL RNase ddH2O (Novagen) and add to the wells. Mix well and place in a qPCR instrument for reaction.

[0086] 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.

[0087] PCR primers: hYJH-012 4P F (SEQ ID NO: 67): NF07_182005, hYJH-012 4P R (SEQ ID NO: 68): NF07_182006,

[0088] Novozymes test kit: batch number 7E750A3

[0089] 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.

[0090] Example 5: Knockdown effect of GalNAc coupling sequence in rat liver

[0091] siRNAs of interest identified from in vitro studies were evaluated in vivo. The pharmacodynamic activity of the following GalNAc-conjugated siRNAs targeting XDH was analyzed in rats following subcutaneous injection of siRNA: Overhangs were generated in some sequences, as indicated in parentheses in the following table.

[0092] Each GalNAc-conjugated siRNA was administered at two dose concentrations (1 mg / kg and 4 mg / kg) weekly for multiple subcutaneous administration. Liver samples were collected 21 days after the start of dosing, and XDH mRNA levels in all samples were analyzed by RT-qPCR. Rat liver tissue was lysed using a lysis buffer (BioFlux) and extracted with chloroform (MREDA). After vortexing and mixing, the supernatant was allowed to stand at room temperature for 2-3 minutes and centrifuged. The supernatant was transferred to a well plate and combined with the binding buffer. Using a nucleic acid extraction instrument, the well plates were arranged in order according to the kit instructions (MagaBio Plus Total RNA Purification Kit II, manufacturer Bioer, batch number C692307003), and total RNA was extracted using the BSC69 procedure.

[0093] 2) Prepare the qPCR system on ice. Add 1 μL One SteP SYBR Green Mix (Novagen), 10 μL 2*One SteP SYBR Green Mix (Novagen), 0.4 μL rYJH-012-2P F (rXDH-2P F), and 0.4 μL rYJH-012-2P R (rXDH-2P R) to each well. Dilute total liver tissue RNA in 8.2 μL RNase-free deionized water (Novagen) and add to the wells. Mix well and place in a qPCR instrument for reaction.

[0094] qPCR reaction conditions were as follows: 50°C for 15 min pre-denaturation, 95°C for 1 min, 95°C annealing for 15 s, and 60°C extension for 1 min, for 39 cycles.

[0095] PCR primers: rYJH-012-2P F (SEQ ID NO: 69): NF09-130069, rYJH-012-2P R (SEQ ID NO: 70): NF09-130070.

[0096] Novozymes test kit: batch number 7E711D3

[0097] As shown in Figure 5, injection of 4 mg / kg of the aforementioned sequences (YJH-012-1609mS2-L96, YJH-012-1609mE1-L96, and YJH-012-3047mE+2-L96) inhibited XDH mRNA expression in rat liver by 42.08%, 49.00%, and 54.90%, respectively. These results demonstrate that injection of GalNAc-conjugated siRNA can downregulate XDH mRNA expression.

[0098] Example 6: Efficacy of GalNAc coupling sequence in mouse hyperuricemia model

[0099] siRNA efficacy was evaluated in a mouse hyperuricemia model. Following subcutaneous injection of siRNA, the pharmacodynamic activity of the following XDH-targeting GalNAc-conjugated siRNAs was analyzed in mice: Overhangs were generated in some sequences, as indicated in parentheses in the sequences in the table below.

[0100] 1) Each GalNAc-coupled siRNA was administered at a single dose concentration (8 mg / kg) subcutaneously 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 daily.

[0101] 2) On the 14th day after the start of the experiment, blood was collected 2 hours after modeling and serum was separated to measure uric acid levels.

[0102] As shown in Figure 6, 8 mg / kg of the aforementioned sequences (YJH-012-1609mS2-L96, YJH-012-1609mE1-L96, and YJH-012-3047mE+2-L96) were injected into hyperuricemia model mice, demonstrating uric acid-lowering effects of 20.15%, 40.82%, and 40.36%, respectively, on day 14 after administration. These results demonstrate that injection of GalNAc-conjugated siRNA can lower blood uric acid levels in hyperuricemia model mice.

[0103] Example 7: Durability of the efficacy of GalNAc coupling sequence in a mouse hyperuricemia model

[0104] The pharmacodynamics of the following GalNAc-conjugated siRNAs targeting XDH were evaluated for their long-lasting efficacy in a mouse model of hyperuricemia. The pharmacodynamic activity of the following XDH-targeting GalNAc-conjugated siRNAs was analyzed in mice following subcutaneous injection of the siRNAs: Overhangs were generated in some sequences, as indicated in parentheses within the sequences in the table below.

[0105] 1) GalNAc-conjugated siRNA was administered subcutaneously at three concentrations (1 mg / kg, 4 mg / kg, and 8 mg / kg) 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 daily.

[0106] 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.

[0107] As shown in Figure 7, a single dose of 4 mg / kg or 8 mg / kg of YJH-012-1609mE1-L96 reduced the blood uric acid level in hyperuricemia model mice for 28 days. However, the 1 mg / kg dose group only showed significant uric acid-lowering effects on day 4 after administration.

[0108] Example 8: Durability of the efficacy of GalNAc conjugated sequences in a cynomolgus monkey hyperuricemia model

[0109] The pharmacodynamics of the following GalNAc-conjugated siRNAs targeting XDH were evaluated for their long-lasting efficacy in a cynomolgus monkey model of hyperuricemia. The pharmacodynamic activity of the following XDH-targeting GalNAc-conjugated siRNAs was analyzed in cynomolgus monkeys following subcutaneous injection of siRNAs: Overhangs were generated in some sequences; the overhangs are indicated in parentheses in the sequences in the table below.

[0110] 1) GalNAc-conjugated siRNA was administered subcutaneously at a single dose concentration (10 mg / kg) on ​​the first day of the experiment. Modeling was performed 30 minutes after administration. Modeling cynomolgus monkeys were intraperitoneally injected with 600 mg / kg potassium oxonate daily.

[0111] 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.

[0112] As shown in FIG8 , a single dose of 10 mg / kg of YJH-012-1609mE1-L96 can reduce the blood uric acid level in the hyperuricemia model cynomolgus monkey for 90 days.

[0113] 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

1. An siRNA for inhibiting the expression of xanthine oxidase gene, characterized in that, It includes a sense strand and an antisense strand; the antisense strand is reverse complementary to a segment on the target gene; 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.

2. The siRNA according to claim 1, wherein At most two additional nucleotides are connected to the 3'-ends of the sense strand and the antisense strand of the nucleotide sequence to form overhangs.

3. The siRNA according to claim 2, wherein The nucleotides of the overhangs are selected from A, C, G, U, T or nucleotides modified from A, C, G, U, T.

4. The siRNA according to claim 1 or 3, characterized in that, At least one nucleotide in the siRNA is modified; the modified nucleotides are selected from one or more of the modification of the sugar moiety at the 2'-position, or at least one phosphate group being a phosphate group containing a modifying group, or nucleotide analogs.

5. The siRNA according to claim 4, wherein The 2'-modified nucleotides include nucleotides modified with 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'-O-DMAP), T-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) or 2'-O-N-methylacetamido (2'-O-NMA).

6. The siRNA according to claim 4, wherein The phosphate group containing a modifying group is specifically a phosphorothioate group formed by replacing at least one oxygen atom in the phosphodiester bond with a sulfur atom.

7. The siRNA according to claim 4, wherein The nucleotide analogs are selected from one of isonucleotides, LNA, ENA, cEt BNA, UNA or GNA.

8. The siRNA according to any one of claims 4-7, wherein The sense strand structure of the modified siRNA molecule is as shown in SEQ ID NO: 35-46; the antisense strand structure of the modified siRNA molecule is as shown in SEQ ID NO: 47-58.

9. The siRNA according to claim 8, wherein The 3'-end of the sense strand is conjugated with a ligand; the ligand includes GalNAc, cholesterol, biotin, vitamins, galactose derivatives or analogs, lactose derivatives or analogs, N-acetylglucosamine derivatives or analogs.

10. The siRNA according to claim 9, wherein The sense strand structure of the siRNA molecule conjugated with a ligand is as shown in SEQ ID NO: 59-61; the antisense strand structure of the modified siRNA molecule is as shown in SEQ ID NO: 62-64.

11. The biological material related to the siRNA according to any one of claims 1-10 is any one of the following: 1) A vector containing the siRNA according to any one of claims 1-10; 2) A reagent or kit containing the siRNA according to any one of claims 1-10 or the vector described in 1); 3) A pharmaceutical composition composed of the siRNA molecule according to any one of claims 1-10 and other pharmaceutically acceptable components.

12. The biomaterial according to claim 11, wherein The dosage form of the pharmaceutical composition can be a liquid preparation or a freeze-dried powder injection.

13. Use of the siRNA according to any one of claims 1-10 or the biological material according to claim 11 in the treatment of hyperuricemia.

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