SiRNA (small interfering Ribonucleic Acid) of targeted urate transporter 1 and application thereof

Abstract

The invention discloses siRNA (small interfering Ribonucleic Acid) of a targeted urate transporter 1 and application of the siRNA. According to the invention, siRNA drug design is carried out on the URAT1 gene to obtain siRNA composed of positive-sense strand RNA and antisense strand RNA, siRNA capable of effectively silencing the gene expression level of URAT1 is screened from the siRNA, and part of siRNA sequences even silence the gene expression level of URAT1 to about 20%. The invention further discloses the modified siRNA which is obtained by chemically modifying the siRNA sequence and can effectively silence the gene expression level of URAT1, and a recombinant vector containing the siRNA or the modified siRNA. The siRNA, the modified siRNA and the recombinant vector containing the siRNA or the modified siRNA provided by the invention have the application in the aspects of preparation of gene therapy medicines for treating hyperuricemia or gout and the like.

Description

Technical Field

[0001] The present invention relates to siRNA, in particular to siRNA targeting urate transporter 1 and application thereof in reducing the expression level of URAT1, and belongs to the field of gene therapy for hyperuricemia or gout. Background Art

[0002] Gout is a common and treatable crystal arthropathy, accounting for 5% of all arthritis. It is an immune inflammation caused by the deposition of monosodium urate crystals (MSU) in non-articular structures in soft tissues and bones within and outside the joints. Characteristic acute arthritis, tophi, and interstitial nephritis caused by hyperuricemia and urate crystals and deposition, as well as joint deformities and dysfunction in severe cases, are often accompanied by uric acid urinary stones. Gout patients often suffer from other diseases at the same time. In about 75% of cases, metabolic syndrome consisting of abdominal obesity, hypertension, insulin resistance and abnormal blood lipid levels occurs simultaneously with gout. Gout is a common complication of diseases such as polycythemia, lead poisoning, renal failure, hemolytic anemia, psoriasis, and organ transplantation.

[0003] Gout is characterized by intermittent attacks of severe and painful arthritis (gout attacks). It is a disorder of purine metabolism. The end product of purine metabolism, uric acid, crystallizes as monosodium urate, precipitates and forms deposits (i.e., tophi) in joints, tendons and surrounding tissues. Tiny tophi may be cleared by a ring of proteins that can hinder the interaction between crystals and cells, thereby avoiding the occurrence of inflammatory responses. Tophi wrapped in proteins may release exposed monosodium urate crystals due to minor joint trauma, drugs, surgical stress or drastic changes in blood uric acid levels. When tophi are released, they trigger a local immune-mediated inflammatory response. In clinical practice and research, hyperuricemia (blood urate concentration exceeds the saturation threshold) usually occurs when serum urate is higher than or equal to 0.42mmol / L (7mg / dL), and the cause of uric acid precipitation is not fully understood. Although uric acid can crystallize when the blood uric acid concentration is normal, uric acid crystals are more likely to form when the blood uric acid concentration is elevated. Other important factors that cause acute gouty arthritis include low temperatures and rapid changes in serum uric acid concentration, pH, synovial fluid composition, and extracellular matrix components (such as proteoglycans, collagen, and chondroitin sulfate). The increased precipitation of uric acid at low temperatures may explain, to some extent, why gout is more likely to occur in the joints of the feet. Many factors can cause rapid changes in uric acid concentration, including external trauma, surgery, chemotherapy, use of diuretics, and the initiation or discontinuation of isopurinol.

[0004] According to statistics, most (about 90%) of hyperuricemia is caused by decreased urate clearance by the kidneys of patients, while less than 10% of hyperuricemia patients produce too much uric acid in the body. The kidneys and intestines play a major role in uric acid excretion, with more than 70% of uric acid excretion occurring in the renal channels. Therefore, the development of agents for increasing uric acid excretion has significant therapeutic potential. Serum urate is regulated by urate transporters in the kidneys and intestines, especially GLUT9 (SLC2A9), URAT1 (SLC22A12), and ABCG2. Monosodium urate crystals play a major role in the activation of NLRP3 inflammasomes and the release of IL-1β in the onset of gout attacks; aggregated neutrophil extracellular traps are important in the resolution stage. Although gout manifests as intermittent attacks, it is a chronic disease. The core strategy for the effective treatment of gout is long-term urate reduction therapy to reverse hyperuricemia, which leads to the dissolution of monosodium urate crystals and long-term prevention of gout attacks. Currently, in addition to drugs such as allopurinol and febuxostat that block xanthine oxidase (XO), inhibition of urate transporter 1 (URAT1) is currently the main treatment for uric acid-lowering therapy.

[0005] Urate transporter 1 (URAT1) is a membrane transporter located in the apical membrane of human renal proximal tubular epithelial cells. It consists of 555 amino acids and belongs to the organic anion transporter (OAT) family. URAT1 is expressed on the luminal side of the proximal tubule and regulates the absorption of uric acid from the tubule to the epithelial cells, mediating most of the reabsorption of urate. Therefore, the drug design of URAT1 inhibitors that target the improvement of renal urate excretion by reducing the reabsorption of urate anions is currently the key to the search for anti-gout drugs. Currently approved URAT1 inhibitors for use in clinical settings include probenecid, benzbromarone, thiopyrazolone, and lesinurad. However, these drugs have side effects and a certain degree of toxicity: probenecid can cause rash, gastrointestinal irritation, hypersensitivity reactions, and hemolytic anemia; benzbromarone can cause severe hepatotoxicity and bone marrow suppression; thiopyrazolone can cause nausea, vomiting, abdominal pain, diarrhea, anemia, and rash; lesinurad has dose-dependent nephrotoxicity and can cause adverse cardiovascular events. URAT1 inhibitors cannot be used in patients with severe renal failure, urate kidney stones, or hematomalnutrition.

[0006] siRNA is a double-stranded RNA of about 21-25bp in length, with a negative charge. It mainly achieves efficient and highly specific binding to the target mRNA through RNA interference (RNAi), and triggers the specific degradation of the target mRNA, thereby regulating gene expression. Screening effective siRNA can reduce the expression level of URAT1 and thus treat related diseases. Summary of the invention

[0007] One of the objects of the present invention is to provide siRNA targeting urate transporter 1 (URAT1).

[0008] The second object of the present invention is to provide a recombinant expression vector containing the siRNA targeting urate transporter 1 (URAT1).

[0009] The third object of the present invention is to provide a host cell containing the recombinant expression vector.

[0010] The fourth object of the present invention is to use the recombinant expression vector containing the siRNA targeting urate transporter 1 (URAT1) to prepare a gene therapy drug for treating hyperuricemia or gout;

[0011] To achieve the above object, the technical solution adopted by the present invention includes:

[0012] One aspect of the present invention is to provide an siRNA targeting urate transporter 1 (URAT1), wherein the siRNA is a double-stranded RNA consisting of a sense strand and an antisense strand complementary to the sense strand, and the siRNA is selected from any one of the double-stranded RNAs described in (1) to (24):

[0013] (1) a double-stranded RNA consisting of a sense strand having a nucleotide sequence as shown in SEQ ID No. 1 and an antisense strand having a nucleotide sequence as shown in SEQ ID No. 2;

[0014] (2) a double-stranded RNA consisting of a sense strand having a nucleotide sequence as shown in SEQ ID No. 3 and an antisense strand having a nucleotide sequence as shown in SEQ ID No. 4;

[0015] (3) a double-stranded RNA consisting of a sense strand having a nucleotide sequence of SEQ ID No. 5 and an antisense strand having a nucleotide sequence of SEQ ID No. 6;

[0016] (4) a double-stranded RNA consisting of a sense strand having a nucleotide sequence of SEQ ID No. 7 and an antisense strand having a nucleotide sequence of SEQ ID No. 8;

[0017] (5) a double-stranded RNA consisting of a sense strand having a nucleotide sequence of SEQ ID No. 9 and an antisense strand having a nucleotide sequence of SEQ ID No. 10;

[0018] (6) a double-stranded RNA consisting of a sense strand having a nucleotide sequence as shown in SEQ ID No. 11 and an antisense strand having a nucleotide sequence as shown in SEQ ID No. 12;

[0019] (7) a double-stranded RNA consisting of a sense strand having a nucleotide sequence as shown in SEQ ID No. 13 and an antisense strand having a nucleotide sequence as shown in SEQ ID No. 14;

[0020] (8) a double-stranded RNA consisting of a sense strand having a nucleotide sequence as shown in SEQ ID No. 15 and an antisense strand having a nucleotide sequence as shown in SEQ ID No. 16;

[0021] (9) a double-stranded RNA consisting of a sense strand having a nucleotide sequence as shown in SEQ ID No. 17 and an antisense strand having a nucleotide sequence as shown in SEQ ID No. 18;

[0022] (10) a double-stranded RNA consisting of a sense strand having a nucleotide sequence of SEQ ID No. 19 and an antisense strand having a nucleotide sequence of SEQ ID No. 20;

[0023] (11) a double-stranded RNA consisting of a sense strand having a nucleotide sequence of SEQ ID No. 21 and an antisense strand having a nucleotide sequence of SEQ ID No. 22;

[0024] (12) a double-stranded RNA consisting of a sense strand having a nucleotide sequence as shown in SEQ ID No. 23 and an antisense strand having a nucleotide sequence as shown in SEQ ID No. 24;

[0025] (13) a double-stranded RNA consisting of a sense strand having a nucleotide sequence of SEQ ID No. 25 and an antisense strand having a nucleotide sequence of SEQ ID No. 26;

[0026] (14) a double-stranded RNA consisting of a sense strand having a nucleotide sequence as shown in SEQ ID No. 27 and an antisense strand having a nucleotide sequence as shown in SEQ ID No. 28;

[0027] (15) a double-stranded RNA consisting of a sense strand having a nucleotide sequence of SEQ ID No. 29 and an antisense strand having a nucleotide sequence of SEQ ID No. 30;

[0028] (16) a double-stranded RNA consisting of a sense strand having a nucleotide sequence of SEQ ID No. 31 and an antisense strand having a nucleotide sequence of SEQ ID No. 32;

[0029] (17) a double-stranded RNA consisting of a sense strand having a nucleotide sequence as shown in SEQ ID No. 33 and an antisense strand having a nucleotide sequence as shown in SEQ ID No. 34;

[0030] (18) a double-stranded RNA consisting of a sense strand having a nucleotide sequence of SEQ ID No. 35 and an antisense strand having a nucleotide sequence of SEQ ID No. 36;

[0031] (19) a double-stranded RNA consisting of a sense strand having a nucleotide sequence of SEQ ID No. 37 and an antisense strand having a nucleotide sequence of SEQ ID No. 38;

[0032] (20) a double-stranded RNA consisting of a sense strand having a nucleotide sequence of SEQ ID No. 39 and an antisense strand having a nucleotide sequence of SEQ ID No. 40;

[0033] (21) a double-stranded RNA consisting of a sense strand having a nucleotide sequence of SEQ ID No. 41 and an antisense strand having a nucleotide sequence of SEQ ID No. 42;

[0034] (22) a double-stranded RNA consisting of a sense strand having a nucleotide sequence as shown in SEQ ID No. 43 and an antisense strand having a nucleotide sequence as shown in SEQ ID No. 44;

[0035] (23) a double-stranded RNA consisting of a sense strand having a nucleotide sequence of SEQ ID No. 45 and an antisense strand having a nucleotide sequence of SEQ ID No. 46;

[0036] (24) A double-stranded RNA consisting of a sense strand having a nucleotide sequence as shown in SEQ ID No.47 and an antisense strand having a nucleotide sequence as shown in SEQ ID No.48.

[0037] Preferably, the siRNA is selected from any one of the following siRNAs:

[0038] An siRNA consisting of the sense RNA shown in SEQ ID No.5 and the antisense RNA shown in SEQ ID No.6; or an siRNA consisting of the sense RNA shown in SEQ ID No.13 and the antisense RNA shown in SEQ ID No.14; or an siRNA consisting of the sense RNA shown in SEQ ID No.17 and the antisense RNA shown in SEQ ID No.18; or an siRNA consisting of the sense RNA shown in SEQ ID No.33 and the antisense RNA shown in SEQ ID No.34; or an siRNA consisting of the sense RNA shown in SEQ ID No.41 and the antisense RNA shown in SEQ ID No.42.

[0039] The siRNA provided by the present invention has a significant inhibitory effect on the expression of URAT1, wherein the siRNA consisting of the sense strand shown in the nucleotide sequence of SEQ ID No.5 and the antisense strand shown in the nucleotide sequence of SEQ ID No.6 can achieve an inhibitory effect of about 80% on the expression of URAT1.

[0040] The present invention further includes modified siRNA obtained by chemically modifying the siRNA.

[0041] For reference, in order to improve the strand selectivity of siRNA for target genes and the silencing effect of target genes, those skilled in the art can use any of the following modification methods to phosphorylate or modify the 5′ end of the antisense strand of the siRNA as required:

[0042]

[0043] At the same time, the two protruding non-paired bases TT at the 3' end of the sense strand or antisense strand of the siRNA sequence are replaced with two other nucleoside or deoxynucleoside bases.

[0044] In order to improve the stability of siRNA in vivo, those skilled in the art can modify the above-mentioned siRNA as needed, including but not limited to the following types of modifications: modifying both ends of the sense strand RNA and the antisense strand RNA of the siRNA with several thiophosphates, or combining the modification of the nucleoside backbone to a thiophosphate bond, a borane phosphate bond or a methyl phosphate bond, which can enhance the stability of the siRNA and reduce the degradation of the siRNA by the exonuclease; or modifying the 2′-OH of the siRNA, for example, replacing the 2′-OH with a methoxy group, a methylethoxy group\fluorine and / or deoxygenating the 2′-OH; or changing the siRNA to a peptide nucleic acid (PNA) type, a locked nucleic acid (LNA) type or an unlocked nucleic acid (UNA) type modification; or modifying and replacing the bases such as 5-methylcytosine, 6-methyladenine, ribavirin, pseudouracil and / or inosine on the nucleotide.

[0045] In a preferred specific embodiment of the present invention, the present invention provides a siRNA obtained by chemically modifying a double-stranded RNA consisting of a sense strand with a nucleotide sequence as shown in SEQ ID No.5 and an antisense strand with a nucleotide sequence as shown in SEQ ID No.6, which consists of the following sense strand and antisense strand:

[0046] Justice Chain: G m *G m *G m G m A m Am C f U m C f U m G f U m C f C m U m A m A m A m A m *dT*dT;

[0047] Antisense strand: Phos-U m *U f *U f U f A m G f G m A f C m A f G m A f G m U f U m C f C m C m C m *dT*dT;

[0048] Among them, m indicates that RNA is 2′-methoxy, f indicates that RNA is 2′-fluoro, * indicates phosphorothioate, dT indicates DNA thymine modification, and Phos indicates 5′ phosphorylation modification.

[0049] Another preferred specific embodiment of the present invention provides a siRNA obtained by chemically modifying a double-stranded RNA consisting of a sense strand with a nucleotide sequence as shown in SEQ ID No.33 and an antisense strand with a nucleotide sequence as shown in SEQ ID No.34, which consists of the following sense strand and antisense strand:

[0050] Justice Chain: C m *C m *U m U m U m G m G f C m U f U m C f A m C f C m U m U m Cm U m U m *dT*dT;

[0051] Justice chain: Phos-A m *A f *G f A f A m G f G m U f G m A f A m G f C m C f A m A f A m G m G m *dT*dT;

[0052] Among them, m indicates that RNA is 2′-methoxy, f indicates that RNA is 2′-fluoro, * indicates phosphorothioate, dT indicates DNA thymine modification, and Phos indicates 5′ phosphorylation modification.

[0053] The second aspect of the present invention is to provide an interference vector or expression vector containing the siRNA; wherein, those skilled in the art can construct the siRNA according to the conventional preparation method in the art to obtain an interference vector or expression vector and sdRNA and shRNA based on the corresponding sequence of the above siRNA and their recombinant expression vectors; preferably, the recombinant expression vector can be a prokaryotic recombinant expression vector or a eukaryotic recombinant expression vector, more preferably, the expression vector is a eukaryotic recombinant expression vector, and particularly preferably, the eukaryotic recombinant expression vector is a recombinant adenovirus expression vector, which is used as a gene therapy drug to reduce URAT1 gene expression and thereby treat hyperuricemia or gout.

[0054] The third aspect of the present invention provides a host cell containing the recombinant expression vector as described above. Preferably, the host cell is a prokaryotic cell or a eukaryotic cell. More preferably, the host cell is a eukaryotic cell. Particularly preferably, the host cell is a mammalian cell.

[0055] The fourth aspect of the present invention provides a pharmaceutical composition for treating hyperuricemia or gout, which is composed of a therapeutically effective amount of siRNA and a carrier or excipient acceptable for pharmaceutical preparations.

[0056] For example, siRNA molecules of the present invention can be delivered to the interior of cells with their native structures using methods known in the art. Alternatively, siRNA molecules of the present invention can be expressed in cells from eukaryotic promoters. Recombinant expression vectors capable of expressing siRNA molecules can be delivered and retained in target cells for expression. After expression, the siRNA molecules interact with target mRNA and produce RNA interference reactions. The siRNA molecules produced in this way are generally referred to as shRNA (short hairpin RNA), because their sense strand and antisense strand are connected by little nucleotide rings. The delivery of the carrier expressing the siRNA molecule can be systemic, such as by intravenous or intramuscular administration, by being administered to target cells explanted from a subject followed by reintroduction into the subject, or by any other mode that allows introduction into the desired target cells.

[0057] For example, siRNA molecules can include delivery vehicles, including liposomes, for administration to subjects. Carriers and diluents and salts thereof can be present in pharmaceutical preparations. Nucleic acid molecules can be administered to cells by a variety of methods known to those skilled in the art, including but not limited to: encapsulated in liposomes, by iontophoresis or by being incorporated into other carriers, such as biodegradable polymers, hydrogels, microspheres, biodegradable nanocapsules and bioadhesive microspheres, or by protein carriers. The intracellular delivery component can also be a viral component, including but not limited to, destroying endosomes to allow nucleic acids to avoid lysosomal decomposition of fusion virus peptides, maintaining expressed viral proteins or viral components that interact with cell surface proteins. Suitable viral intracellular delivery components include but are not limited to, retroviruses, herpes simplex viruses, adenoviruses and preferably adenovirus-associated viruses, etc.

[0058] The form of the pharmaceutical preparation of the present invention depends in part on the use or route of entry, such as oral, percutaneous or by injection. The present invention also includes a composition prepared for storage or administration, which contains a pharmaceutically effective amount of the desired compound in a pharmaceutical carrier or diluent. Carriers or diluents that can be used for therapeutic use are known in the pharmaceutical field. For example, preservatives, stabilizers, dyes and flavorings can be provided. These include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. In addition, antioxidants and suspending agents can be used.

[0059] The formulations or siRNAs of the present invention and described herein can be administered in unit dose formulations containing conventional non-toxic pharmaceutical carriers, adjuvants and / or excipients. The form of the formulation can be suitable for oral use, and the composition for oral use can be prepared according to any method known in the art for preparing a pharmaceutical composition and such a composition can contain one or more such sweeteners, flavoring agents, coloring agents or preservatives to provide a pharmaceutically good and palatable formulation.

[0060] Unless otherwise indicated, the nucleic acid sequences referred to herein are written in the 5' to 3' direction. The term "nucleic acid" refers to DNA or RNA or a modified form thereof, which contains (adenine "A", cytosine "C", guanine "G", thymine "T") present in DNA or (adenine "A", cytosine "C", guanine "G", uracil "U") purine or pyrimidine bases present in RNA. The interfering RNA provided herein may contain a "T" base, for example at the 3' end, even if the "T" base does not naturally occur in RNA. In some cases, these bases may appear as "dT" to distinguish deoxyribonucleotides present in ribonucleotide chains.

[0061] The term "interfering RNA" or "RNAi" or "interfering RNA sequence" refers to double-stranded RNA (i.e., duplex RNA) that can reduce or inhibit target gene expression (i.e., by regulating the degradation of mRNAs that are complementary to the sequence of the interfering RNA) when the interfering RNA is in the same cell as the target gene. Therefore, interfering RNA refers to double-stranded RNA formed by two complementary chains or by a single, self-complementary chain. The interfering RNA may have substantial or complete identity with the target gene, or may include mismatch regions. The sequence of the interfering RNA may be consistent with the full-length target gene or a subsequence thereof. Interfering RNA includes "small-interfering RNA" or "siRNA", for example, having a length of about 15-60, 15-50 or 15-40 duplex nucleotides. Among them, examples of siRNA in the present invention include, but are not limited to, a double-stranded polynucleotide molecule assembled from two single-stranded molecules, one of which is a sense strand and the other is a complementary antisense strand; a double-stranded polynucleotide molecule assembled from single-stranded molecules, wherein the sense and antisense regions are connected by a nucleic acid-based or non-nucleic acid-based linker; a double-stranded polynucleotide molecule with a hairpin secondary structure having self-complementary sense and antisense regions; and a circular single-stranded polynucleotide molecule with two or more loop structures and a stem having self-complementary sense and antisense regions, wherein the circular polynucleotide can be processed in vivo or in vitro to produce an active double-stranded siRNA molecule.

[0062] An "effective amount" or "therapeutically effective amount" of siRNA in the present invention is an amount sufficient to produce the desired effect, such as inhibition of target sequence expression compared to normal expression levels detected in the absence of the siRNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 The experimental results of the gene regulation effect of URAT1 siRNA (U-1 to U-29); Note: The experimental method is URAT1-GFP 300ng+RFP 200ng, siRNA 10nM, Lipo 3000 1.5μL / well.

[0064] Figure 2 The IC50 results of U-4 are shown in Table 1.

[0065] Figure 3 The figure shows the IC50 determination result of U-8.

[0066] Figure 4 The figure shows the IC50 determination result of U-12.

[0067] Figure 5 The IC50 results of U-23 are shown in Table 1.

[0068] Figure 6 The IC50 results of U-27 are shown in Table 1.

[0069] Figure 7 The figure shows the IC50 determination result of U-4-M.

[0070] Figure 8 The figure shows the IC50 determination result of U-23-M. DETAILED DESCRIPTION

[0071] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0072] Experimental instruments, reagents, consumables and plasmids

[0073] 1. Experimental instruments, reagents and consumables

[0074] Table 1 Experimental instruments, reagents and consumables

[0075]

[0076]

[0077] 2. Plasmid

[0078] Ordered from Fenghui Bio, SLC22A12 (NM_144585) pcDNA3.1-T2A-EGFP is a plasmid vector containing URAT1 gene and EGFP gene.

[0079] Experimental Example 1 Design of siRNA sequence

[0080] The plasmid expression vector for co-expression of fluorescent protein was constructed as URAT1, and siRNA was designed for the coding region of URAT1, and TT was added to the 3′ end of the siRNA as a protruding base to increase the stability of the siRNA double-stranded complex. All the sequences of the designed siRNA were screened, and the specific nucleotide sequences of the designed siRNA are shown in Table 2.

[0081] Table 2 URAT1a siRNA sequences

[0082]

[0083]

[0084] Note: m indicates RNA is 2′-methoxy, f indicates RNA is 2′-fluoro, * indicates phosphorothioate, dT or T indicates DNA thymine modification, and Phos indicates 5′ phosphorylation modification.

[0085] Experimental Example 2 Screening experiment of siRNA sequence inhibiting URAT1 gene expression

[0086] 1 Experimental methods

[0087] 1.1 Cell culture

[0088] (1) DMEM (+, +) medium preparation

[0089] 50 mL FBS (sterile-filtered) and 5 mL double antibodies (penicillin and streptomycin) were added to 445 mL DMEM medium to prepare DMEM (+, +) containing 10% FBS and 1% double antibodies and stored in a 4° C. refrigerator for cell culture.

[0090] (2) DMEM (+, -) medium configuration

[0091] 450 mL of DMEM medium was added with 50 mL of FBS (sterile-filtered) to prepare DMEM (+, -) containing 10% FBS and stored in a refrigerator at 4° C. for cell culture.

[0092] (3) Cell proliferation, passaging and plating

[0093] HEK 293T cells were revived and cultured in 75 cm 2 15 mL of DMEM (+, +) medium was added to a breathable cell culture flask, and the cells were placed in a cell culture incubator at a constant temperature of 37° C. and a CO 2 concentration of 5% for proliferation.

[0094] When the cell density in the culture flask reaches about 95%, cell passage is performed. Discard the culture medium in the cell culture flask, wash twice with 1×PBS (about 3 mL / time), add 3 mL of trypsin and digest in the cell incubator until the cells become round and a small amount of cells float up (about 3 minutes), then add 6 mL of DMEM (+, +) to stop digestion. Use a 1 mL pipette to gently blow the cells down from the bottom. Be careful not to create bubbles in this step. Transfer the cell suspension to a 15 mL centrifuge tube and centrifuge at 1000 g / min for 5 minutes. Discard the supernatant, resuspend the cells with fresh DMEM (+, +), take about 1 / 3 of the cell suspension, add 15 mL of DMEM (+, +), mix well, and add a new 75 cm 2 The cells continued to proliferate in the gas-permeable cell culture flask in the incubator.

[0095] Count the cells using a cell counting plate and prepare 2 × 10 5 / mL of cell resuspension, add 0.5mL of cell resuspension to each well of the 24-well plate, shake well by the cross method, and then culture in a cell incubator for 24 hours.

[0096] 1.2 Cell transfection

[0097] After culturing in a 24-well plate cell incubator for 24 hours, a cell transfection experiment was performed when the cell density was about 70%. The cell transfection used a commercial transfection reagent to transfect nucleic acids. The steps of the transfection experiment were as follows:

[0098] (1) Preparation of Lipofectamine 3000 TM +Opti-MEM stock solution

[0099] Lipofectamine 3000 in 24-well plates TM 1.5μL / well, take 37.5μL and add 600μL Opti-MEM medium to dilute to 637.5μL Lipofectamine 3000 TM +Opti-MEM stock solution, mix with a pipette and incubate at room temperature for 5 min.

[0100] (2) Prepare nucleic acid + plasmid transfection stock solution

[0101] Prepare plasmid stock solution: 300ng URAT1-GFP + 200ng RFP per well, dilute to 600μL with Opti-MEM medium, add 37.5μL p3000, mix well with a pipette, and incubate at room temperature for 2min.

[0102] The siRNA sequence was a 10 μM stock solution (1×PBS), and the siRNA was diluted into the plasmid Opti-MEM culture medium stock solution according to the transfection concentration, and 25 μL was configured per well.

[0103] (3) Prepare transfection stock solution

[0104] Lipofectamine 3000 after incubation in step (1) TM Add an equal volume of +Opti-MEM stock solution (25 μL per well) to the nucleic acid + plasmid transfection stock solution prepared in step (2), pipette 10-20 times to mix, and incubate at room temperature for 10 min.

[0105] (4) siRNA transfection

[0106] Remove the DMEM (+, +) medium in the 24-well plate and add 450 μL DMEM (+, -) medium to each well; add 50 μL of the transfection stock solution prepared in step (3) to each well of the 24-well plate according to the experimental design and gently shake to mix. After completion, place the plate in a cell incubator and evaluate 48 hours after the start of transfection.

[0107] 1.3 Flow cytometry evaluation

[0108] 48 hours after the transfection experiment, the cells in the 24-well plate were digested with 0.5 mL of trypsin, and the digestion was terminated with 1 mL of culture medium and transferred to a centrifuge tube. The cells were collected at 2000 g for 5 min, washed with 1× PBS, and resuspended with 1× PBS. The degree of GFP gene silencing was detected by flow cytometry. RFP gene expression was used as an internal control, GFP used the FITC channel, and RFP used the PE channel. Statistical analysis was performed using CytExpert software.

[0109] 2 Experimental results

[0110] 2.1 siRNA sequence screening experimental results

[0111] This experiment screened the siRNA sequence of URAT1, and the experimental method mainly included URAT1-GFP 300ng+RFP 200ng, siRNA 10nM, Lipo 3K 1.5μL / well.

[0112] Figure 1 and Table 3 show the experimental results of the gene regulation effect of URAT1 siRNA (U-1 to U-29).

[0113] Table 3 Results of gene regulation experiments of URAT1 sRNA

[0114] siRNA sequence name Relative expression of remaining GFP protein (%) U-1 81.63 U-2 83.33 U-3 55.53 U-4 27.87 U-5 87.91 U-6 75.88 U-7 74.68 U-8 32.35 U-9 76.94 U-10 81.54 U-11 82.45 U-12 35.65 U-13 75.52 U-14 72.05 U-15 67.91 U-16 47.83 U-17 77.46 U-18 76.14 U-19 64.76 U-20 40.86 U-21 77.65 U-22 42.45 U-23 37.87 U-24 89.09 U-25 73.46 U-26 73.22 U-27 45.84 U-28 76.15 U-29 78.07 RB-2 38.53

[0115] according to Figure 1The experimental results in Table 3 show that U-4, U-8, U-12, U-23 and U-27 have significant inhibitory effects on the expression of URAT1. After screening 29 siRNA sequences, U-4, U-8, U-12, U-23 and U-27 showed better inhibition of URAT1 gene expression, among which U-4 can achieve an inhibitory effect of about 80%, and can be used as siRNA for the treatment of gout.

[0116] 2.2 IC50 determination results of siRNA and chemically modified siRNA

[0117] The concentrations of 0.005, 0.01, 0.05, 0.1, 0.2, 0.5, 1, 5, 25, 50, and 100 nM were selected to conduct IC50 test experiments on URAT1 protein knockdown of siRNA of U-4, U-8, U-12, U-23 (human and mouse homologous sequences), and U-27. At the same time, this experiment took U-4 and U-23 as examples, and chemically modified their skeletons and nucleosides to obtain U-4-M and U-23-M (specific sequences are shown in Table 2), and conducted IC50 test experiments on their efficacy in knocking down URAT1 protein levels.

[0118] Table 4 IC50 determination results of siRNA

[0119] siRNA U-4 U-8 U-12 U-23 U-27 U-4-M U-23-M IC50(nM) 0.458 0.494 0.685 0.433 0.470 0.301 0.514

[0120] According to Table 4 and Figure 2-8 Judging from the IC50 values ​​of the 7 siRNA sequences for protein knockdown, the data all reached the subnanomolar level.

Claims

1. An siRNA targeting urate transporter 1, characterized in that: The siRNA is selected from any one of the double-stranded RNAs described in (1) to (24): (1) a double-stranded RNA consisting of a sense strand having a nucleotide sequence as shown in SEQ ID No. 1 and an antisense strand having a nucleotide sequence as shown in SEQ ID No. 2; (2) a double-stranded RNA consisting of a sense strand having a nucleotide sequence as shown in SEQ ID No. 3 and an antisense strand having a nucleotide sequence as shown in SEQ ID No. 4; (3) a double-stranded RNA consisting of a sense strand having a nucleotide sequence of SEQ ID No. 5 and an antisense strand having a nucleotide sequence of SEQ ID No. 6; (4) a double-stranded RNA consisting of a sense strand having a nucleotide sequence of SEQ ID No. 7 and an antisense strand having a nucleotide sequence of SEQ ID No. 8; (5) a double-stranded RNA consisting of a sense strand having a nucleotide sequence of SEQ ID No. 9 and an antisense strand having a nucleotide sequence of SEQ ID No. 10; (6) a double-stranded RNA consisting of a sense strand having a nucleotide sequence as shown in SEQ ID No. 11 and an antisense strand having a nucleotide sequence as shown in SEQ ID No. 12; (7) a double-stranded RNA consisting of a sense strand having a nucleotide sequence as shown in SEQ ID No. 13 and an antisense strand having a nucleotide sequence as shown in SEQ ID No. 14; (8) a double-stranded RNA consisting of a sense strand having a nucleotide sequence as shown in SEQ ID No. 15 and an antisense strand having a nucleotide sequence as shown in SEQ ID No. 16; (9) a double-stranded RNA consisting of a sense strand having a nucleotide sequence as shown in SEQ ID No. 17 and an antisense strand having a nucleotide sequence as shown in SEQ ID No. 18; (10) a double-stranded RNA consisting of a sense strand having a nucleotide sequence of SEQ ID No. 19 and an antisense strand having a nucleotide sequence of SEQ ID No. 20; (11) a double-stranded RNA consisting of a sense strand having a nucleotide sequence of SEQ ID No. 21 and an antisense strand having a nucleotide sequence of SEQ ID No. 22; (12) a double-stranded RNA consisting of a sense strand having a nucleotide sequence as shown in SEQ ID No. 23 and an antisense strand having a nucleotide sequence as shown in SEQ ID No. 24; (13) a double-stranded RNA consisting of a sense strand having a nucleotide sequence of SEQ ID No. 25 and an antisense strand having a nucleotide sequence of SEQ ID No. 26; (14) a double-stranded RNA consisting of a sense strand having a nucleotide sequence as shown in SEQ ID No. 27 and an antisense strand having a nucleotide sequence as shown in SEQ ID No. 28; (15) a double-stranded RNA consisting of a sense strand having a nucleotide sequence of SEQ ID No. 29 and an antisense strand having a nucleotide sequence of SEQ ID No. 30; (16) a double-stranded RNA consisting of a sense strand having a nucleotide sequence of SEQ ID No. 31 and an antisense strand having a nucleotide sequence of SEQ ID No. 32; (17) a double-stranded RNA consisting of a sense strand having a nucleotide sequence as shown in SEQ ID No. 33 and an antisense strand having a nucleotide sequence as shown in SEQ ID No. 34; (18) a double-stranded RNA consisting of a sense strand having a nucleotide sequence of SEQ ID No. 35 and an antisense strand having a nucleotide sequence of SEQ ID No. 36; (19) a double-stranded RNA consisting of a sense strand having a nucleotide sequence of SEQ ID No. 37 and an antisense strand having a nucleotide sequence of SEQ ID No. 38; (20) a double-stranded RNA consisting of a sense strand having a nucleotide sequence of SEQ ID No. 39 and an antisense strand having a nucleotide sequence of SEQ ID No. 40; (21) a double-stranded RNA consisting of a sense strand having a nucleotide sequence of SEQ ID No. 41 and an antisense strand having a nucleotide sequence of SEQ ID No. 42; (22) a double-stranded RNA consisting of a sense strand having a nucleotide sequence as shown in SEQ ID No. 43 and an antisense strand having a nucleotide sequence as shown in SEQ ID No. 44; (23) a double-stranded RNA consisting of a sense strand having a nucleotide sequence of SEQ ID No. 45 and an antisense strand having a nucleotide sequence of SEQ ID No. 46; (24) a double-stranded RNA consisting of a sense strand having a nucleotide sequence of SEQ ID No. 47 and an antisense strand having a nucleotide sequence of SEQ ID No. 48; Preferably, the siRNA is selected from any one of the following siRNAs: An siRNA consisting of the sense RNA shown in SEQ ID No.5 and the antisense RNA shown in SEQ ID No.6; or an siRNA consisting of the sense RNA shown in SEQ ID No.13 and the antisense RNA shown in SEQ ID No.14; or an siRNA consisting of the sense RNA shown in SEQ ID No.17 and the antisense RNA shown in SEQ ID No.18; or an siRNA consisting of the sense RNA shown in SEQ ID No.33 and the antisense RNA shown in SEQ ID No.34; or an siRNA consisting of the sense RNA shown in SEQ ID No.41 and the antisense RNA shown in SEQ ID No.

42.

2. A siRNA, characterized in that The nucleotide sequence of the siRNA contains 15 or more consecutive nucleotide sequences identical to those in the siRNA sequence of claim 1.

3. A modified siRNA, characterized in that: The modified siRNA is a modified siRNA obtained by subjecting the siRNA according to claim 1 or 2 to any one of backbone modification, sugar ring modification and / or base modification; The backbone modification includes but is not limited to: thio-modification and / or amino-modification of the phosphate backbone of the sense strand RNA and the antisense strand RNA of the siRNA; The sugar ring modification includes but is not limited to: replacing the 2′-OH in the nucleotide with methoxy, methylethoxy and / or fluorine; or deoxygenating the 2′-OH in the nucleotide; or modifying the nucleotide in the siRNA to a peptide nucleic acid type, a locked nucleic acid type or an unlocked nucleic acid type; The base modification includes but is not limited to: base modification or substitution of 5-methylcytosine, 6-methyladenine, ribavirin, pseudouracil and / or inosine of the nucleoside base.

4. The siRNA according to claim 3, characterized in that The siRNA is selected from any one of the following (1) or (2): (1) siRNA consisting of the following sense strand and antisense strand: Sense strand: G m *G m *G m G m A m A m C f U m C f U m G f U m C f C m U m A m A m A m A m *dT*dT; Antisense strand: Phos-U m *U f *U f U f A m G f G m A f C m A f G m A f G m U f U m C f C m C m C m *dT*dT; (2) siRNA consisting of the following sense strand and antisense strand: Sense strand: C m *C m *U m U m U m G m G f C m U f U m C f A m C f C m U m U m C m U m U m *dT*dT; Sense strand: Phos-A m *A f *G f A f A m G f G m U f G m A f A m G f C m C f A m A f A m G m G m *dT*dT; Among them, m indicates that RNA is 2′-methoxy, f indicates that RNA is 2′-fluoro, * indicates phosphorothioate, dT indicates DNA thymine modification, and Phos indicates 5′ phosphorylation modification.

5. The siRNA according to claim 3, characterized in that The modified siRNA is obtained by modifying the siRNA of claim 1 or 2 in the following manner using any of the following modification methods: replacing the two unpaired continuous protruding base sequences TT at the 3' end of the forward / antisense strand of the siRNA with any other two nucleosides or deoxynucleosides and performing any of the following phosphorylation or phosphorylation analog modification on the 5' end of the antisense strand:

6. A shRNA or sdRNA, characterized in that The nucleotide sequence of the shRNA contains 15 or more consecutive nucleotide sequences identical to those of the siRNA sequence of claim 1; the nucleotide sequence of the sdRNA contains 15 or more consecutive nucleotide sequences identical to those of the siRNA sequence of claim 1.

7. A recombinant expression vector expressing the siRNA according to any one of claims 1 to 5, or a recombinant expression vector expressing the shRNA or sdRNA according to claim 6; preferably, the recombinant expression vector is a eukaryotic recombinant expression vector, and more preferably, the eukaryotic recombinant expression vector is a recombinant adenovirus expression vector.

8. A recombinant host cell containing the recombinant expression vector according to claim 7; preferably, the recombinant host cell is a mammalian cell.

9. Use of the siRNA according to any one of claims 1 to 5, the shRNA or sdRNA according to claim 6, the recombinant expression vector according to claim 7 or the recombinant host cell according to claim 8 in the preparation of a drug for treating hyperuricemia or gout.

10. A pharmaceutical composition for treating hyperuricemia or gout, characterized in that: The invention is composed of a therapeutically effective amount of an active ingredient and a carrier or excipient acceptable for pharmaceutical preparations; wherein the active ingredient is selected from the siRNA according to any one of claims 1 to 5, the shRNA or sdRNA according to claim 6, or the recombinant expression vector according to claim 7.

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