Sirna for inhibiting XDH, and modifier and use thereof

Abstract

The present invention belongs to the field of biotechnology, and relates to an siRNA for inhibiting XDH, and a modifier and the use thereof. Provided is the siRNA for inhibiting XDH. The siRNA contains a sense strand and an antisense strand, and the sense strand can be at least partially reversely complementary to the antisense strand to form a double-stranded region.

Description

siRNA for inhibiting XDH and its modified products and applications

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. 2023118202736, filed with the Chinese Patent Office on December 27, 2023, entitled “siRNA for inhibiting XDH, its modifications and applications,” the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present disclosure relates to siRNA for inhibiting XDH and its modified products and applications, and belongs to the field of biotechnology. Background Art

[0004] Reduced renal clearance of uric acid is a result of multiple factors, including defects in urate transporters such as SLC2A9 and ABCG2; decreased renal excretion due to renal disease, hypothyroidism, volume contraction and failure, acidosis, lead poisoning, and familial nephropathy caused by uromodulin deposition; and altered renal clearance due to hyperinsulinemia or insulin resistance in diabetes. Increased uric acid synthesis is associated with: hyperuricemia plus hyperuricosuria; inborn errors of metabolism such as Lesch Nyhan / HPRT deficiency, PRPP synthase overactivity, and glucose-6-phosphate dehydrogenase deficiency (Von Gierke disease / glycogen storage disease type Ia); certain conditions with high cell turnover (such as tumor lysis syndrome); and certain conditions with high ATP turnover (such as glycogen storage diseases, tissue ischemia). In addition, conditions such as chronic kidney disease, hypertension, metabolic syndrome, and high fructose intake may lead to increased uric acid synthesis and reduced uric acid clearance.

[0005] Gout is a progressive inflammatory arthritis caused by hyperuricemia (elevated serum uric acid levels) and the deposition of monosodium urate crystals in joints and tendons. It is estimated to affect 0.6% of the world's population, with much higher prevalence in certain geographic regions and ethnic groups. Gout patients who do not receive urate-lowering treatment experience recurrent gout flares (inflammatory responses) and eventually develop advanced gout, characterized by chronic joint pain and limited mobility.

[0006] Xanthine dehydrogenase (XDH) is a molybdenum-containing hydroxylase that catalyzes the production of uric acid from xanthine. XDH is highly expressed in the liver and gastrointestinal tract. Hepatocyte-specific ablation of XDH or global inhibition of XDH activity can reverse the hyperuricemia phenotype in animal models.

[0007] Small interfering RNA (siRNA), typically a double-stranded RNA of 20 to 25 nucleotides in length, specifically regulates gene expression through the RNA interference (RNAi) mechanism to treat disease. Therefore, developing an siRNA to inhibit XDH production would be an effective treatment for gout and its various complications.

[0008] Summary of the Invention

[0009] To solve the above problems, the present disclosure provides an siRNA for inhibiting XDH, wherein the siRNA contains a sense strand and an antisense strand, wherein the sense strand and the antisense strand are at least partially reverse-complementary to form a double-stranded region.

[0010] wherein, the sense strand comprises the nucleotide sequence shown in SEQ ID NO.1, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.2, or,

[0011] Wherein, the sense strand comprises the nucleotide sequence shown in SEQ ID NO.3, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.4, or,

[0012] Wherein, the sense strand comprises the nucleotide sequence shown in SEQ ID NO.5, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.6, or,

[0013] Wherein, the sense strand comprises the nucleotide sequence shown in SEQ ID NO.7, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.8, or,

[0014] wherein the sense strand comprises the nucleotide sequence shown in SEQ ID NO.9, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.10, or,

[0015] wherein the sense strand comprises the nucleotide sequence shown in SEQ ID NO.11, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.12, or,

[0016] wherein the sense strand comprises the nucleotide sequence shown in SEQ ID NO.13, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.14, or,

[0017] wherein the sense strand comprises the nucleotide sequence shown in SEQ ID NO.15, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.16, or,

[0018] wherein the sense strand comprises the nucleotide sequence shown in SEQ ID NO.17, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.18, or

[0019] wherein the sense strand comprises the nucleotide sequence shown in SEQ ID NO.19, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.20, or,

[0020] wherein the sense strand comprises the nucleotide sequence shown in SEQ ID NO.21, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.22, or,

[0021] wherein the sense strand comprises the nucleotide sequence shown in SEQ ID NO.23, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.24, or,

[0022] wherein the sense strand comprises the nucleotide sequence shown in SEQ ID NO.25, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.26, or,

[0023] wherein the sense strand comprises the nucleotide sequence shown in SEQ ID NO.27, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.28, or,

[0024] wherein the sense strand comprises the nucleotide sequence shown in SEQ ID NO.29, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.30, or,

[0025] wherein the sense strand comprises the nucleotide sequence shown in SEQ ID NO.31, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.32, or,

[0026] wherein the sense strand comprises the nucleotide sequence shown in SEQ ID NO.33, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.34, or,

[0027] wherein the sense strand comprises the nucleotide sequence shown in SEQ ID NO.35, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.36, or,

[0028] wherein the sense strand comprises the nucleotide sequence shown in SEQ ID NO.37, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.38, or,

[0029] wherein the sense strand comprises the nucleotide sequence shown in SEQ ID NO.39, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.40, or,

[0030] wherein the sense strand comprises the nucleotide sequence shown in SEQ ID NO.41, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.42, or,

[0031] The positive strand comprises the nucleotide sequence shown in SEQ ID NO.43, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.44.

[0032] In one embodiment of the present disclosure, taking the sense strand and antisense strand as shown in SEQ ID NO.1 and SEQ ID NO.2 as examples, the sense strand comprises: a nucleotide sequence as shown in SEQ ID NO.1 or a nucleotide sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity with the nucleotide sequence as shown in SEQ ID NO.1 and retains the biological function of the sequence from which it is derived, and the antisense strand comprises: a nucleotide sequence as shown in SEQ ID NO.2 or a nucleotide sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity with the nucleotide sequence as shown in SEQ ID NO.2 and retains the biological function of the sequence from which it is derived. The nucleotide sequences of the remaining sense strand and antisense strand are nucleotide sequences that have at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity and retain the biological function of the sequence from which it is derived.

[0033] The present disclosure also provides a product for inhibiting XDH, which comprises an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is the above-mentioned siRNA or the above-mentioned modified siRNA.

[0034] In one embodiment of the present disclosure, the product is a pharmaceutical composition or a kit.

[0035] The present disclosure also provides the use of the above siRNA or the above product in the preparation of a product for preventing, diagnosing and / or treating a pathological condition or disease caused by XDH.

[0036] In one embodiment of the present disclosure, the disease associated with XDH is gout.

[0037] The present disclosure also provides the use of the above-mentioned siRNA or the above-mentioned product in preventing, diagnosing and / or treating pathological conditions or diseases caused by XDH.

[0038] The technical solution disclosed in this disclosure has the following advantages:

[0039] The present disclosure provides siRNAs for inhibiting XDH. Experiments have shown that all siRNAs have high inhibitory activity against XDH. The present disclosure also provides modified siRNAs for inhibiting XDH, including methoxy modification, fluoro modification, phosphorothioate linkage, LNA modification, and methoxyethyl modification. The psiCHECK™-2 plasmid was used to construct and test the plasmids, and the results were determined to be highly inhibitory at a concentration of 10 nM against a sequence consisting of a sense strand as shown in SEQ ID NO. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, or 23 and an antisense strand as shown in SEQ ID NO. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24.

[0040] Further experiments were conducted on the chemically modified siRNAs, confirming their targeting relationship with the target gene fragment. The modified siRNAs also exhibited a strong XDH inhibitory effect at a concentration of 0.1 nM. Following GalNAc conjugation, single-dose in vivo validation in mice demonstrated that, at a dose of 9 mg / kg, the modified siRNAs chemically modified with the sense strand of SEQ ID NO. 15 and the antisense strand of SEQ ID NO. 16, as well as the modified siRNAs chemically modified with the sense strand of SEQ ID NO. 17 and the antisense strand of SEQ ID NO. 18, exhibited highly significant inhibitory effects (inhibition rates exceeding 70%). At a dose of 3 mg / kg, the modified siRNAs chemically modified with the sense strand of SEQ ID NO. 15 or 17 and the antisense strand of SEQ ID NO. 16 or 18, again exhibited highly significant inhibitory effects (inhibition rates exceeding 70% after 10 days).

[0041] The above siRNA or the above product has good application prospects in the preparation of products for preventing, diagnosing and / or treating pathological conditions or diseases caused by XDH. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1: On-target activity of unmodified sequences (XDH001UM to XDH022UM) used to inhibit XDH.

[0043] Figure 2: qPCR detection results of a single-dose study (9 mg / kg) of modified siRNA conjugates (RD34XDH007G, RD34XDH008G, RD34XDH009G and RD34XDH022G) for inhibiting XDH in mice.

[0044] Figure 3: qPCR detection results of a single-dose study (3 mg / kg) of modified siRNA conjugates (RD34XDH008G, RD34XDH009G, RD34XDH008-1G, RD34XDH008-2G, RD34XDH009-1G and RD34XDH009-2G) for inhibiting XDH in mice. DETAILED DESCRIPTION

[0045] In the following examples, XDH mRNA refers to mRNA having the sequences shown in GeneBank Accession Nos. NM_011723.3, NM_000379.4, and XM_005576183.3. Further, unless otherwise specified, the term "target gene" used in this disclosure refers to the gene that transcribes the above-mentioned XDH mRNA, and the term "target mRNA" refers to the above-mentioned XDH mRNA.

[0046] If no specific experimental steps or conditions are specified in the following examples, the experiments were carried out according to the conventional experimental steps or conditions described in the literature in the field. If no manufacturer is specified for the reagents or instruments used, they are all commercially available conventional reagents.

[0047] In the following examples, capital letters C, G, U, and A represent ribonucleotides; lowercase letter m indicates that the nucleotide adjacent to the left of letter m is a methoxy-modified nucleotide; lowercase letter f indicates that the nucleotide adjacent to the left of letter f is a fluorinated-modified nucleotide; lowercase letter s indicates that the nucleotides adjacent to the left and right of letter s are modified with a phosphorothioate group.

[0048] In the following examples, a "modified nucleotide" refers to a nucleotide or nucleotide analog in which the hydroxyl group at the 2' position of the ribose group of a nucleotide is replaced by another group, or a nucleotide in which the base on the nucleotide is a modified base. A "fluorinated nucleotide" refers to a nucleotide in which the hydroxyl group at the 2' position of the ribose group of a nucleotide is replaced by fluorine, and a "non-fluorinated nucleotide" refers to a nucleotide or nucleotide analog in which the hydroxyl group at the 2' position of the ribose group of a nucleotide is replaced by a non-fluorinated group. A "nucleotide analog" refers to a group that can replace a nucleotide in a nucleic acid but has a structure different from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine deoxyribonucleotide. Examples include isonucleotides, bridged nucleic acids (BNAs), or acyclic nucleotides. A "methoxy-modified nucleotide" refers to a nucleotide in which the 2'-hydroxyl group of the ribose group is replaced by a methoxy group.

[0049] In the following examples, the terms "complementary" and "reverse complementary" are used interchangeably and have the meanings known to those skilled in the art, i.e., in a double-stranded nucleic acid molecule, the bases of one strand pair with bases on the other strand in a complementary manner. In DNA, the purine base adenine (A) always pairs with the pyrimidine base thymine (T) (or uracil (U) in RNA); the purine base guanine (C) always pairs with the pyrimidine base cytosine (G). Each base pair consists of a purine and a pyrimidine. When adenine on one strand always pairs with thymine (or uracil) on the other strand, and guanine always pairs with cytosine, the two strands are considered to be complementary to each other, and the sequence of a strand can be inferred from the sequence of its complementary strand.

[0050] In the following embodiments, particularly when describing the preparation method of siRNA of the present disclosure, pharmaceutical composition or siRNA conjugate, unless otherwise stated, nucleoside monomer (nucleoside monomer) refers to, according to the kind and order of nucleotide in the siRNA for preparation or the siRNA conjugate, the modification used in the phosphoramidite solid phase synthesis or unmodified nucleoside phosphoramidite monomer (unmodified or modified RNA phosphoramidites, RNA phosphoramidites is also referred to as Nucleoside phosphoramidites sometimes).Phosphoramidite solid phase synthesis is the method used in RNA synthesis known to those skilled in the art.The nucleoside monomer used in the disclosure all can be commercially available.

[0051] In the following embodiments, "coupling" refers to the covalent attachment of two or more chemical moieties, each with a specific function, to one another; accordingly, "conjugate" refers to a compound formed by covalent attachment of these chemical moieties. Furthermore, "siRNA conjugate" refers to a compound formed by covalent attachment of one or more chemical moieties with a specific function to an siRNA. The term "siRNA conjugate" should be understood, depending on the context, as a general term for multiple siRNA conjugates or as a siRNA conjugate represented by a certain chemical formula. In the context of the present disclosure, a "conjugated molecule" should be understood as a specific compound that can be coupled to an siRNA through a reaction to ultimately form the siRNA conjugate of the present disclosure.

[0052] In the following embodiments, "optional" or "optionally" means that the event or situation described thereafter may or may not occur, and the description includes both instances where the event or situation occurs and instances where it does not occur. For example, "optionally substituted" "alkyl" includes "alkyl" and "substituted alkyl" as defined below. It will be understood by those skilled in the art that for any group containing one or more substituents, these groups are not intended to introduce any substitution or substitution pattern that is sterically impractical, synthetically unfeasible, and / or inherently unstable.

[0053] In the following examples, "treating," "alleviating," or "improving" are used interchangeably herein. These terms refer to an approach for obtaining beneficial or desired results, including but not limited to a therapeutic benefit. "Therapeutic benefit" means eradication or amelioration of the underlying disorder being treated. Furthermore, a therapeutic benefit is achieved by eradication or amelioration of one or more physiological symptoms associated with the underlying disorder, such that an improvement is observed in the subject, even though the subject may still be afflicted with the underlying disorder.

[0054] In the following examples, "prevent" and "prevent" are used interchangeably. These terms refer to an approach to obtaining a beneficial or desired result, including but not limited to a prophylactic benefit. To obtain a "prophylactic benefit," a composition can be administered to a subject at risk for a particular disease, or to a subject reporting one or more pathological symptoms of a disease, even though a diagnosis of the disease may not have yet been made.

[0055] To solve the above problems, the present disclosure provides an siRNA for inhibiting XDH, wherein the siRNA contains a sense strand and an antisense strand, wherein the sense strand and the antisense strand are at least partially reverse-complementary to form a double-stranded region.

[0056] wherein, the sense strand comprises the nucleotide sequence shown in SEQ ID NO.1, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.2, or,

[0057] Wherein, the sense strand comprises the nucleotide sequence shown in SEQ ID NO.3, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.4, or,

[0058] Wherein, the sense strand comprises the nucleotide sequence shown in SEQ ID NO.5, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.6, or,

[0059] Wherein, the sense strand comprises the nucleotide sequence shown in SEQ ID NO.7, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.8, or,

[0060] wherein the sense strand comprises the nucleotide sequence shown in SEQ ID NO.9, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.10, or,

[0061] wherein the sense strand comprises the nucleotide sequence shown in SEQ ID NO.11, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.12, or,

[0062] wherein the sense strand comprises the nucleotide sequence shown in SEQ ID NO.13, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.14, or,

[0063] wherein the sense strand comprises the nucleotide sequence shown in SEQ ID NO.15, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.16, or,

[0064] wherein the sense strand comprises the nucleotide sequence shown in SEQ ID NO.17, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.18, or

[0065] wherein the sense strand comprises the nucleotide sequence shown in SEQ ID NO.19, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.20, or,

[0066] wherein the sense strand comprises the nucleotide sequence shown in SEQ ID NO.21, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.22, or,

[0067] wherein the sense strand comprises the nucleotide sequence shown in SEQ ID NO.23, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.24, or,

[0068] wherein the sense strand comprises the nucleotide sequence shown in SEQ ID NO.25, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.26, or,

[0069] wherein the sense strand comprises the nucleotide sequence shown in SEQ ID NO.27, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.28, or,

[0070] wherein the sense strand comprises the nucleotide sequence shown in SEQ ID NO.29, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.30, or,

[0071] wherein the sense strand comprises the nucleotide sequence shown in SEQ ID NO.31, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.32, or,

[0072] wherein the sense strand comprises the nucleotide sequence shown in SEQ ID NO.33, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.34, or,

[0073] wherein the sense strand comprises the nucleotide sequence shown in SEQ ID NO.35, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.36, or,

[0074] wherein the sense strand comprises the nucleotide sequence shown in SEQ ID NO.37, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.38, or,

[0075] wherein the sense strand comprises the nucleotide sequence shown in SEQ ID NO.39, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.40, or,

[0076] Wherein, the sense strand comprises the nucleotide sequence shown in SEQ ID NO.41, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.42, or,

[0077] The positive strand comprises the nucleotide sequence shown in SEQ ID NO.43, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.44.

[0078] In one embodiment of the present disclosure, taking the sense strand and antisense strand as shown in SEQ ID NO.1 and SEQ ID NO.2 as examples, the sense strand comprises: a nucleotide sequence as shown in SEQ ID NO.1 or a nucleotide sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity with the nucleotide sequence as shown in SEQ ID NO.1 and retains the biological function of the sequence from which it is derived, and the antisense strand comprises: a nucleotide sequence as shown in SEQ ID NO.2 or a nucleotide sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity with the nucleotide sequence as shown in SEQ ID NO.2 and retains the biological function of the sequence from which it is derived. The nucleotide sequences of the remaining sense strand and antisense strand are nucleotide sequences that have at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity and retain the biological function of the sequence from which it is derived.

[0079] In one embodiment of the present disclosure, siRNA is prepared by solid phase synthesis or liquid phase synthesis.

[0080] In one embodiment of the present disclosure, the nucleotides in the siRNA are each independently a modified or unmodified nucleotide.

[0081] In one embodiment of the present disclosure, each nucleotide in the siRNA is an unmodified nucleotide.

[0082] In one embodiment of the present disclosure, some or all of the nucleotides in the siRNA are modified nucleotides, and these modifications on the nucleotide groups do not significantly weaken or lose the function of the siRNA of the present disclosure in inhibiting XDH gene expression.

[0083] In one embodiment of the present disclosure, at least one nucleotide in the sense strand or the antisense strand of the siRNA is a modified nucleotide.

[0084] In one embodiment of the present disclosure, at least one phosphate group in the sense strand or the antisense strand of the siRNA is a phosphate group having a modified group.

[0085] In one embodiment of the present disclosure, at least a portion of the phosphate group and / or ribose group in the phosphate-sugar backbone of at least one single strand in the sense strand and the antisense strand of the siRNA is a phosphate group having a modified group and / or a ribose group having a modified group.

[0086] In one embodiment of the present disclosure, all nucleotides in the sense strand and / or antisense strand of the siRNA are modified nucleotides, and these modifications on the nucleotide groups do not significantly weaken or lose the function of the siRNA of the present disclosure in inhibiting XDH gene expression.

[0087] In one embodiment of the present disclosure, each nucleotide in the sense strand and the antisense strand of the siRNA is independently a fluorine-modified nucleotide or a non-fluorine-modified nucleotide.

[0088] In one embodiment of the present disclosure, the modification is a chemical modification, and the chemical modification is selected from one or more of methoxy modification, fluorination modification, or phosphorothioate linkage.

[0089] In one embodiment of the present disclosure, a "fluorinated nucleotide" refers to a nucleotide in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is substituted with fluorine, and has a structure represented by the following formula (1). Non-fluorinated nucleotides are independently selected from nucleotides or nucleotide analogs in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is substituted with a non-fluorinated group.

[0090] In one embodiment of the present disclosure, nucleotides in which the hydroxyl group at the 2' position of the ribose group is replaced by a non-fluorinated group are well known to those skilled in the art, and these nucleotides can be selected from 2'-alkoxy-modified nucleotides, 2'-substituted alkoxy-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-substituted alkyl-modified nucleotides, 2'-amino-modified nucleotides, 2'-substituted amino-modified nucleotides, and 2'-deoxynucleotides.

[0091] In one embodiment of the present disclosure, the 2'-alkoxy modified nucleotide is a 2'-methoxy (2'-OMe) modified nucleotide, as shown in formula (2); the 2'-substituted alkoxy modified nucleotide may be, for example, a 2'-O-methoxyethyl (2'-MOE) modified nucleotide, as shown in formula (3); the 2'-amino (2'-NH2) modified nucleotide is as shown in formula (4); and the 2'-deoxynucleotide (DNA) is as shown in formula (5):

[0092] In one embodiment of the present disclosure, the fluorinated modified nucleotides are located in the antisense strand and the sense strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the 7th, 8th, and 9th nucleotides of the sense strand are fluorinated modified nucleotides, and at least the 2nd, 6th, 14th, and 16th nucleotides of the antisense strand are fluorinated modified nucleotides.

[0093] In one embodiment of the present disclosure, the fluorinated-modified nucleotides are located in the sense strand and the antisense strand, the number of fluorinated-modified nucleotides in the sense strand is no more than 5, and, in the direction from the 5' end to the 3' end, at least the 7th, 8th, and 9th nucleotides of the sense strand are fluorinated-modified nucleotides, the number of fluorinated-modified nucleotides in the antisense strand is no more than 7, and, at least the 2nd, 6th, 14th, and 16th nucleotides of the antisense strand are fluorinated-modified nucleotides.

[0094] In one embodiment of the present disclosure, in the direction from the 5' end to the 3' end, in the sense chain, the nucleotides at positions 7, 8, and 9 of the sense chain are fluorinated-modified nucleotides, and the nucleotides at the remaining positions in the sense chain are non-fluorinated-modified nucleotides; in the antisense chain, the nucleotides at positions 2, 6, 14, and 16 of the antisense chain are fluorinated-modified nucleotides, and the nucleotides at the remaining positions in the antisense chain are non-fluorinated-modified nucleotides.

[0095] In one embodiment of the present disclosure, the methoxy-modified nucleotides are located in the antisense strand and the sense strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, and 21st nucleotides of the sense strand are methoxy-modified nucleotides, and at least the 1st, 3rd, 4th, 5th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 15th, 17th, 18th, 19th, 20th, and 23rd nucleotides of the antisense strand are methoxy-modified nucleotides.

[0096] In one embodiment of the present disclosure, the methoxy-modified nucleotides are located in the antisense strand and the sense strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the 1st, 2nd, 3rd, 4th, 5th, 6th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, and 19th nucleotides of the sense strand are methoxy-modified nucleotides, and at least the 1st, 3rd, 4th, 5th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 15th, 17th, 18th, 19th nucleotides of the antisense strand are methoxy-modified nucleotides.

[0097] In one embodiment of the present disclosure, the methoxyethyl-modified nucleotide is located in the sense strand of the nucleotide sequence, and, from the 5' end to the 3' end, at least the first nucleotide of the sense strand is a methoxyethyl-modified nucleotide.

[0098] In one embodiment of the present disclosure, a nucleotide analog refers to a group that can replace a nucleotide in a nucleic acid but has a structure different from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide or thymine deoxyribonucleotide.

[0099] In one embodiment of the present disclosure, the nucleotide analog may be an isonucleotide, a bridged nucleotide, or an acyclic nucleotide.

[0100] In one embodiment of the present disclosure, a bridged nucleic acid (BNA) refers to a constrained or inaccessible nucleotide. BNA may contain a five-membered ring, a six-membered ring, or a seven-membered ring with a "fixed" C3'-endo sugar condensation bridge structure. The bridge is usually incorporated into the 2'- and 4'-positions of the ribose to provide a 2',4'-BNA nucleotide.

[0101] In one embodiment of the present disclosure, the BNA may be an LNA, an ENA, a cET BNA, etc., wherein the LNA is as shown in formula (6), the ENA is as shown in formula (7), and the cET BNA is as shown in formula (8):

[0102] In one embodiment of the present disclosure, at least a portion of the phosphate groups in the phosphate-sugar backbone of at least one single strand of the sense strand and the antisense strand of the siRNA is a phosphate group having a modified group.

[0103] In one embodiment of the present disclosure, the phosphate group having a modified group is a phosphorothioate group formed by replacing at least one oxygen atom in the phosphodiester bond in the phosphate group with a sulfur atom.

[0104] In one embodiment of the present disclosure, the phosphate group having a modifying group is a thiophosphate group having a structure as shown in formula (9):

[0105] In one embodiment of the present disclosure, the phosphorothioate linkage is present at at least one of the following positions: between the first and second nucleotides at either end of the sense strand or the antisense strand; between the second and third nucleotides at either end of the sense strand or the antisense strand; or any combination thereof.

[0106] In one embodiment of the present disclosure, phosphorothioate linkages are present at all of the aforementioned positions except the 5' end of the sense strand.

[0107] In one embodiment of the present disclosure, phosphorothioate linkages are present at all of the aforementioned positions except the 3' end of the sense strand.

[0108] In one embodiment of the present disclosure, the phosphorothioate linkage is present in at least one of the following positions:

[0109] Between the first and second nucleotides at the 5' end of the sense strand;

[0110] Between the second and third nucleotides at the 5' end of the sense strand;

[0111] Between the first and second nucleotides at the 3' end of the sense strand;

[0112] Between the second and third nucleotides at the 3' end of the sense strand;

[0113] between the first and second nucleotides at the 5' end of the antisense strand;

[0114] between the second and third nucleotides from the 5' end of the antisense strand;

[0115] Between the first and second nucleotides from the 3' end of the antisense strand; and

[0116] Between the second and third nucleotides from the 3' end of the antisense strand.

[0117] In one embodiment of the present disclosure, nucleotides linked by thiophosphate groups are located in the antisense strand and the sense strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the 1st and 2nd positions, and the 2nd and 3rd positions of the sense strand are nucleotides linked by thiophosphate groups; and in the direction from the 5' end to the 3' end, at least the 1st and 2nd positions, the 2nd and 3rd positions, the 19th and 20th positions, and the 20th and 21st positions of the antisense strand are nucleotides linked by thiophosphate groups.

[0118] In one embodiment of the present disclosure, siRNA incorporates modified nucleotides by using nucleomonomers with corresponding modifications.

[0119] The present disclosure also provides a product for inhibiting XDH, which comprises an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is the above-mentioned siRNA or the above-mentioned modified siRNA.

[0120] In one embodiment of the present disclosure, the product is a pharmaceutical composition or a kit.

[0121] In one embodiment of the present disclosure, the product is a pharmaceutical composition, and the pharmaceutically acceptable carrier can be a carrier conventionally used in the field of siRNA administration, such as, but not limited to, magnetic nanoparticles (such as nanoparticles based on Fe3O4 or Fe2O3), carbon nanotubes, mesoporous silicon, calcium phosphate nanoparticles, polyethylenimine (PEI), polyamidoamine (PAMAM) dendrimer, poly(L-lysine, PLL), chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), poly (D&L-lactic / glycolic acid) copolymer (PLGA), poly (2-aminoethyl ethylene phosphate), and the like. phosphate), PPEEA) and poly (methacrylate-N, N-dimethylaminoethyl ester) (poly (2-dimethylaminoethylmethacrylate), PDMAEMA) and one or more of their derivatives.

[0122] In one embodiment of the present disclosure, there is no particular requirement for the content of siRNA and pharmaceutically acceptable carriers, and the conventional content of each component can be used.

[0123] In one embodiment of the present disclosure, in the pharmaceutical composition, the weight ratio of the active ingredient to the pharmaceutically acceptable carrier is 1:(1-500).

[0124] In one embodiment of the present disclosure, in the pharmaceutical composition, the weight ratio of the active ingredient to the pharmaceutically acceptable carrier is 1:(1-50).

[0125] In one embodiment of the present disclosure, the pharmaceutical composition may further comprise other pharmaceutically acceptable excipients, which may be one or more of various preparations or compounds conventionally used in the art.

[0126] In one embodiment of the present disclosure, other pharmaceutically acceptable excipients may include at least one of a pH buffer, a protective agent, and an osmotic pressure regulator.

[0127] In one embodiment of the present disclosure, the pH buffer may be a tris hydrochloride buffer with a pH value of 7.5 to 8.5 and / or a phosphate buffer with a pH value of 5.5 to 8.5.

[0128] In one embodiment of the present disclosure, the protective agent may be at least one of inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose and glucose.

[0129] In one embodiment of the present disclosure, the content of the protective agent may be 0.01 to 30 weight % based on the total weight of the pharmaceutical composition.

[0130] In one embodiment of the present disclosure, the osmotic pressure regulator may be sodium chloride and / or potassium chloride.

[0131] In one embodiment of the present disclosure, the content of the osmotic pressure regulator makes the osmotic pressure of the pharmaceutical composition 200-700 milliosm / liter (mOSM / L). According to the required osmotic pressure, those skilled in the art can easily determine the content of the osmotic pressure regulator.

[0132] In one embodiment of the present disclosure, the pharmaceutical composition may be a liquid preparation, such as an injection; or it may be a lyophilized powder injection, which is mixed with a liquid excipient to prepare a liquid preparation during administration.

[0133] In one embodiment of the present disclosure, the liquid preparation can be used for, but is not limited to, subcutaneous, intramuscular or intravenous administration, and can also be administered to the lungs by spray, or administered to other organs (such as the liver) through the lungs by spray.

[0134] In one embodiment of the present disclosure, the pharmaceutical composition is for intravenous administration.

[0135] In one embodiment of the present disclosure, the pharmaceutical composition may be in the form of a liposomal formulation.

[0136] In one embodiment of the present disclosure, the pharmaceutically acceptable carrier used in the liposome formulation comprises an amine-containing transfection compound (hereinafter also referred to as an organic amine), a helper lipid and / or a PEGylated lipid.

[0137] In one embodiment of the present disclosure, the organic amine, the helper lipid and the PEGylated lipid can be selected from one or more of the amine-containing transfection compounds or pharmaceutically acceptable salts or derivatives thereof, the helper lipid and the PEGylated lipid described in CN108220295B (incorporated herein by reference in its entirety).

[0138] In one embodiment of the present disclosure, the pharmaceutically acceptable targeting group in the siRNA conjugate can be galactose or N-acetylgalactosamine (GalNAc). N-acetylgalactosamine is a ligand that binds to the asialoglycoprotein receptor (ASGPR) on the liver surface. The asialoglycoprotein receptor is an endocytic receptor specifically expressed by hepatocytes. N-acetylgalactosamine serves as a targeting molecule to deliver small RNA to the liver.

[0139] In one embodiment of the present disclosure, the galactose or N-acetylgalactosamine molecule can be monovalent, divalent, trivalent, or tetravalent; monovalent, divalent, trivalent, and tetravalent respectively refer to that after the siRNA molecule forms an siRNA conjugate with a coupling group containing a galactose or N-acetylgalactosamine molecule as a targeting group, the molar ratio of the siRNA molecule to the galactose or N-acetylgalactosamine molecule in the siRNA conjugate is 1:1, 1:2, 1:3, or 1:4.

[0140] In one embodiment of the present disclosure, when siRNA is coupled to a coupling group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent or tetravalent.

[0141] In one embodiment of the present disclosure, when siRNA is conjugated to a coupling group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent.

[0142] In one embodiment of the present disclosure, the targeting group can be connected to the siRNA molecule via a suitable linker. Those skilled in the art can select a suitable linker according to the specific type of the targeting group.

[0143] In one embodiment of the present disclosure, the types of linkers and targeting groups and the connection methods with siRNA can be found in WO2015006740A2, the entire contents of which are incorporated herein by reference.

[0144] In one embodiment of the present disclosure, the siRNA conjugate formed by GalNAc and siRNA molecules has a structure as shown in the following formula (10):

[0145] In one embodiment of the present disclosure, the kit further comprises a pharmaceutically acceptable carrier and / or excipient.

[0146] In one embodiment of the present disclosure, the siRNA, pharmaceutically acceptable carrier and / or excipient in the kit may exist alone, in the form of a mixture of two or more thereof, or in the form of a final pharmaceutical composition.

[0147] In one embodiment of the present disclosure, in the kit, the pharmaceutically acceptable carrier is an amine-containing compound, a helper lipid, and a PEGylated lipid.

[0148] In one embodiment of the present disclosure, in the kit, the pharmaceutically acceptable carrier is a mixture or exists independently.

[0149] In one embodiment of the present disclosure, the siRNA, pharmaceutically acceptable carrier and / or excipients in the kit are provided in liquid form, dry form or lyophilized form.

[0150] In one embodiment of the present disclosure, the siRNA, pharmaceutically acceptable carriers and / or excipients in the kit are substantially pure and / or sterile.

[0151] In one embodiment of the present disclosure, the kit comprises a container for providing siRNA, one or more containers for providing an amine-containing compound, a helper lipid, and a PEGylated lipid, and optionally, a container for providing an excipient.

[0152] In one embodiment of the present disclosure, the kit further comprises one or more components necessary or beneficial for a specific application, the components being selected from:

[0153] one or more components for achieving the desired cell transfection;

[0154] One or more components used to achieve the diagnosis, treatment, or prevention of a specific disease or disorder;

[0155] one or more buffers;

[0156] Positive or negative control samples;

[0157] excipients, stabilizers or preservatives.

[0158] In one embodiment of the present disclosure, one or more components used to achieve diagnosis, treatment or prevention of a particular disease or disorder are one or more additional therapeutic compounds or compositions, one or more diagnostic agents.

[0159] In one embodiment of the present disclosure, the kit further comprises one or more of sterile water, physiological saline and PBS.

[0160] The present disclosure also provides the use of the above siRNA or the above product in the preparation of a product for preventing, diagnosing and / or treating a pathological condition or disease caused by XDH.

[0161] In one embodiment of the present disclosure, the disease associated with XDH is gout.

[0162] The present disclosure also provides the use of the above-mentioned siRNA or the above-mentioned product in preventing, diagnosing and / or treating pathological conditions or diseases caused by XDH.

[0163] Unless otherwise specified, the reagents and culture media used in the following examples are commercially available products, and the nucleic acid electrophoresis, real-time PCR and other operations used are performed with reference to the methods described in Molecular Biology (4th Edition) (Alexander McLennan et al., 2019).

[0164] The experimental cells involved in the following examples are 293T, A549, Hela, Hep3B, and SK-BR3, which were purchased from Frontier Biopharmaceuticals (Nanjing) Co., Ltd.

[0165] The siRNA involved in the following examples is a siRNA sequence synthesized by phosphoramidite solid phase.

[0166] The insertion sequences involved in the following examples are DNA sequences custom-synthesized by Suzhou GeneGene Co., Ltd., specifically:

[0167] For transfection of cells with the siRNAs and siRNA conjugates targeting the XDH gene, or the negative control siRNAs and siRNA conjugates described in the following examples, Lipofectamine 2000 (purchased from Invitrogen) was used as the transfection reagent, and specific procedures were performed according to the manufacturer's instructions. For qPCR detection, HiScript III RT SuperMix for qPCR (purchased from Vazyme) was used as the reverse transcription reagent, and specific procedures were performed according to the manufacturer's instructions.

[0168] Unless otherwise stated, the reagent ratios provided below are calculated by volume (v / v).

[0169] Example 1: A siRNA for inhibiting XDH

[0170] This example provides an siRNA for inhibiting XDH. The nucleotide sequence of the siRNA is designed based on the target mRNA, as shown in Table 1. The siRNA molecule with the following sequence was synthesized by Suzhou Genetron Health Co., Ltd.

[0171] Table 1. Nucleotide sequences of siRNAs that inhibit XDH

[0172] Experimental Example 1: On-target activity detection of unmodified siRNA for inhibiting XDH

[0173] This experimental example provides an on-target activity assay for unmodified siRNAs used to inhibit XDH. The assay uses the psiCHECK2 vector, a plasmid construct that can monitor changes in the expression of a target gene fused to a reporter gene. The psiCHECK2 vector uses Renilla luciferase as the primary reporter gene. The target fragment is cloned into the multiple cloning site downstream of the translation stop codon of Renilla luciferase. RNAi targeting the target gene, triggered by the synthesized siRNA, results in cleavage and subsequent degradation of the fusion mRNA. Changes in Renilla luciferase activity can be used to determine whether the siRNA and target gene fragment are on-target. The experimental procedure is as follows:

[0174] Step 1: Construct detection plasmid XDH-psiCHECK2

[0175] Using psiCHECK TM -2(Promega TM ) plasmid construction test plasmid, the test plasmid contains the insertion sequence shown in SEQ ID NO: 45, the insertion sequence is obtained by splicing the target sequence that is completely complementary to all nucleotide sequences in the antisense strand of the siRNA shown in Table 1, and a single copy of the spliced ​​sequence is cloned into psiCHECK TM -2 plasmid Xho I / Not I site, to obtain the detection plasmid XDH-psiCHECK2;

[0176] Step 2: Cell culture and transfection

[0177] siRNA was added to a 96-well plate at a volume of 5 μL per well, Opti-MEM containing 20 ng of XDH-psiCHECK2 detection plasmid was added at a volume of 12.5 μL per well, Opti-MEM (Gibco) was added at a volume of 32.5 μL per well, and Lipofectamine 2000 (purchased from Invitrogen, catalog number 11668-019) was added at a volume of 0.3 μL per well, and then incubated at room temperature (22°C) for 15 minutes to obtain a mixture. To the above mixture, a solution containing 1×10 4 293T cells were cultured in complete DMEM medium (purchased from Transgen Biotech, catalog number FI101-01) at 37°C for 24 hours for subsequent dual-luciferase assays. Single-dose experiments were performed at a final siRNA concentration of 10 nM.

[0178] Step 3: Dual luciferase assay

[0179] The 5× lysis buffer in the dual-luciferase assay kit (purchased from Promega, catalog number E2940) was diluted with water to 1× lysis buffer. Take the cells obtained by culturing in step 2, discard the supernatant, dilute and wash twice with PBS buffer (purchased from Hyclone, product number SH30256.01) per well, add 1× lysis buffer to each cell plate at a volume of 50 μL per well, and lyse at room temperature (22°C) for 20 minutes to obtain a lysed cell plate; 30 μL / well of lysate was respectively aspirated from the lysed cell plate and added to an opaque 96-well detection plate, take a dual luciferase detection kit, prepare substrate 1 and substrate 2 according to the instructions, and add substrate 1 and substrate 2 to the opaque 96-well detection plate at a volume of 30 μL per well, respectively, and detect with a multifunctional microplate reader after each addition of the substrate to obtain the numerical results of firefly luciferase and Renilla luciferase, respectively.

[0180] The luminescence ratio of each well on the ELISA plate was calculated as Renilla / Firefly. The luminescence ratio of each test or control group was the average of the luminescence ratios of three culture wells. The luminescence ratio of each test group was normalized to the luminescence ratio of the control group. The ratio R (luminescence ratio (test) / luminescence ratio (control)) was calculated to represent the expression level of the Renilla reporter gene, i.e., the relative residual activity. The inhibition rate of siRNA was calculated as (1-R) ​​× 100%.

[0181] The on-target activity results of the 22 siRNAs are shown in FIG1 . It can be seen that all 22 siRNAs have high inhibitory activity, and except for XDH004UM, XDH014UM, and XDH020UM, the inhibition rates are all greater than 80%.

[0182] Example 2: A modified siRNA for inhibiting XDH

[0183] This embodiment provides a modified siRNA for inhibiting XDH. Based on the results of Experimental Example 1, XDH001UM~XDH003UM, XDH005UM~XDH0012UM, and XDH015UM~XDH022UM in Example 1 were modified, and the sequences were allosterically modified to obtain the corresponding modified siRNAs: RD34XDH001~RD34XDH003, RD34XDH005~RD34XDH012, and RD34XDH015~RD34XDH022.

[0184] The positive strands of RD34XDH001 to RD34XDH003 and RD34XDH005 to RD34XDH007 are obtained by chemically modifying the sequences selected from SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.9, SEQ ID NO.11, and SEQ ID NO.13, respectively. In the direction from the 5' end to the 3' end, the sequences from positions 1 to 19 are retained, the nucleotides at positions 1 and 2, and at positions 2 and 3 are linked by thiophosphate groups, the nucleotides at positions 1, 2, 3, 4, 5, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19 are methoxy-modified nucleotides, and the nucleotides at positions 7, 8, and 9 are fluorine-modified nucleotides. The antisense strands of RD34XDH001 to RD34XDH003 and RD34XDH005 to RD34XDH007 are obtained by chemically modifying the sequences selected from SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 10, SEQ ID NO. 12, and SEQ ID NO. 14, respectively. From the 5' end to the 3' end, the nucleotides at positions 1 and 2, positions 2 and 3, positions 19 and 20, and positions 20 and 21 are linked by thiophosphate groups, the nucleotides at positions 1, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, and 21 are methoxy-modified nucleotides, and the nucleotides at positions 2, 6, 14, and 16 are fluorine-modified nucleotides.

[0185] The positive strands of RD34XDH008 to RD34XDH012 and RD34XDH015 to RD34XDH018 are obtained by chemically modifying the sequences selected from SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, and SEQ ID NO. 35, respectively. From the 5' end to the 3' end, the nucleotides at positions 1 and 2, and at positions 2 and 3 are linked by phosphorothioate groups, the nucleotides at positions 1, 2, 3, 4, 5, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19 are methoxy-modified nucleotides, and the nucleotides at positions 7, 8, and 9 are fluorine-modified nucleotides. The antisense strands of RD34XDH008 to RD34XDH012 and RD34XDH015 to RD34XDH018 are obtained by chemically modifying the sequences selected from SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, and SEQ ID NO. 36, respectively. From the 5' end to the 3' end, the nucleotides at positions 1 and 2, positions 2 and 3, positions 19 and 20, and positions 20 and 21 are linked by phosphorothioate groups, the nucleotides at positions 1, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, and 21 are methoxy-modified nucleotides, and the nucleotides at positions 2, 6, 14, and 16 are fluorine-modified nucleotides.

[0186] The positive strands of RD34XDH019 to RD34XDH022 are obtained by chemically modifying the sequences selected from SEQ ID NO.37, SEQ ID NO.39, SEQ ID NO.41, and SEQ ID NO.43, respectively. From the 5' end to the 3' end, the nucleotides at positions 1 and 2, and 2 and 3 are nucleotides linked by thiophosphate groups, the nucleotides at positions 1, 2, 3, 4, 5, 6, 7, 8, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 are methoxy-modified nucleotides, and the nucleotides at positions 9, 10, and 11 are fluorine-modified nucleotides. The antisense strands of RD34XDH019 to RD34XDH022 are obtained by chemically modifying the sequences selected from SEQ ID NO.38, SEQ ID NO.40, SEQ ID NO.42, SEQ ID NO. The sequence shown in NO.44 is obtained after chemical modification, in the direction from 5' end to 3' end, the nucleotides at positions 1 and 2, positions 2 and 3, positions 21 and 22, and positions 22 and 23 are nucleotides linked by thiophosphate groups, the nucleotides at positions 1, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, and 23 are methoxy-modified nucleotides, and the nucleotides at positions 2, 6, 14, and 16 are fluorine-modified nucleotides.

[0187] Experimental Example 2: Detection of on-target activity of modified siRNA for inhibiting XDH

[0188] This experimental example provides an on-target activity detection experiment for modified siRNA used to inhibit FGL1. Based on Experimental Example 1, steps 1 to 3 are retained, and the 22 siRNAs described in Example 1 in step 2 are replaced with the 18 modified siRNAs described in Example 2. The single-dose experiment is performed at final siRNA concentrations of 10 nM, 1 nM, and 0.1 nM.

[0189] The residual activity results of 18 siRNAs in Hep3B cells are shown in Table 2. It can be seen that the modified siRNAs all have a high inhibitory effect. RD34XDH001, RD34XDH002, RD34XDH005, RD34XDH006, RD34XDH007, RD34XDH008, RD34XDH009, RD34XDH010, RD34XDH011, RD34XDH012, RD34XDH015, RD34XDH016, RD34XDH017 and RD34XDH022 have a good inhibitory effect at a concentration of 0.1 nM.

[0190] Table 2. On-target activity of modified siRNAs

[0191] Experimental Example 3: Single-dose study of modified siRNA in mice (9 mg / kg)

[0192] This experimental example provides a single-dose study of modified siRNA in mice at a dose level of 9 mg / kg. The experimental procedure is as follows:

[0193] Alnylam Pharmaceuticals, Inc. first reported that siRNA based on GalNAc conjugation technology exerted interference activity in mice (Nair et al., J. Am. Chem. Soc., 2014, 136, 16958-16961). The literature reported that siRNA conjugated to three clusters of GalNAc showed good delivery activity in both in vivo and in vitro experiments. Referring to the preparation method in the above literature, according to the results of Experimental Example 2, the siRNA described in Example 2 was conjugated with GalNAc. The GalNAc-conjugated siRNAs RD34XDH007, RD34XDH008, RD34XDH009, and RD34XDH022 were obtained. Four C57BL / 6J mice (female, 6-8 weeks) in each group were subcutaneously administered a single dose of 9 mg / kg of GalNAc-conjugated siRNA or saline as a control. The administration requirements are shown in Table 3.

[0194] Table 3. 9 mg / kg single dose test administration requirements

[0195] Seven days after administration, the mice were sacrificed, liver samples were collected and snap-frozen in liquid nitrogen, and liver mRNA was extracted and analyzed by RT-qPCR. The RT-qPCR detection steps were as follows:

[0196] Step 1: RNA extraction:

[0197] 1) 20 mg of mouse liver tissue was collected and lysed by adding 1 mL of Trizol Lysis Buffer (Life Technologies, Cat. No. 410701). The tissue was then ground and lysed. The completely dissolved mixture was transferred to an RNase-free 1.5 mL centrifuge tube. The mixture was shaken vigorously for approximately 15 seconds to fully lyse the tissue cells and allowed to stand at room temperature (25°C) for 5 minutes.

[0198] 2) Carefully open the tube cap and add 200 μL of chloroform (purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd., catalog number 20140925); shake vigorously for 20 seconds, let stand at room temperature (25°C) for 3 minutes; and centrifuge at 4°C, 12,000 × g, for 20 minutes.

[0199] 3) After centrifugation, carefully remove the centrifuge tube to a centrifuge tube rack, aspirate the supernatant aqueous phase into a new 2.0 mL centrifuge tube, add 1.5 times the volume of anhydrous ethanol (purchased from Jiangsu Qiangsheng Functional Chemical Co., Ltd., catalog number 20210802) of the supernatant aqueous phase, and mix by inversion.

[0200] 4) Add 700 μL of the mixture from step 3) to a purification column with a collection tube (purchased from VWI, catalog number 11822AG0627). Let stand for 2 minutes. Centrifuge at 4°C, 10,000 × g, for 1 minute, and discard the filtrate. Repeat the above steps with the remaining mixture.

[0201] 5) Add 700 μL of 80% (v / v) ethanol to the purification column, centrifuge at 10,000 × g at 4°C for 1 min, and discard the filtrate;

[0202] 6) Add 700 μL of 80% (v / v) ethanol to the purification column, centrifuge at 10,000 × g at 4°C for 1 min, and discard the filtrate;

[0203] 7) Centrifuge the column at 4°C, 10,000 × g for 2 min.

[0204] 8) After centrifugation, carefully remove the purification column with the collection tube (if there is liquid in the collection tube, be careful not to splash the liquid onto the purification column), discard the collection tube, place the purification column in a new 1.5 mL centrifuge tube, add 100 μL of DEPC water to the purification column, let it stand at room temperature (25°C) for 2 minutes, and then centrifuge at 4°C, 10,000 × g, for 1 minute.

[0205] 9) Collecting the RNA solution from step 8) for subsequent experiments;

[0206] Step 2: RNA reverse transcription

[0207] HiScript III RT SuperMix for qPCR (purchased from Novozymes, catalog number R323-01) was used according to the product instructions. A 20 μL reverse transcription reaction system was prepared according to the reverse transcription protocol in the kit instructions, and total cellular RNA was reverse transcribed. The reverse transcription conditions were as follows: the reverse transcription reaction system was incubated at 37°C for 15 min, followed by incubation at 85°C for 5 s. 80 μL of DEPC water was added to each reverse transcription reaction system to obtain a solution containing cDNA.

[0208] Step 3: qPCR reaction system configuration

[0209] For each reverse transcription reaction system, 4 μL of the above-mentioned cDNA-containing solution was taken as a template, and the reagents provided by the AceQ Universal SYBR qPCR Master Mix kit (purchased from Vazyme, catalog number Q511-02) were used to prepare 20 μL of the qPCR reaction system on an ice box according to Table 4. Among them, Primer1 and Primer2 were the PCR primer sequences for amplifying the target gene XDH and the internal reference gene GAPDH, respectively (as shown in Table 5). Each qPCR reaction system was placed in an ABI StepOnePlus Real-Time On the PCR instrument, a three-step method was used for amplification. The amplification program was pre-denaturation at 95°C for 10 minutes, then denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds. After repeating the above denaturation, annealing, and extension process 40 times, a product W containing the amplified target gene XDH and the internal reference gene GAPDH was obtained; the product W was then incubated at 95°C for 15 seconds, 60°C for 1 minute, and 95°C for 15 seconds. The real-time fluorescence quantitative PCR instrument collected the melting curves of the target gene XDH and the internal reference gene GAPDH in the product W, respectively, and the Ct values ​​of the target gene XDH and the internal reference gene GAPDH were obtained.

[0210] Table 4. RNA amplification reaction system

[0211] Table 5. Primer information

[0212] The comparative Ct (ΔΔCt) method was used to calculate the relative quantification of the target gene XDH in each test group. The calculation method is as follows:

[0213] ΔCt(test group) = Ct(test group target gene) – Ct(test group reference gene)

[0214] ΔCt(control group) = Ct(control group target gene) – Ct(control group internal reference gene)

[0215] ΔΔCt(test group)=ΔCt(test group)-ΔCt(control group average)

[0216] ΔΔCt(control group) = ΔCt(control group) - ΔCt(control group average)

[0217] Wherein, ΔCt(control group average) is the arithmetic mean of the ΔCt(control group) of each of the four samples in the control group; thus, each sample in the test group and the control group corresponds to a ΔΔCt value.

[0218] The expression level of XDH mRNA in the test group was normalized with the control group as the benchmark, and the expression level of XDH mRNA in the control group was defined as 100%.

[0219] Relative expression level of XDH mRNA in the test group = 2-ΔΔCt (test group) × 100%

[0220] XDH mRNA levels were compared with the internal reference gene GAPDH, and the values ​​were normalized to the mean of the saline control group. The data were expressed as a percentage relative to the saline control group and presented as the mean plus the standard deviation.

[0221] The results are shown in Figure 2. In mice after a single dose, RD34XDH008G and RD34XDH009G showed significant inhibitory effects at a dose of 9 mg / kg.

[0222] Example 3: A modified siRNA for inhibiting XDH

[0223] This embodiment provides a modified siRNA for inhibiting XDH, wherein the modified siRNA includes RD34XDH008-1G, RD34XDH008-2G, RD34XDH009-1G and RD34XDH009-2G.

[0224] The positive strands of RD34XDH008-1G and RD34XDH009-1G are obtained by chemically modifying the sequences shown in SEQ ID NO. 15 and SEQ ID NO. 17, respectively. From the 5' end to the 3' end, the nucleotides at positions 1 and 2, and 2 and 3 are linked by phosphorothioate groups, the nucleotides at positions 2, 3, 4, 5, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19 are methoxy-modified nucleotides, the nucleotides at positions 7, 8, and 9 are fluorine-modified nucleotides, and the nucleotide at position 1 is an LNA-modified nucleotide. The antisense strands of RD34XDH008-1G and RD34XDH009-1G are obtained by chemically modifying the sequences selected from SEQ ID NO. 16 and SEQ ID NO. 18, respectively. From the 5' end to the 3' end, the nucleotides at positions 1 and 2, positions 2 and 3, positions 19 and 20, and positions 20 and 21 are linked by phosphorothioate groups, the nucleotides at positions 1, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, and 21 are methoxy-modified nucleotides, and the nucleotides at positions 2, 6, 14, and 16 are fluorine-modified nucleotides.

[0225] The positive strands of RD34XDH008-2G and RD34XDH009-2G are obtained by chemically modifying the sequences selected from SEQ ID NO. 15 and SEQ ID NO. 17, respectively. From the 5' end to the 3' end, the nucleotides at positions 1 and 2, and 2 and 3 are linked by thiophosphate groups, the nucleotides at positions 2, 3, 4, 5, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19 are methoxy-modified nucleotides, the nucleotides at positions 7, 8, and 9 are fluorine-modified nucleotides, and the nucleotide at position 1 is a 2'-O-methoxyethyl (2'-MOE)-modified nucleotide. The antisense strands of RD34XDH008-2G and RD34XDH009-2G are obtained by chemically modifying the sequences selected from SEQ ID NO. 16 and SEQ ID NO. 18, respectively. From the 5' end to the 3' end, the nucleotides at positions 1 and 2, positions 2 and 3, positions 19 and 20, and positions 20 and 21 are linked by phosphorothioate groups, the nucleotides at positions 1, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, and 21 are methoxy-modified nucleotides, and the nucleotides at positions 2, 6, 14, and 16 are fluorine-modified nucleotides.

[0226] Table 6 Sequence information

[0227] Experimental Example 4: Single-dose study of modified siRNA in mice (3 mg / kg)

[0228] This experimental example provides a single-dose study of modified siRNA in mice at a dose level of 3 mg / kg. The experimental procedure is as follows:

[0229] According to the results of Experimental Example 3, GalNAc was used to couple DRD34XDH008 and RD34XDH009 shown in Example 2 to obtain GalNAc-coupled siRNAs: RD34XDH008G and RD34XDH009G. GalNAc was used to couple RD34XDH008-1G, RD34XDH008-2G, RD34XDH009-1G and RD34XDH009-2G shown in Example 3 to obtain GalNAc-coupled siRNAs: RD34XDH008-1G, RD34XDH008-2G, RD34XDH009-1G and RD34XDH009-2G. Three C57BL / 6J mice were used in each group. Mice (female, 6-8 weeks) were subcutaneously administered a single dose of 3 mg / kg of GalNAc-conjugated siRNA or saline, or GRD342260 and GRD342217 controls. The administration requirements are shown in Table 7. On the 10th day after administration, the mice were sacrificed, and liver samples were collected and quick-frozen in liquid nitrogen. Liver mRNA was extracted and analyzed by RT-qPCR.

[0230] Table 7 3 mg / kg single dose test administration requirements

[0231] According to Figure 3, in mice after a single dose, at a dose of 3 mg / kg, all sequences had a good inhibitory effect, but XDH009G still had a very prominent inhibitory effect, with a 10-day inhibition rate of more than 75%.

[0232] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present disclosure. Industrial Applicability

[0233] The present disclosure provides siRNA for inhibiting XDH. Experiments have shown that the siRNAs have high inhibitory activity against XDH.

Claims

1. An siRNA for inhibiting XDH, characterized in that, The siRNA contains a sense strand and an antisense strand, and the sense strand can at least partially reverse complementarily pair with the antisense strand to form a double-stranded region. Wherein, the sense strand comprises the nucleotide sequence shown in SEQ ID NO.1, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.2, or, Wherein, the sense strand comprises the nucleotide sequence shown in SEQ ID NO.3, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.4, or, Wherein, the sense strand comprises the nucleotide sequence shown in SEQ ID NO.5, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.6, or, Wherein, the sense strand comprises the nucleotide sequence shown in SEQ ID NO.7, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.8, or, Wherein, the sense strand comprises the nucleotide sequence shown in SEQ ID NO.9, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.10, or, Wherein, the sense strand comprises the nucleotide sequence shown in SEQ ID NO.11, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.12, or, Wherein, the sense strand comprises the nucleotide sequence shown in SEQ ID NO.13, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.14, or, Wherein, the sense strand comprises the nucleotide sequence shown in SEQ ID NO.15, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.16, or, Wherein, the sense strand comprises the nucleotide sequence shown in SEQ ID NO.17, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.18, or, Wherein, the sense strand comprises the nucleotide sequence shown in SEQ ID NO.19, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.20, or, Wherein, the sense strand comprises the nucleotide sequence shown in SEQ ID NO.21, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.22, or, Wherein, the sense strand comprises the nucleotide sequence shown in SEQ ID NO.23, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.24, or, Wherein, the sense strand comprises the nucleotide sequence shown in SEQ ID NO.25, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.26, or, Wherein, the sense strand comprises the nucleotide sequence shown in SEQ ID NO.27, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.28, or, Wherein, the sense strand comprises the nucleotide sequence shown in SEQ ID NO.29, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.30, or, Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO.31, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.32, or, Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO.33, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.34, or, Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO.35, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.36, or, Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO.37, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.38, or, Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO.39, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.40, or, Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO.41, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.42, or, Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO.43, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.

44.

2. The siRNA according to claim 1, wherein At least one nucleotide in the sense strand or antisense strand of the siRNA is a modified nucleotide.

3. The siRNA according to claim 2, wherein The modification is a chemical modification, and the chemical modification is selected from one or more of methoxy modification, fluoro modification, phosphorothioate modification, LNA modification, and methoxyethyl modification.

4. The siRNA according to any one of claims 2 to 3, characterized in that, The fluorine-modified nucleotides are located in the antisense strand and sense strand of the nucleotide sequence. And, in the direction from the 5'-end to the 3'-end, at least the 9th, 10th, and 11th nucleotides of the sense strand are fluorine-modified nucleotides, and at least the 2nd, 6th, 14th, and 16th nucleotides of the antisense strand are fluorine-modified nucleotides; or, The fluorine-modified nucleotides are located in the antisense strand and sense strand of the nucleotide sequence. And, in the direction from the 5'-end to the 3'-end, at least the 7th, 8th, and 9th nucleotides of the sense strand are fluorine-modified nucleotides, and at least the 2nd, 6th, 14th, and 16th nucleotides of the antisense strand are fluorine-modified nucleotides.

5. The siRNA according to any one of claims 2 to 3, characterized in that, The methoxy-modified nucleotides are located in the antisense strand and sense strand of the nucleotide sequence. And, in the direction from the 5'-end to the 3'-end, at least the 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st nucleotides of the sense strand are methoxy-modified nucleotides, and at least the 1st, 3rd, 4th, 5th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 15th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd nucleotides of the antisense strand are methoxy-modified nucleotides; or, The methoxy-modified nucleotides are located in the antisense strand and the sense strand of the nucleotide sequence. And, in the direction from the 5'-end to the 3'-end, the nucleotides at least at the 1st, 2nd, 3rd, 4th, 5th, 6th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th positions of the sense strand are methoxy-modified nucleotides, and the nucleotides at least at the 1st, 3rd, 4th, 5th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 15th, 17th, 18th, 19th, 20th, 21st positions of the antisense strand are methoxy-modified nucleotides.

6. The siRNA according to any one of claims 2 to 3, characterized in that, The phosphorothioate-linked nucleotides are located in the antisense strand and the sense strand of the nucleotide sequence. And, in the direction from the 5'-end to the 3'-end, the nucleotides between at least the 1st and 2nd positions, and between the 2nd and 3rd positions of the sense strand are linked by a phosphorothioate group, and the nucleotides between at least the 1st and 2nd positions, between the 2nd and 3rd positions, between the 21st and 22nd positions, and between the 22nd and 23rd positions of the antisense strand are linked by a phosphorothioate group; or, The phosphorothioate-linked nucleotides are located in the antisense strand and the sense strand of the nucleotide sequence. And, in the direction from the 5'-end to the 3'-end, the nucleotides between at least the 1st and 2nd positions, and between the 2nd and 3rd positions of the sense strand are linked by a phosphorothioate group, and the nucleotides between at least the 1st and 2nd positions, between the 2nd and 3rd positions, between the 19th and 20th positions, and between the 20th and 21st positions of the antisense strand are linked by a phosphorothioate group.

7. The siRNA according to any one of claims 2 to 3, characterized in that, The methoxyethyl-modified nucleotides are located in the sense strand of the nucleotide sequence. And, in the direction from the 5'-end to the 3'-end, the nucleotide at least at the 1st position of the sense strand is a methoxyethyl-modified nucleotide.

8. The siRNA according to any one of claims 2 to 3, characterized in that, The LNA-modified nucleotides are located in the sense strand of the nucleotide sequence. And, in the direction from the 5'-end to the 3'-end, the nucleotide at least at the 1st position of the sense strand is an LNA-modified nucleotide.

9. The siRNA according to any one of claims 2 to 3, characterized in that, The 3'-end of the sense strand of the siRNA is conjugated with a ligand, and the ligand is GalNAc.

10. A product for inhibiting XDH, characterized in that, The product contains an active ingredient and a pharmaceutically acceptable carrier, and the active ingredient is the siRNA according to any one of claims 1 to 9.

11. The product according to claim 10, wherein, The product is a pharmaceutical composition or a kit.

12. Use of the siRNA according to any one of claims 1 to 9 or the product according to any one of claims 10 to 11 in the preparation of a product for preventing, diagnosing and / or treating a pathological condition or disease caused by XDH.

13. Use of the siRNA according to any one of claims 1 to 9 or the product according to any one of claims 10 to 11 in preventing, diagnosing and / or treating a pathological condition or disease caused by XDH.

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