Compositions and methods for inhibiting xanthine dehydrogenase (XDH)

Abstract

Provided are xanthine dehydrogenase (XDH) dsRNA agents, and compositions thereof with xanthine dehydrogenase (XDH) antisense polynucleotide agents and / or non-xanthine dehydrogenase (XDH) dsRNA therapeutic agents, useful for reducing the expression of xanthine dehydrogenase (XDH) in cells and subjects, useful for inhibiting xanthine dehydrogenase (XDH) gene expression and for treating xanthine dehydrogenase (XDH) related diseases and disorders.

Description

Compositions and methods for inhibiting xanthine dehydrogenase (XDH) Technical Field

[0001] The present invention relates to compositions and methods useful for inhibiting xanthine dehydrogenase (XDH). Background Art

[0002] Uric acid (also referred to herein as urate) is the final metabolite of endogenous and dietary purine metabolism. Xanthine oxidase (also referred to herein as "XO") (EC 1.1.3.22) and xanthine dehydrogenase (also referred to herein as "XDH") (EC 1.17.1.4) catalyze the oxidation of hypoxanthine to xanthine and xanthine to uric acid, respectively. These enzymes are molybdenum pterin-containing flavoproteins composed of two identical subunits of approximately 145 kDa. These enzymes are synthesized from mammals, including humans, in the form of dehydrogenases, but can be easily converted to the oxidase form by oxidation or proteolysis of sulfhydryl residues. XDH is primarily expressed in the intestine and liver, but can also be expressed in other tissues, including adipose tissue.

[0003] Chronic elevation of serum uric acid (chronic hyperuricemia), generally defined as serum urate levels above 6.8 mg / dL (greater than 360 mmol / dL), above which the physiological saturation threshold is exceeded (Mandell, Cleve. Clin. Med. 75: S5-S8, 2008), is associated with many diseases, such as gout, which is characterized by recurrent episodes of acute inflammatory arthritis, usually due to insufficient renal clearance of uric acid or excessive uric acid production, leading to an inflammatory response to uric acid crystals in the joints. Fructose-related gout is associated with variants of transport proteins expressed in the kidney, intestine, and liver. Chronic elevated uric acid is also associated with nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), metabolic disorders, cardiovascular disease, type 2 diabetes, and diseases related to oxidative stress, chronic low-grade inflammation, and insulin resistance (Xu et al., J. Hepatol. 62:1412-1419, 2015; Cardoso et al., J. Pediatr. 89:412-418, 2013; Sertoglu et al., Clin. Biochem., 47:383-388, 2014).

[0004] Allopurinol and febuxostat Inhibitors of the enzyme XDH are commonly used to treat gout. However, their use is contraindicated in patients with comorbidities of gout, particularly those with decreased renal function (e.g., due to chronic kidney disease or hepatic insufficiency). Their use may be limited in patients with metabolic syndrome, hypertension, dyslipidemia, nonalcoholic steatohepatitis (NASH) or nonalcoholic fatty liver disease (NAFLD), cardiovascular disease, or diabetes (type 1 or type 2) due to organ function limitations caused by the disease or condition or adverse drug interactions with medications used to treat such conditions.

[0005] Currently, treatment options for XDH-related diseases or conditions do not fully meet the needs of patients. Therefore, there is a need for additional therapies for subjects who would benefit from reduced XDH gene expression, such as subjects with gout.

[0006] Summary of the Invention

[0007] According to one aspect of the present invention, a double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of a xanthine dehydrogenase (XDH) gene is provided, the dsRNA agent comprising a sense strand and an antisense strand, comprising a region complementary to a xanthine dehydrogenase (XDH) gene RNA transcript at nucleotide positions 2 to 18 in the antisense strand, wherein the complementary region comprises at least 15 consecutive nucleotides that differ from one of the antisense sequences listed in Tables 1-3 by 0, 1, 2 or 3 nucleotides, and optionally comprises a targeting ligand.

[0008] In some embodiments, the region complementary to the xanthine dehydrogenase (XDH) gene RNA transcript comprises at least 15, 16, 17, 18, or 19 consecutive nucleotides that differ by no more than 3 nucleotides from one of the antisense sequences listed in Tables 1-3.

[0009] In some embodiments, the antisense strand of the dsRNA is at least substantially complementary to any target region of SEQ ID NO: 1 and provided in one of Tables 1-3.

[0010] In some embodiments, the antisense strand of the dsRNA is fully complementary to any target region of SEQ ID NO: 1 and provided in one of Tables 1-3.

[0011] In some embodiments, the dsRNA agent comprises any one of the sense strand sequences listed in Tables 1-3, wherein the sense strand sequence is at least substantially complementary to the antisense strand sequence in the dsRNA agent.

[0012] In some embodiments, the dsRNA agent comprises any one of the sense strand sequences listed in Tables 1-3, wherein the sense strand sequence is fully complementary to the antisense strand sequence in the dsRNA agent.

[0013] In some embodiments, the dsRNA agent comprises any one of the antisense strand sequences listed in Tables 1-3.

[0014] In some embodiments, the dsRNA agent comprises any one of the sequences listed as duplex sequences in Tables 1-3.

[0015] In some embodiments, the dsRNA agent comprises at least one modified nucleotide.

[0016] In some embodiments, all or substantially all of the nucleotides of the antisense strand are modified nucleotides.

[0017] In some embodiments, all or substantially all nucleotides of the sense and antisense strands are modified nucleotides.

[0018] In some embodiments, the at least one modified nucleotide comprises a 2'-methoxy nucleotide, a 2'-fluoro nucleotide, a 2'-deoxy nucleotide, a 2',3'-seco nucleotide mimetic, a locked nucleotide, an unlocked nucleic acid nucleotide (UNA), a glycol nucleic acid nucleotide (GNA), a 2'-F-arabino nucleotide, a 2'-methoxyethyl nucleotide, an abasic nucleotide, a ribitol, an inverted nucleotide, an inverted abasic nucleotide, an inverted 2'-OMe nucleotide, an inverted 2'-deoxy nucleotide, an isomannite nucleotide, a 2'-amino modified nucleotide, a 2'-alkyl modified nucleotide, a morpholino nucleotide, and a 3'-OMe nucleotide, a nucleotide comprising a 5'-phosphorothioate group, or a terminal nucleotide linked to a cholesterol derivative or a dodecanoic acid bisdecylamide group, a 2'-amino modified nucleotide, a phosphoramidate, or an unnatural base containing nucleotide.

[0019] In some embodiments, the dsRNA agent includes an E-vinylphosphonate nucleotide at the 5' end of the guide strand.

[0020] In some embodiments, the dsRNA agent comprises at least one phosphorothioate internucleoside linkage.

[0021] In some embodiments, the sense strand comprises at least one phosphorothioate internucleoside linkage.

[0022] In some embodiments, the antisense strand comprises at least one phosphorothioate internucleoside linkage.

[0023] In some embodiments, the sense strand comprises 1, 2, 3, 4, 5, or 6 phosphorothioate internucleoside linkages.

[0024] In some embodiments, the antisense strand comprises 1, 2, 3, 4, 5, or 6 phosphorothioate internucleoside linkages.

[0025] In some embodiments, the antisense strand comprises at least 15 modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoro nucleotides, wherein fewer than 6 are 2'-fluoro nucleotide modified nucleotides. In certain embodiments, the antisense strand comprises 3 or 5 2'-fluoro nucleotides, preferably, the antisense strand comprises 5 2'-fluoro nucleotides.

[0026] In some embodiments, the sense strand comprises at least 15 modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoro nucleotides, wherein fewer than 4 are 2'-fluoro nucleotide modified nucleotides, and in certain embodiments, the sense strand comprises 3 2'-fluoro nucleotides.

[0027] In some embodiments, the antisense strand comprises 2'-fluorine-modified nucleotides at positions 2, 7, 12, 14, and 16 of the antisense strand, starting from the first paired nucleotide at the 5' end of the antisense strand in the direction from the 5' end to the 3' end, and each nucleotide at other positions in the antisense strand is independently a non-fluorine-modified nucleotide.

[0028] In some embodiments, the sense strand comprises nucleotides at positions 9, 11, and 13 of the sense strand, counted from the first paired nucleotide at the 3' end of the sense strand in the direction from the 3' end to the 5' end, that are 2'-fluorine-modified nucleotides, and each nucleotide at the other position in the sense strand is independently a non-fluorine-modified nucleotide.

[0029] In some embodiments, the antisense strand comprises 15 or more modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoro nucleotides, wherein the 2nd, 7th, 12th, 14th and / or 16th positions from the 5' end of the antisense strand are nucleotides modified with 2'-fluoro nucleotides, and the rest are nucleotides modified with 2'-O-methyl nucleotides.

[0030] In some embodiments, the sense strand comprises 15 or more modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoro nucleotides, wherein the 9th, 11th and / or 13th position from the first matching position at the 3' end of the sense strand is a nucleotide modified with a 2'-fluoro nucleotide, and the rest are nucleotides modified with a 2'-O-methyl nucleotide.

[0031] In some embodiments, the modified sense strand is a modified sense strand sequence listed in Table 2 and / or Table 3.

[0032] In some embodiments, the modified antisense strand is a modified antisense strand sequence listed in Table 2 and / or Table 3.

[0033] In some embodiments, the sense strand is complementary or substantially complementary to the antisense strand, and the length of the complementary region is between 16 and 23 nucleotides. In some embodiments, the length of the complementary region is 19-21 nucleotides. In some embodiments, the length of the complementary region is 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.

[0034] In some embodiments, each strand is no more than 40 nucleotides in length.

[0035] In some embodiments, each strand is no more than 30 nucleotides in length.

[0036] In some embodiments, each strand is no more than 25 nucleotides in length.

[0037] In some embodiments, each strand is no more than 23 nucleotides in length.

[0038] In some embodiments, each strand is no more than 21 nucleotides in length.

[0039] In some embodiments, a dsRNA agent comprises at least one modified nucleotide and further comprises one or more targeting groups or linking groups.

[0040] In some embodiments, one or more targeting groups or linking groups are conjugated to the sense strand. In some embodiments, the targeting group or linking group comprises N-acetyl-galactosamine (GalNAc).

[0041] In some embodiments, the targeting group or linking group has a structure as shown in formula (X), comprising a targeting moiety, a linker, and a linker W, wherein the targeting moiety is selected from an N-acetyl-galactosamine derivative (GalNAc), which is linked to the linker W via a linker, and the linker W has a structure as shown in formula (XI), X is selected from O, NH2 or S, and Y ﹣ Selected from: O ﹣ 、S ﹣ , methyl or NR a R b , R a and R b are independently selected from hydrogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, or R a and R bTogether with the attached atoms, it forms a 3-12 membered heterocycloalkyl group containing 1-3 heteroatoms of N, O, and S. In some preferred embodiments, in the substituted or unsubstituted C1-C6 alkyl group or the substituted or unsubstituted C3-C6 cycloalkyl group, the substituent is selected from hydroxyl and amino.

[0042] In some embodiments, the linker in the targeting group is selected from polyethylene glycol, optionally substituted C2-C 12 Alkyl, substituted or unsubstituted C3-C 12 Cycloalkyl, substituted or unsubstituted C3-C 12 Heterocycloalkyl, substituted or unsubstituted C3-C 12 In some embodiments, the substituted or unsubstituted C2-C 12 Alkyl, substituted or unsubstituted C3-C 12 Cycloalkyl, substituted or unsubstituted C3-C 12 Heterocycloalkyl, substituted or unsubstituted C3-C 12 In the amide, the substituent is selected from hydroxyl and carbonyl.

[0043] In some embodiments, the linker in the targeting group is preferably selected from the following fragments:

[0044] Each m is an integer from 1 to 6, each n, o, and p is 0 or 1, and each q is an integer from 1 to 6. 1 With q 2 Each independently is 0, 1 or 2.

[0045] In some embodiments, the linker in the targeting group is more preferably selected from the following fragments:

[0046] In some embodiments, the targeting moiety in the targeting group has the following structural fragment,

[0047] n' is 1 or 2.

[0048] In some embodiments, the targeting group has the following structure:

[0049] In some embodiments, the dsRNA agent comprises a targeting group conjugated to the 5 '-end of the sense strand.

[0050] In some embodiments, the dsRNA agent comprises a targeting group conjugated to the 3 '-end of the sense strand.

[0051] In some embodiments, the antisense strand comprises an inverted abasic residue at the 3'-terminus.

[0052] In some embodiments, the sense strand comprises one or two inverted abasic residues at the 3' or / and 5' termini.

[0053] In some embodiments, the sense strand comprises one or two imann residues at the 3' or / and 5' termini.

[0054] In some embodiments, the 3' and 5' ends of the sense strand each independently comprise an imann residue.

[0055] In some embodiments, the dsRNA agent has two blunt ends.

[0056] In some embodiments, at least one strand comprises a 3' overhang of at least 1 nucleotide in length. In some embodiments, at least one strand comprises a 3' overhang of at least 2 nucleotides in length. In one embodiment, the sense strand has an overhang of 1-10 nucleotides at the 3'-end and / or the 5'-end, such as an overhang of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

[0057] Another aspect of the present invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of a xanthine dehydrogenase (XDH) gene, the dsRNA agent comprising a sense strand and an antisense strand, wherein each strand is 14 to 30 nucleotides in length, and comprises a region complementary to a xanthine dehydrogenase (XDH) gene RNA transcript at nucleotide positions 2 to 18 in the antisense strand, wherein the complementary region comprises at least 15 consecutive nucleotides that differ from one of the double-stranded ribonucleic acid (dsRNA) agent antisense sequences listed in Formula I by 0, 1, 2 or 3 nucleotides, and optionally comprises a targeting ligand.

[0058] The antisense strand of the double-stranded ribonucleic acid (dsRNA) reagent is shown in Formula I: 5'-N a -(A1A2A3A4) i -N b –(B1B2B3B4)-N b -(C1C2C3C4C5) j -N a -3' (Formula I)

[0059] Wherein: i and j are each independently selected from 0 or 1;

[0060] Each N a and N bEach independently represents an oligonucleotide 0-17, said oligonucleotide being optionally modified by a chemical modification as used herein; preferably, said chemical modification is selected from 2'-OMe or 2'-fluorine modification;

[0061] A1A2A3A4 represents a motif in which four consecutive nucleotides are modified by lowercase 2'-OMe, uppercase 2'-fluorine, lowercase 2'-OMe, and lowercase 2'-OMe, respectively. Preferably, A1 and A2, and A2 and A3 further contain phosphorothioate internucleoside linkages.

[0062] B1B2B3B4 represents a motif in which four consecutive nucleotides are modified with one uppercase 2'-fluorine and three consecutive lowercase 2'-OMe;

[0063] C1C2C3C4C5 represents a motif in which five consecutive nucleotides are modified with lowercase 2'-OMe, preferably further comprising phosphorothioate internucleoside linkages between C3 and C4 and between C4 and C5.

[0064] In some embodiments, N a Each independently represents 0-2 oligonucleotides, N b Each independently represents 2-5 oligonucleotides that have or have not been chemically modified as used herein.

[0065] In some embodiments, N a Each independently represents 0 oligonucleotide, N b Each independently represents 2-5 chemically modified oligonucleotides used herein.

[0066] In some embodiments, N a Each independently represents 0 oligonucleotide, N b Each independently represents 2-5 oligonucleotides chemically modified with 2'-OMe or 2'-fluorine.

[0067] Furthermore, in some embodiments, formula (I) is further represented by formula (II): 5'-xYxxxxYxxxxYxYxYxxxxx-3' (Formula II)

[0068] X represents a lowercase 2'-OMe modified oligonucleotide, and Y represents an uppercase 2'-fluorine modified oligonucleotide. Preferably, X and Y and / or X and X further comprise phosphorothioate internucleoside linkages.

[0069] Furthermore, in some embodiments, the sense strand of the double-stranded ribonucleic acid (dsRNA) reagent is represented by formula (III): 5′-(N′ L ) n′ N′ L N′ L N′L N′ N1 N′ N2 N′ N3 N′ L N′ F N′ L N′ N4 N′ N5 N′ N6 N′ L N′ L N′ L (N′ L ) m′ -3′ Formula (III)

[0070] Among them, N' F Represents a 2'-fluoro modified nucleotide; N' N1 ,N′ N2 ,N′ N3 ,N′ N4 ,N′ N5 ,and N′ N6 Each independently represents a modified or unmodified nucleotide; N' N1 N′ N2 N′ N3 and N′ N4 N′ N5 N′ N6 Each independently represents a nucleotide fragment comprising at least two different modifications; N' L Each independently represents a modified or unmodified nucleotide, wherein the modified nucleotide is not a 2'-fluorine-modified nucleotide; m' and n' each independently represent an integer from 0 to 7.

[0071] In some embodiments, n′ is 1 and m′ is 1, or n′ is 1 and m′ is 2, or n′ is 1 and m′ is 3, or n′ is 1 and m′ is 4, or n′ is 1 and m′ is 5, or n′ is 3 and m′ is 1, or n′ is 3 and m′ is 2, or n′ is 3 and m′ is 3, or n′ is 5 and m′ is 1.

[0072] In some embodiments, the antisense strand of the dsRNA is at least substantially complementary to any target region of SEQ ID NO: 1. In some embodiments, the antisense strand of the dsRNA is fully complementary to any target region of SEQ ID NO: 1. In some embodiments, the sense strand sequence of any of the dsRNAs is at least substantially complementary to the antisense strand sequence of the dsRNA agent. In some embodiments, the sense strand sequence of any of the dsRNAs is fully complementary to the antisense strand sequence of the dsRNA agent.

[0073] In some embodiments, the region complementary to SEQ ID NO: 1 comprises at least 15, 16, 17, 18, or 19 contiguous nucleotides that differ by no more than 3 nucleotides from one of the antisense sequences listed in Tables 1-3.

[0074] In some embodiments, the antisense strand of the dsRNA is at least substantially complementary to any target region of SEQ ID NO: 1 and provided in one of Tables 1-3.

[0075] In some embodiments, the antisense strand of the dsRNA is fully complementary to any target region of SEQ ID NO: 1 and provided in one of Tables 1-3.

[0076] In some embodiments, the dsRNA agent comprises any one of the sense strand sequences listed in Tables 1-3, wherein the sense strand sequence is at least substantially complementary to the antisense strand sequence in the dsRNA agent.

[0077] In some embodiments, the dsRNA agent comprises any one of the sense strand sequences listed in Tables 1-3, wherein the sense strand sequence is fully complementary to the antisense strand sequence in the dsRNA agent.

[0078] In some embodiments, the dsRNA agent comprises any one of the antisense strand sequences listed in Tables 1-3.

[0079] In some embodiments, the dsRNA agent comprises any one of the sequences listed as duplex sequences in Tables 1-3.

[0080] In some embodiments, the sense strand comprises one or two inverted abasic residues at the 3' or / and 5' termini.

[0081] In some embodiments, the sense strand comprises one or two imann residues at the 3' or / and 5' termini.

[0082] In some embodiments, the 3' and 5' ends of the sense strand each independently comprise an imann residue.

[0083] In some embodiments, the dsRNA agent has two blunt ends. In some embodiments, at least one strand comprises a 3' overhang of at least 1 nucleotide in length. In some embodiments, at least one strand comprises a 3' overhang of at least 2 nucleotides in length. In one embodiment, the sense strand has a 1-10 nucleotide overhang at the 3'-end and / or the 5'-end, such as an overhang of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

[0084] In some embodiments, the dsRNA agent comprises at least one modified nucleotide and also comprises one or more targeting groups or linking groups. In some embodiments, one or more targeting groups or linking groups are conjugated to the sense strand. In some embodiments, the targeting group or linking group comprises N-acetyl-galactosamine (GalNAc). In some embodiments, the targeting group is as used herein, any of GLS-1, GLS-2, GLS-3, GLS-4, GLS-5, GLS-6, GLS-7, GLS-8, GLS-9, GLS-10, GLS-11, GLS-12, GLS-13, GLS-14, GLS-15, GLS-16, GLO-1, GLO-2, GLO-3, GLO-4, GLO-5, GLO-6, GLO-7, GLO-8, GLO-9, GLO-10, GLO-11, GLO-12, GLO-13, GLO-14, GLO-15 and GLO-16.

[0085] In some embodiments, the sense strand independently comprises an imann residue at the 3' and 5' ends, wherein any one of the 3' and 5' terminal residues is optionally further linked to a targeting group, preferably the aforementioned GLS-15.

[0086] According to another aspect of the present invention, a composition is provided, comprising any embodiment of the above-mentioned dsRNA agent aspect of the present invention.

[0087] In certain embodiments, the composition further comprises a pharmaceutically acceptable carrier.In some embodiments, the composition further comprises one or more additional therapeutic agents.

[0088] In certain embodiments, the composition is packaged in a kit, container, package, dispenser, prefilled syringe, or vial.

[0089] In some embodiments, the composition is formulated for subcutaneous administration or is formulated for intravenous (IV) administration.

[0090] According to another aspect of the present invention, a cell is provided, comprising any embodiment of the above-mentioned dsRNA agent of the present invention.

[0091] In some embodiments, the cell is a mammalian cell, optionally a human cell.

[0092] According to another aspect of the present invention, a method for inhibiting xanthine dehydrogenase (XDH) gene expression in a cell is provided, the method comprising: (i) preparing a cell comprising an effective amount of the above dsRNA agent or any embodiment of the above composition.

[0093] In certain embodiments, the method further comprises: (ii) maintaining the prepared cells for a sufficient time to obtain degradation of the mRNA transcript of the xanthine dehydrogenase (XDH) gene, thereby inhibiting the expression of the xanthine dehydrogenase (XDH) gene in the cells.

[0094] In some embodiments, the cells are in a subject and the dsRNA agent is administered subcutaneously to the subject.

[0095] In some embodiments, the cells are in a subject and the dsRNA agent is administered to the subject by IV administration.

[0096] In certain embodiments, the method further comprises assessing inhibition of the xanthine dehydrogenase (XDH) gene after administering the dsRNA agent to the subject, wherein the means for assessing comprises: (i) determining one or more physiological characteristics of a xanthine dehydrogenase (XDH)-related disease or condition in the subject, and (ii) comparing the determined physiological characteristics to a baseline pre-treatment physiological characteristic of the xanthine dehydrogenase (XDH)-related disease or condition and / or a control physiological characteristic of the xanthine dehydrogenase (XDH)-related disease or condition, wherein the comparison indicates the presence or absence of inhibition of xanthine dehydrogenase (XDH) gene expression in the subject.

[0097] In some embodiments, the physiological characteristic determined is uric acid level in the blood;

[0098] In some embodiments, a reduction in uric acid levels in the blood indicates a reduction in xanthine dehydrogenase (XDH) gene expression in the subject.

[0099] According to another aspect of the present invention, a method for inhibiting xanthine dehydrogenase (XDH) gene expression in a subject is provided, comprising administering to the subject an effective amount of the aforementioned dsRNA agent embodiment or the aforementioned composition embodiment.

[0100] In some embodiments, the dsRNA agent is administered subcutaneously to the subject. In certain embodiments, the dsRNA agent is administered to the subject via IV administration. In some embodiments, the method further comprises: assessing the suppression of the xanthine dehydrogenase (XDH) gene after administering the dsRNA agent, wherein the assessment means comprises: (i) determining one or more physiological characteristics of the xanthine dehydrogenase (XDH)-related disease or condition of the subject; (ii) comparing the determined physiological characteristics with the baseline pre-treatment physiological characteristics of the xanthine dehydrogenase (XDH)-related disease or condition and / or the control physiological characteristics of the xanthine dehydrogenase (XDH)-related disease or condition; wherein the comparison result indicates the presence or absence of suppression of xanthine dehydrogenase (XDH) gene expression in the subject. In some embodiments, the determined physiological characteristic is the uric acid level in the blood. A reduction in the uric acid level in the blood indicates a reduction in xanthine dehydrogenase (XDH) gene expression in the subject.

[0101] According to another aspect of the present invention, there is provided a use of a drug for preparing a drug for treating a disease or condition associated with xanthine dehydrogenase (XDH) protein, wherein the drug comprises any embodiment of the aforementioned dsRNA agent of the present invention or any embodiment of the aforementioned composition of the present invention, and the drug can inhibit the expression of the xanthine dehydrogenase (XDH) gene.

[0102] According to another aspect of the present invention, there is provided a method for treating a disease or condition associated with xanthine dehydrogenase (XDH) protein, comprising: administering to a subject an effective amount of any embodiment of the aforementioned dsRNA agent of the present invention or any embodiment of the aforementioned composition of the present invention to inhibit xanthine dehydrogenase (XDH) gene expression.

[0103] In certain embodiments, the xanthine dehydrogenase (XDH)-associated disease or condition is selected from the group consisting of hyperuricemia, gout, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), metabolic disorders, insulin resistance, cardiovascular disease, type 2 diabetes, Lesch Nyhan syndrome, glycogen storage disease (GSD), such as type I GSD, and diseases associated with oxidative stress, such as chronic low-grade inflammation; or other XDH-associated diseases.

[0104] In certain embodiments, the disease associated with XDH is gout. In certain embodiments, the disease associated with XDH is hyperuricemia. In certain embodiments, the disease associated with XDH is NASH or NAFLD.

[0105] In some embodiments, the method further comprises administering to the subject an additional therapeutic regimen.

[0106] In some embodiments, the additional treatment regimen includes the treatment of a xanthine dehydrogenase (XDH)-related disease or condition. In certain embodiments, the additional treatment regimen includes: administering one or more xanthine dehydrogenase (XDH) antisense polynucleotides of the present invention to the subject; administering a non-xanthine dehydrogenase (XDH) dsRNA therapeutic agent to the subject; and performing behavioral changes in the subject. In some embodiments, the non-xanthine dehydrogenase (XDH) dsRNA therapeutic agent is one of the following additional therapeutic agents: for example, allopurinol, hydroxypurine, febuxostat or interleukin-1β (IL-1β) antagonists, preferably, the interleukin-1β antagonist is selected from canakinumab or rilonacept, thereby treating a subject whose disease will benefit from a reduction in xanthine dehydrogenase (XDH).

[0107] In some embodiments, the dsRNA reagent is administered subcutaneously to the subject. In certain embodiments, the dsRNA reagent is administered to the subject by IV administration. In some embodiments, the method also includes determining the efficacy of the double-stranded ribonucleic acid (dsRNA) reagent administered in the subject. In some embodiments, determining the means for treating the efficacy of the subject includes: (i) determining one or more physiological characteristics of xanthine dehydrogenase (XDH) related diseases or conditions in the subject; (ii) comparing the physiological characteristics determined with the baseline pre-treatment physiological characteristics of xanthine dehydrogenase (XDH) related diseases or conditions, wherein the comparison result indicates that the efficacy of the double-stranded ribonucleic acid (dsRNA) reagent administered to the subject exists, does not exist, and one or more of the levels. In some embodiments, the physiological characteristic determined is the uric acid level in the blood. The reduction of uric acid levels in the blood indicates the presence of the effectiveness of the double-stranded ribonucleic acid (dsRNA) reagent administered to the subject.

[0108] According to another aspect of the present invention, there is provided a method for reducing the level of xanthine dehydrogenase (XDH) protein in a subject compared to the baseline pre-treatment level of xanthine dehydrogenase (XDH) protein in the subject, comprising administering to the subject an effective amount of any of the embodiments of the aforementioned dsRNA agents of the present invention or any of the embodiments of the aforementioned compositions of the present invention to reduce the level of xanthine dehydrogenase (XDH) gene expression. In some embodiments, the dsRNA agent is administered subcutaneously to the subject or is administered to the subject by IV.

[0109] According to another aspect of the present invention, there is provided a method for changing the physiological characteristics of a xanthine dehydrogenase (XDH) related disease or condition in a subject compared to the baseline physiological characteristics before treatment of the xanthine dehydrogenase (XDH) related disease or condition in the subject, the method comprising administering to the subject any embodiment of the aforementioned dsRNA reagent of the present invention or any embodiment of the aforementioned composition of the present invention in an effective amount to change the physiological characteristics of the xanthine dehydrogenase (XDH) related disease or condition in the subject. In some embodiments, the dsRNA reagent is administered subcutaneously to the subject or is administered to the subject by IV. In some embodiments, the physiological characteristic determined is a reduction in uric acid.

[0110] Sequence Description

[0111] Duplexes AV00206 to AV00292 are shown in Table 1 and their sense strand sequences are shown.

[0112] Duplexes AV00206 to AV00292 are shown in Table 1 and their antisense strand sequences are shown.

[0113] SEQ ID NO: 1 is human xanthine dehydrogenase (XDH) mRNA [NCBI Reference Sequence: NM_000379.4].

[0114] In the sequences shown in Table 2, chemical modifications are indicated as: upper case: 2'-fluoro; lower case: 2'-OMe; phosphorothioate: *.

[0115] In the sequences shown in Table 3, the delivery molecules used in the in vivo studies are indicated as "GLX-n" at the 3' or 5' end of each sense strand. Chemical modifications are indicated as follows: uppercase: 2'-fluoro; lowercase: 2'-OMe; phosphorothioate: *; Invab = inverted abasic; imann: at the end of each strand: Or when further coupled with a delivery molecule or targeting group: DETAILED DESCRIPTION

[0116] Some embodiments of the present invention include RNAi agents capable of inhibiting the expression of the xanthine dehydrogenase (XDH) gene, such as, but not limited to, double-stranded (ds) RNAi agents. The xanthine dehydrogenase (XDH) dsRNA agents of the present invention can target xanthine dehydrogenase (XDH) transcripts, resulting in inhibition of xanthine dehydrogenase (XDH) protein expression. Some embodiments of the present invention also include compositions comprising xanthine dehydrogenase (XDH) gene RNAi agents and methods of using the compositions. The xanthine dehydrogenase (XDH) gene RNAi agents disclosed herein can be attached to a delivery compound for delivery to cells, including delivery to hepatocytes. The pharmaceutical compositions of the present invention may include at least one xanthine dehydrogenase (XDH) dsRNA agent and a delivery compound. In some embodiments of the present invention, the delivery compound is a GalNAc-containing delivery compound. The xanthine dehydrogenase (XDH) gene RNAi agent delivered to a cell can inhibit the expression of the xanthine dehydrogenase (XDH) gene, thereby reducing the activity of the gene's xanthine dehydrogenase (XDH) protein product in the cell. The dsRNAi agents of the present invention can be used to treat xanthine dehydrogenase (XDH)-related diseases and conditions. Such dsRNAi agents include, for example, duplexes AV00206 to AV00292 shown in Table 1. In some embodiments, preferred dsRNAi agents include, for example, duplexes AV00227, AV00260, AV00285, AV00266, AV00280, AV00238, AV00249, AV00289, AV00233, AV00225, AV00273, or AV00283. In other embodiments, preferred dsRNAi agents in Table 2 include, for example, duplexes AV00227, AV00260, AV00285, AV00266, or AV00280. In other embodiments, preferred dsRNAi agents of Table 3 include, for example, duplexes AD00353, AD00356, AD00358, AD00364, AD00365, AD00366, AD00368, AD00370, AD00430, AD00431, AD00432, AD00521, AD00521-1, AD00364-1, AD00365-2, AD00366-1, and AD00432-1.

[0117] In some embodiments of the present invention, reducing xanthine dehydrogenase (XDH) expression in a cell or subject treats a disease or condition associated with xanthine dehydrogenase (XDH) expression in a cell or subject, respectively. Non-limiting examples of diseases and conditions that can be treated by reducing xanthine dehydrogenase (XDH) activity are: hyperuricemia, gout, NAFLD, NASH, metabolic disorders, insulin resistance, cardiovascular disease, type 2 diabetes, Lesch Nyhan syndrome, glycogen storage disease (GSD), such as type I GSD, or diseases associated with oxidative stress, such as chronic low-grade inflammation; or other XDH-related diseases.

[0118] Described below are compositions comprising single-stranded (ssRNA) and double-stranded (dsRNA) xanthine dehydrogenase (XDH) agents to inhibit xanthine dehydrogenase (XDH) gene expression, as well as compositions and methods for treating diseases and conditions caused or regulated by xanthine dehydrogenase (XDH) gene expression. As used herein, diseases and / or conditions caused or regulated by the presence and / or level of xanthine dehydrogenase (XDH) gene expression are referred to as "xanthine dehydrogenase (XDH)-related diseases and / or conditions." The term "RNAi" is also known in the art and may be referred to as "siRNA."

[0119] As used herein, "G", "C", "A" and "U" each generally represent a nucleotide containing guanine, cytosine, adenine and uracil as a base, respectively. However, it is understood that the term "ribonucleotide" or "nucleotide" may also refer to a modified nucleotide, as further described below, or an alternative replacement moiety. It is understood by those skilled in the art that guanine, cytosine, adenine and uracil can be replaced by other moieties without substantially changing the base pairing properties of the oligonucleotide containing the nucleotide of such replacement moiety. For example, but not limited to, a nucleotide containing inosine as its base can base pair with a nucleotide containing adenine, cytosine or uracil. Therefore, in the nucleotide sequences of the present invention, a nucleotide containing uracil, guanine or adenine can be replaced by a nucleotide containing, for example, inosine. Sequences containing such replacement moieties are embodiments of the present invention.

[0120] As used herein, the term "RNAi" refers to an agent comprising RNA and mediating the targeted cutting of RNA transcripts by RNA-induced silencing complex (RISC) pathways. As known in the art, the RNAi target region refers to a continuous portion of the nucleotide sequence of the RNA molecule formed during gene transcription, including messenger RNA (mRNA), which is a processed product of primary transcript RNA. The target portion of the sequence will be at least long enough to be used as a substrate for RNAi directed cutting at or near this portion. The target sequence can be 8-30 nucleotides long (including end values), 10-30 nucleotides long (including end values), 12-25 nucleotides long (including end values), 15-23 nucleotides long (including end values), 16-23 nucleotides long (including end values), or 18-23 nucleotides long (including end values), and includes all shorter lengths within each specified range. In some embodiments of the invention, the target sequence is 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides long. In certain embodiments, the length of the target sequence is between 9 and 26 nucleotides (including the end values), including all subranges and integers therebetween. For example, although not intended to be limiting, in certain embodiments of the invention, the target sequence is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides long, and the sequence is completely or at least substantially complementary to at least a portion of the RNA transcript of the xanthine dehydrogenase (XDH) gene.

[0121] Some aspects of the present invention include pharmaceutical compositions comprising one or more xanthine dehydrogenase (XDH) dsRNA agents and a pharmaceutically acceptable carrier. In certain embodiments of the present invention, xanthine dehydrogenase (XDH) gene RNAi as described herein inhibits the expression of xanthine dehydrogenase (XDH) protein.

[0122] In some embodiments of the present invention, the pharmaceutical composition includes one, two, three or more independent anti-xanthine dehydrogenase (XDH) dsRNA agents, and may also include one or more independently selected delivery compounds. In some embodiments, two, three, four or more xanthine dehydrogenase (XDH) dsRNAs that can target one, two, three, four or more different locations / regions of xanthine dehydrogenase (XDH) mRNA are administered to a cell or object.

[0123] As used herein, "dsRNA agent" refers to a composition comprising RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecules that are capable of degrading or inhibiting the translation of a target mRNA transcript. Although not wishing to be limited to a particular theory, the dsRNA agents of the present invention may act via an RNA interference mechanism (i.e., by inducing RNA interference by interacting with the RNA interference pathway machinery of mammalian cells (RNA-induced silencing complex or RISC)), or by any alternative mechanism or pathway. Methods for achieving gene silencing in plant, invertebrate, and vertebrate cells are well known in the art (see, e.g., Sharp et al., Genes Dev. 2001, 15:485; Bernstein, et al., (2001) Nature 409:363; Nykanen, et al., (2001) Cell 107:309; and Elbashir, et al., (2001) Genes Dev. 15:188), the disclosures of each of which are incorporated herein by reference in their entirety. Gene silencing approaches known in the art can be used in conjunction with the disclosure provided herein to achieve inhibition of xanthine dehydrogenase (XDH) expression.

[0124] The dsRNA agents disclosed herein are composed of a sense strand and an antisense strand, and include, but are not limited to, short interfering RNA (siRNA), RNAi agents, microRNA (miRNA), short hairpin RNA (shRNA), and Dicer substrates. The antisense strand of the dsRNA agents described herein is at least partially complementary to the targeted mRNA, and it is understood in the art that dsRNA duplex structures of various lengths can be used to inhibit target gene expression. For example, dsRNAs with duplex structures of 19, 20, 21, 22, and 23 base pairs are known to effectively induce RNA interference (Elbashir et al., EMBO 2001, 20: 6877-6888). It is also known in the art that shorter or longer RNA duplex structures can also effectively induce RNA interference. Xanthine dehydrogenase (XDH) dsRNA in certain embodiments of the present invention can comprise at least one strand of at least 21 nt in length, or the duplex can have a length based on one of the sequences listed in Tables 1-3 minus 1, 2, 3 nt, or less. Compared to the dsRNA listed in Tables 1-3, respectively, a reduction of 4 nucleotides at one or both ends can also be effective. In some embodiments of the present invention, the xanthine dehydrogenase (XDH) dsRNA agent may have a partial sequence of at least 15, 16, 17, 18, 19, 20 or more consecutive nucleotides from one or more sequences in Tables 1-3, and their ability to inhibit xanthine dehydrogenase (XDH) gene expression differs by no more than 5%, 10%, 15%, 20%, 25% or 30% from the level of inhibition produced by the dsRNA comprising the full sequence (also referred to herein as the "parent" sequence). The sense sequences, antisense sequences, and duplexes disclosed in Tables 1-3 may be referred to herein as "parent" sequences, meaning that the sequences disclosed in Tables 1-3 can be modified, shortened, extended, include substitutions, etc., as described herein, and the resulting sequences retain all or at least some of the efficacy of their parent sequences in the methods and compositions of the present invention.

[0125] Certain embodiments of the compositions and methods of the present invention include single-stranded RNA in the composition and / or administer the single-stranded RNA to a subject. For example, the antisense strands listed in any one of Tables 1-3 can be used as a composition or within a composition that, when administered to a subject, reduces xanthine dehydrogenase (XDH) polypeptide activity and / or xanthine dehydrogenase (XDH) gene expression in the subject. Tables 1-3 show the antisense strand and sense strand core extension base sequences of certain xanthine dehydrogenase (XDH) dsRNA agents. Single-stranded antisense molecules that can be included in certain compositions of the present invention and / or administered in certain methods of the present invention are referred to herein as "single-stranded antisense agents" or "antisense polynucleotide agents." Single-stranded sense molecules that can be included in certain compositions of the present invention and / or administered in certain methods of the present invention are referred to herein as "single-stranded sense agents" or "sense polynucleotide agents." The term "base sequence" herein refers to a polynucleotide sequence without chemical modification or delivery compound. For example, the sense strands shown in Table 1 correspond to the corresponding base sequences in Table 3; however, the corresponding sequences in Table 3 show their respective chemical modifications and delivery compounds. Sequences disclosed herein may be assigned identifiers. For example, a single-stranded sense sequence may be identified by "sense strand SS#"; a single-stranded antisense sequence may be identified by "antisense strand AS#"; and a duplex comprising a sense strand and an antisense strand may be identified by "duplex AD#."

[0126] Table 1 includes a sense strand and an antisense strand, and provides identification numbers for duplexes formed by the sense strand and the antisense strand on the same row in Table 1. In certain embodiments of the present invention, the antisense sequence comprises nucleobase u or nucleobase a at position 1 thereof. In certain embodiments of the present invention, the antisense sequence comprises nucleobase u at position 1 of the antisense sequence. As used herein, the term "matching position" refers in a sense to the position in each strand that "pairs" with each other when the two strands are as a duplex. For example, in a 21 nucleobase sense strand and a 21 nucleobase antisense strand, the nucleobase at position 1 of the sense strand is in a "matching position" with the nucleobase at position 21 of the antisense strand. In another non-limiting example, for a 23 nucleobase sense strand and a 23 nucleobase antisense strand, the nucleobase at position 2 of the sense strand is in a matching position with the nucleobase at position 22 of the antisense strand. In another non-limiting example, in an 18-nucleobase sense strand and an 18-nucleobase antisense strand, nucleobase position 1 of the sense strand is in a matching position with nucleobase position 18 of the antisense strand; and nucleobase position 4 of the sense strand is in a matching position with nucleobase position 15 of the antisense strand. A skilled artisan will understand how to identify matching positions between the sense and antisense strands of duplexes and paired strands.

[0127] The first column in Table 1 represents the duplex AV# of a duplex that includes both the sense and antisense sequences in the same row of the table. For example, Table 1 discloses a duplex designated "Duplex AV00206," which includes corresponding sense and antisense strand sequences. Thus, each row in Table 1 identifies a duplex of the present invention, each including the sense and antisense sequences shown in the same row, with the designated identifier for each duplex shown in the first column of that row.

[0128] In some embodiments of the methods of the present invention, an RNAi agent comprising a polynucleotide sequence shown in Table 1 is administered to a subject. In some embodiments of the present invention, the RNAi agent administered to a subject comprises a duplex comprising at least one of the base sequences listed in Table 1 and comprising 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 sequence modifications. In some embodiments of the methods of the present invention, the RNAi agent comprising a polynucleotide sequence shown in Table 1 is further coupled to a delivery molecule, a non-limiting example of which is a delivery compound comprising GalNAc.

[0129] Table 1: Antisense and sense strand sequences of unmodified xanthine dehydrogenase (XDH) gene RNAi reagents

[0130] All sequences in Table 1 are shown in the 5' to 3' direction. Duplex AV# is the number assigned to the duplexes of the two strands in the same row of the table. The "SEQ ID NO" in the third column of Table 1 is the sequence number of the sense sequence shown in the second column, and the "SEQ ID NO" in the fifth column of Table 1 is the sequence number of the antisense sequence shown in the fourth column.

[0131] Table 2 shows certain chemically modified xanthine dehydrogenase (XDH) gene RNAi agent antisense strand and sense strand sequences of the present invention. In some embodiments of the inventive method, the RNAi agent with the polynucleotide sequence shown in Table 2 is applied to cells and / or objects. In some embodiments of the inventive method, the RNAi agent with the polynucleotide sequence shown in Table 2 is applied to objects. In some embodiments of the present invention, the RNAi agent applied to the object is included in the duplex marked in the first column of Table 2, and comprises the sequence modifications in the sense and antisense strand sequences shown in the third and sixth columns of the same row in Table 2, respectively. In some embodiments of the inventive method, the sequence shown in Table 2 can be connected to (also referred to as "conjugated to" in this article) and can be delivered to the cells and / or tissues of the object. Non-limiting examples of delivery compounds that can be used in certain embodiments of the present invention are compounds containing GalNAc. In Table 2, the first column represents the duplex AV# of the base sequence, corresponding to Table 1. For the base sequences identified by duplex AV#, not only are the base sequences included in the sense and antisense strands indicated, but also the chemical modifications designated as shown in the same row of Table 2. For example, the first row of Table 1 shows the sense and antisense single-stranded base sequences, which together form a duplex, designated as duplex AV#AV00206. In contrast, the duplex AV#AV00206 listed in Table 2 contains the base sequences AV00206-SS and AV00206-AS as a duplex, and also includes the chemical modifications designated as shown in the third and sixth columns of the sense and antisense sequences, respectively. The "Sense Strand SS#" in the second column of Table 2 designates the sense sequence (including modifications) shown in the third column of the same row. The "Antisense Strand AS#" in the fifth column of Table 2 designates the antisense sequence (including modifications) shown in the sixth column. The "SEQ ID NO" in the fourth column of Table 2 designates the sequence number of the sense sequence (including modifications) shown in the third column of the same row. The "SEQ ID NO" in the seventh column of Table 2 is the sequence number of the antisense sequence (including modifications) shown in the sixth column.

[0132] Table 3 shows certain chemically modified xanthine dehydrogenase (XDH) gene RNAi agent antisense and sense strand sequences of the present invention. In some embodiments of the present invention, the RNAi agent shown in Table 3 is applied to cells and / or subjects. In some embodiments of the present invention, the RNAi agent with the polynucleotide sequence shown in Table 3 is applied to subjects. In some embodiments of the present invention, the RNAi agent administered to the subject comprises the duplex identified in the first column of Table 3 and comprises the sequence modification and / or delivery compound shown in the sense and antisense strand sequences of the third and sixth columns of the same row in Table 3, respectively. This sequence is used for certain in vivo testing studies described elsewhere herein. In some embodiments of the present invention, the sequence shown in Table 3 can be connected to (also referred to herein as "conjugated to") a compound for delivery, a non-limiting example of which is a GalNAc-containing compound, i.e., a delivery compound identified as "GLX-n" on the sense strand of the third column in Table 3. Certain embodiments of the delivery compound are identified as "GLS-5," "GLS-15," or "GLX-n" on the sense strand of the third column in Table 3. As used herein and as shown in Table 3, "GLS-5," "GLS-15," and "GLX-n" refer to compounds containing GalNAc. As used herein, "GLX" is used to refer to "GLS" or "GLO" delivery compounds ("X" can be "S" or "O"), and GLX-n can be any GLS or GLO delivery compound that can be attached to the 3'- or 5'-end of an oligonucleotide during synthesis. For example, but not limited to, GLX-13 and GLX-14 can be attached to the 3'-end of an oligonucleotide of the present invention during synthesis; GLX-5 and GLX-15 can be attached to the 5'-end of an oligonucleotide of the present invention during synthesis. In some embodiments, as used herein and as shown in Table 3, "GLX-n" is used to indicate that the attached GalNAc-containing compound is replaced with any one of compounds GLS-1, GLS-2, GLS-3, GLS-4, GLS-5, GLS-6, GLS-7, GLS-8, GLS-9, GLS-10, GLS-11, GLS-12, GLS-13, GLS-14, GLS-15, GLS-16, GLO-1, GLO-2, GLO-3, GLO-4, GLO-5, GLO-6, GLO-7, GLO-8, GLO-9, GLO-10, GLO-11, GLO-12, GLO-13, GLO-14, GLO-15, and GLO-16.In some embodiments, GLX-n represents, for example but not limited to, the GalNAc-containing compounds disclosed in the prior art (Jayaprakash, et al., (2014) J. Am. Chem. Soc., 136, 16958-16961) that can be used for attachment, which compounds were used in certain in vivo testing studies described elsewhere herein. Those skilled in the art will be able to prepare and use the dsRNA compounds of the present invention, wherein the attached delivery compounds include, but are not limited to: GLS-1, GLS-2, GLS-3, GLS-4, GLS-5, GLS-6, GLS-7, GLS-8, GLS-9, GLS-10, GLS-11, GLS-12, GLS-13, GLS-14, GLS-15, GLS-16, GLO-1, GLO-2, GLO-3, GLO-4, GLO-5, GLO-6, GLO-7, GLO-8, GLO-9, GLO-10, GLO-11, GLO-12, GLO-13, GLO-14, GLO-15 and GLO-16. The structures of each of these are provided elsewhere herein. The first column of Table 3 provides the duplex AD# assigned to the duplex of the sense and antisense sequences in that row of the table. For example, duplex AD#AD00352 is a duplex consisting of sense strand AD00352-SS and antisense strand AD00352-AS. Each row in Table 3 provides a sense strand and an antisense strand and discloses the duplex consisting of the sense strand and antisense strand shown. The "sense strand SS#" in the second column of Table 3 is the designated identifier of the sense sequence (including modifications) shown in the third column of the same row. The "antisense strand AS#" in the fifth column of Table 3 is the designated identifier of the antisense sequence (including modifications) shown in the sixth column. The identifier of certain linked GalNAc-containing GLO compounds is shown as GLX-n, and it should be understood that another of the GLO-n or GLS-n compounds can replace the compound shown as GLX-n, and the resulting compounds are also included in the embodiments of the methods and / or compositions of the present invention. In Table 3, SEQ ID NO: 350 to SEQ ID NO: 397, SEQ ID NO: 446 to SEQ ID NO: 451 are the sequences of the sense strand, and SEQ ID NO: 398 to SEQ ID NO: 445, SEQ ID NO: 452 to SEQ ID NO: 457 are the sequences of the antisense strand. In Table 3, the delivery molecule is indicated as "GLS-5," "GLS-15," or "GLX-n" at the 3' or 5' end of the sense strand NO: 350 to SEQ ID NO: 397, SEQ ID NO: 446 to SEQ ID NO: 451.Chemical modifications are indicated as follows: uppercase: 2'-fluoro; lowercase: 2'-methoxy; phosphorothioate: *; Invab: inverted abasic; imann: at the end of each strand:. Or when further coupled with a delivery molecule or targeting group:

[0133] mismatch

[0134] It is known to those skilled in the art that mismatches are tolerable for the efficacy of dsRNA, especially when the mismatch is in the terminal region of the dsRNA. Certain mismatches are better tolerated, for example, mismatches with wobble base pairs G:U and A:C are better tolerated for efficacy (Du et el., A systematic analysis of the silencing effects of an active siRNA at all single-nucleotide mismatched target sites. Nucleic Acids Res. 2005 Mar 21; 33(5): 1671-7. Doi: 10.1093 / nar / gki312. Nucleic Acids Res. 2005; 33(11): 3698). In some embodiments of the methods and compounds of the present invention, the xanthine dehydrogenase (XDH) dsRNA agent may contain one or more mismatches with the xanthine dehydrogenase (XDH) target sequence. In some embodiments, the xanthine dehydrogenase (XDH) dsRNA agent of the present invention does not contain mismatches. In certain embodiments, the xanthine dehydrogenase (XDH) dsRNA agents of the invention contain no more than one mismatch. In some embodiments, the xanthine dehydrogenase (XDH) dsRNA agents of the invention contain no more than two mismatches. In certain embodiments, the xanthine dehydrogenase (XDH) dsRNA agents of the invention contain no more than three mismatches. In some embodiments of the invention, the antisense strand of the xanthine dehydrogenase (XDH) dsRNA agent contains a mismatch with a xanthine dehydrogenase (XDH) target sequence that is not located at the center of the region of complementarity. In some embodiments, the antisense strand of the xanthine dehydrogenase (XDH) dsRNA agent contains one, two, three, four, or more mismatches that are located within the last five, four, three, two, or one nucleotide of either or both of the 5' or 3' ends of the region of complementarity. The methods described herein and / or methods known in the art can be used to determine whether a xanthine dehydrogenase (XDH) dsRNA agent that contains a mismatch with a xanthine dehydrogenase (XDH) target sequence is effective in inhibiting the expression of a xanthine dehydrogenase (XDH) gene.

[0135] Complementarity

[0136] As used herein, unless otherwise indicated, the term "complementarity" when used to describe the relatedness of a first nucleotide sequence (e.g., a xanthine dehydrogenase (XDH) dsRNA agent sense strand or a target xanthine dehydrogenase (XDH) mRNA) to a second nucleotide sequence (e.g., a xanthine dehydrogenase (XDH) dsRNA agent antisense strand or a single-stranded antisense polynucleotide) refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide comprising the second nucleotide sequence [forming hydrogen bonds between base pairs under mammalian physiological conditions (or similar conditions in vitro)] and to form a duplex or duplex structure under certain conditions. Other conditions may also apply, such as physiologically relevant conditions that may be encountered in an organism. A skilled person will be able to determine the most appropriate set of conditions for testing the complementarity of two sequences based on the ultimate application of the hybridizing nucleotides. Complementary sequences include Watson-Crick base pairs or non-Watson-Crick base pairs, and include natural or modified nucleotides or nucleotide mimetics, as long as at least the above hybridization requirements are met. Sequence identity or complementarity is independent of modification.

[0137] For example, the complementary sequence within a xanthine dehydrogenase (XDH) dsRNA as described herein comprises base pairing of an oligonucleotide or polynucleotide comprising a first nucleotide sequence with an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the full length of one or both nucleotide sequences. Such sequences may be referred to herein as being "fully complementary" to one another. It should be understood that in embodiments where two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs are not considered herein as mismatches determined based on complementarity. For example, a xanthine dehydrogenase (XDH) dsRNA reagent comprises an oligonucleotide of 19 nucleotides in length and another oligonucleotide of 20 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 19 nucleotides that is fully complementary to the shorter oligonucleotide. For purposes described herein, this may be referred to as "fully complementary." Therefore, as used herein, "fully complementary" means that all (100%) bases in the contiguous sequence of a first polynucleotide will hybridize to the same number of bases in the contiguous sequence of a second polynucleotide. The contiguous sequence may comprise all or part of the first or second nucleotide sequence.

[0138] As used herein, the term "substantially complementary" means that in a hybridization pair of nucleobase sequences, at least about 85% (but not all) of the bases in the contiguous sequence of a first polynucleotide will hybridize to the same number of bases in the contiguous sequence of a second polynucleotide. If the two sequences contain one or more mismatched base pairs upon hybridization, such as at least 1, 2, 3, 4, or 5 mismatched base pairs, the term "substantially complementary" can be used to refer to a first sequence that forms a duplex of up to 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs (bp) relative to the second sequence while retaining the ability to hybridize under conditions most relevant to its ultimate application, for example, inhibition of xanthine dehydrogenase (XDH) gene expression by the RISC pathway. The term "partially complementary" can be used herein to refer to a hybridization pair of nucleobase sequences in which at least 75% (but not all) of the bases in the contiguous sequence of a first polynucleotide will hybridize to the same number of bases in the contiguous sequence of a second polynucleotide. In some embodiments, "partially complementary" refers to at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the bases in the contiguous sequence of a first polynucleotide will hybridize to the same number of bases in the contiguous sequence of a second polynucleotide.

[0139] The terms "complementary," "fully complementary," "substantially complementary," and "partially complementary" as used herein may refer to base matches between the sense and antisense strands of a xanthine dehydrogenase (XDH) dsRNA agent, base matches between the antisense strand of a xanthine dehydrogenase (XDH) dsRNA agent and the sequence of a target xanthine dehydrogenase (XDH) mRNA, or base matches between a single-stranded antisense oligonucleotide and a target xanthine dehydrogenase (XDH) mRNA sequence. It should be understood that the term "antisense strand of a xanthine dehydrogenase (XDH) dsRNA agent" can refer to the same sequence as a "xanthine dehydrogenase (XDH) antisense polynucleotide agent."

[0140] As used herein, the terms "substantially identical" or "substantial identity" used in reference to nucleic acid sequences refer to nucleic acid sequences comprising sequences having at least about 85% or higher sequence identity, preferably at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity, compared to a reference sequence. The percentage of sequence identity is determined by comparing the optimal alignment of the two sequences over the comparison window. The percentage is calculated by determining the number of positions at which the same nucleic acid base appears in the two sequences to produce the number of matching positions; dividing the number of matching positions by the total number of positions in the comparison window and then multiplying the result by 100 to derive the percentage of sequence identity. The invention disclosed herein includes nucleotide sequences substantially identical to those disclosed herein (e.g., in Tables 1-3). In some embodiments, the nucleotide sequence is identical to a sequence disclosed herein (e.g., in Tables 1-3), or has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0141] As used herein, the term "chain comprising a sequence" refers to an oligonucleotide comprising a nucleotide chain described by a sequence referred to using standard nucleotide nomenclature. As used herein, the term "double-stranded RNA" or "dsRNA" refers to a sequence comprising an RNA molecule or RNAi molecule complex, wherein the molecule or complex has a hybridized double-stranded region comprising two antiparallel and substantially or completely complementary nucleic acid chains, which are referred to as having "sense" and "antisense" directions relative to the target xanthine dehydrogenase (XDH) gene RNA, respectively. The double-stranded region can have any desired length that allows for specific degradation of the target xanthine dehydrogenase (XDH) gene RNA by the RISC pathway, but is typically 9 to 30 base pairs in length, such as 15-30 base pairs in length. Contemplating duplexes between 9 and 30 base pairs, the duplexes can be any length within this range, for example, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs, and any subranges therein, including but not limited to 15-30 base pairs, 15-26 base pairs; 15-23 base pairs, 15-22 base pairs, 15-21 base pairs, 15-20 base pairs, 15-19 base pairs, 15-18 base pairs, 15-17 base pairs, 18-30 base pairs, 18-26 base pairs, 18-27 base pairs, 18-30 base pairs, 18-28 base pairs, 18-30 base pairs, 18-26 base pairs, 18-27 base pairs, 18-28 base pairs, 18-29 base pairs, 18-30 base pairs, 18-29 base pairs, 18-30 base pairs, 18-26 .... -23 base pairs, 18-22 base pairs, 18-21 base pairs, 18-20 base pairs, 19-30 base pairs, 19-26 base pairs, 19-23 base pairs, 19-22 base pairs, 19-21 base pairs, 19-20 base pairs, 20-30 base pairs, 20-26 base pairs, 20-25 base pairs, 20-24 base pairs, 20-23 base pairs, 20-22 base pairs, 20-21 base pairs, 21-30 base pairs, 21-26 base pairs, 21-25 base pairs, 21-24 base pairs, 21-23 base pairs or 21-22 base pairs. The length of xanthine dehydrogenase (XDH) dsRNA agents produced in cells by processing with Dicer and similar enzymes is generally in the range of 19-22 base pairs. One chain of the double-stranded region of the xanthine dehydrogenase (XDH) dsDNA agent comprises a sequence that is substantially complementary to the region of the target xanthine dehydrogenase (XDH) gene RNA. The two chains forming the duplex structure can be from a single RNA molecule having at least one self-complementary region, or can be formed by two or more independent RNA molecules. In the case where the double-stranded region is formed by a single molecule, the molecule can have a duplex structure (referred to herein as a "hairpin loop") formed by a single-stranded nucleotide chain at one 3'-end and another chain at the corresponding 5'-end.In some embodiments of the invention, the hairpin configuration comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more unpaired nucleotides. When the two substantially complementary strands of the xanthine dehydrogenase (XDH) dsRNA reagent consist of separate RNA molecules, these molecules do not need to be covalently linked, but can also be covalently linked. When the two strands are covalently linked by means other than a hairpin loop, the connecting structure is referred to as a "linker." The term "siRNA" is also used herein to refer to dsRNA reagents as described herein.

[0142] In some embodiments of the invention, a xanthine dehydrogenase (XDH) dsRNA agent may comprise a sense and antisense sequence having unpaired nucleotides or nucleotide analogs at one or both ends of the dsRNA agent. An end without unpaired nucleotides is referred to as a "blunt end" and has no nucleotide overhangs. If both ends of a dsRNA agent are blunt-ended, the dsRNA is referred to as "blunt-ended." In some embodiments of the invention, the first end of the dsRNA agent is blunt-ended, in some embodiments, the second end of the dsRNA agent is blunt-ended, and in certain embodiments of the invention, both ends of the xanthine dehydrogenase (XDH) dsRNA agent are blunt-ended.

[0143] In some embodiments of the dsRNA reagent of the present invention, the dsRNA does not have one or two blunt ends. In this case, there is at least one unpaired nucleotide at the end of one chain of the dsRNA reagent. For example, when the 3'-end of one chain of the dsRNA extends beyond the 5'-end of the other chain, there is a nucleotide overhang, and vice versa. The dsRNA may comprise an overhang of at least 1, 2, 3, 4, 5, 6 or more nucleotides. The nucleotide overhang may comprise or be composed of nucleotide / nucleoside analogs, including deoxynucleotides / nucleosides. It should be understood that in some embodiments, the nucleotide overhang is on the sense strand of the dsRNA reagent, on the antisense strand of the dsRNA reagent, or at both ends of the dsRNA reagent, and the nucleotides of the overhang may be present at the 5' end, 3' end, or both ends of the antisense strand or sense strand of the dsRNA. In certain embodiments of the present invention, one or more nucleotides in the overhang are replaced by nucleoside phosphorothioates.

[0144] As used herein, the term "antisense strand" or "guide strand" refers to the strand of a xanthine dehydrogenase (XDH) dsRNA agent that comprises a region that is substantially complementary to a xanthine dehydrogenase (XDH) target sequence. As used herein, the term "sense strand" or "passenger strand" refers to the strand of a xanthine dehydrogenase (XDH) dsRNA agent that comprises a region that is substantially complementary to a region of the antisense strand of a xanthine dehydrogenase (XDH) dsRNA agent.

[0145] Modification

[0146] In some embodiments of the present invention, the RNA of xanthine dehydrogenase (XDH) gene RNAi agent is chemically modified to obtain enhanced stability and / or one or more other beneficial properties. The nucleic acid in certain embodiments of the present invention can be synthesized and / or modified by methods well known in the art, for example, see "Current protocols in Nucleic Acid Chemistry," Beaucage, SL et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference. Modifications that may be present in certain embodiments of the xanthine dehydrogenase (XDH) dsRNA agents of the present invention include, for example: (a) terminal modifications, such as 5'-terminal modifications (phosphorylation, conjugation, reverse ligation, etc.), 3'-terminal modifications (conjugation, DNA nucleotides, reverse ligation, etc.); (b) base modifications, such as substitution with stabilizing bases, destabilizing bases, or bases that base-pair with an expanded library of partners, deletion of bases (abasic nucleotides), or conjugated bases; (c) sugar modifications (e.g., at the 2' or 4' position) or replacement of sugars; and (d) backbone modifications, including modification or replacement of phosphodiester bonds. Specific examples of RNA compounds useful in certain embodiments of the xanthine dehydrogenase (XDH) dsRNA agents, xanthine dehydrogenase (XDH) antisense polynucleotides, and xanthine dehydrogenase (XDH) sense polynucleotides of the present invention include, but are not limited to, RNAs comprising modified backbones or lacking natural internucleoside linkages. As a non-limiting example, RNAs with backbone modifications may not have phosphorus atoms in the backbone. RNAs that do not have a phosphorus atom in their internucleoside backbone can be referred to as oligonucleosides. In certain embodiments of the invention, the modified RNA has a phosphorus atom in its internucleoside backbone.

[0147] It should be understood that the term "RNA molecule" or "RNA" or "ribonucleic acid molecule" includes not only RNA molecules expressed or found in nature, but also analogs and derivatives of RNA, which include one or more ribonucleotides / ribonucleoside analogs or derivatives as described herein or known in the art. The terms "ribonucleoside" and "ribonucleotide" are used interchangeably herein. RNA molecules can be modified in the core base structure or the ribose-phosphate backbone structure (e.g., as described below), and molecules containing ribonucleoside analogs or derivatives must retain the ability to form duplexes. As non-limiting examples, the RNA molecule can also include at least one modified ribonucleoside, which includes but is not limited to 2'-methoxy-modified nucleosides, nucleosides comprising 5' thiophosphate groups, terminal nucleosides connected to cholesterol derivatives or dodecanoic acid bisdecylamide groups, locked nucleosides, abasic nucleosides, 2'-deoxy-2'-fluoro modified nucleosides, 2'-amino-modified nucleosides, 2'-alkyl-modified nucleosides, morpholino nucleosides, phosphoramidates or the non-natural bases comprising nucleosides, or any combination thereof. In some embodiments of the invention, the RNA molecule includes the modified ribonucleoside of following number: at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or up to the total length of the ribonucleoside of xanthine dehydrogenase (XDH) dsRNA agent molecule. For each of the multiple modified ribonucleosides in this RNA molecule, modification need not be identical.

[0148] In some embodiments, the dsRNA reagents, xanthine dehydrogenase (XDH) antisense polynucleotides and / or xanthine dehydrogenase (XDH) sense polynucleotides of the present invention can include one or more independently selected modified nucleotides and / or one or more independently selected non-phosphodiester bonds. The term "independent selection" as used herein refers to selected elements, such as modified nucleotides, non-phosphodiester bonds, etc., and refers to two or more selected elements that can be identical to each other but do not necessarily have to be identical to each other. As used herein, "nucleotide bases", "nucleotides" or "nucleobases" are heterocyclic pyrimidine or purine compounds, which are standard components of all nucleic acids and include bases that form nucleotides: adenine (a), guanine (g), cytosine (c), thymine (t) and uracil (u). Nucleobases can be further modified to include (but are not intended to be limiting): universal bases, hydrophobic bases, mixed bases, bases with expanded size and fluorinated bases. The terms "ribonucleotides" or "nucleotides" can be used herein to refer to unmodified nucleotides, modified nucleotides or alternative moieties. Those skilled in the art will recognize that guanine, cytosine, adenine, and uracil may be replaced by other moieties without significantly altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such a replacement moiety.

[0149] In one embodiment, the RNA of the modification contemplated for use in the methods and compositions described herein is a peptide nucleic acid (PNA) having the ability to form a desired duplex structure and allow or mediate the specific degradation of target RNA via RISC pathway. In certain embodiments of the invention, xanthine dehydrogenase (XDH) gene RNA interfering agents include single-stranded RNA that interacts with target xanthine dehydrogenase (XDH) gene RNA sequence to guide target xanthine dehydrogenase (XDH) gene RNA cutting.

[0150] The modified RNA backbone can include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates (including 3'-alkylenephosphonates and chiral phosphonates), phosphinates, phosphoramidates (including 3'-aminophosphoramidates and aminoalkylphosphoramidates), thiophosphoramidates, thioalkylphosphonates, thioalkylphosphotriesters, and borate phosphates (which have normal 3'-5' linkages, as well as 2'-5' linkage analogs of these, and those with inverted polarity, wherein adjacent nucleoside unit pairs are linked in a 3'-5' to 5'-3' or 2'-5' to 5'-2' form). Various salts, mixed salts, and free acid forms are also included. Methods for preparing phosphorus-containing bonds are routinely practiced in the art, and such methods can be used to prepare certain modified xanthine dehydrogenase (XDH) dsRNA agents, certain modified xanthine dehydrogenase (XDH) antisense polynucleotides and / or certain modified xanthine dehydrogenase (XDH) sense polynucleotides of the present invention.

[0151] The modified RNA backbone that does not contain a phosphorus atom has a backbone formed by a short-chain alkyl or cycloalkyl nucleoside linkage, a mixed heteroatom and alkyl or cycloalkyl nucleoside linkage, or one or more short-chain heteroatom or heterocyclic nucleoside linkages. It includes those with morpholine bonds (partially formed by the sugar portion of the nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; methyl and thiomethyl backbones; methylenemethyl and thiomethyl backbones; backbones containing olefins; sulfamate backbones; methyleneimino and methylenehydrazine backbones; sulfonate and sulfonamide backbones; amide backbones; and other parts mixed with N, O, S and CH2 components. Methods for preparing modified RNA backbones that do not contain phosphorus atoms are conventional practices in the art, and such methods can be used to prepare certain modified xanthine dehydrogenase (XDH) dsRNA reagents of the present invention, certain modified xanthine dehydrogenase (XDH) antisense polynucleotides and / or certain modified xanthine dehydrogenase (XDH) sense polynucleotides.

[0152] In certain embodiments of the present invention, RNA mimics are included in xanthine dehydrogenase (XDH) dsRNA, xanthine dehydrogenase (XDH) antisense polynucleotides and / or xanthine dehydrogenase (XDH) sense polynucleotides, such as, but not limited to, replacing the sugar and internucleoside linkages (i.e., backbones) of nucleotide units with new groups. In such embodiments, the base units are retained to hybridize with suitable xanthine dehydrogenase (XDH) nucleic acid target compounds. One such oligomeric compound (an RNA mimic that has been shown to have excellent hybridization properties) is referred to as peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced by an amide-containing backbone, particularly an aminoethylglycine backbone. The core base is retained and is bound directly or indirectly to the nitrogen atoms of the backbone amide portion. Methods for preparing RNA mimics are conventional practices in the art, and such methods can be used to prepare xanthine dehydrogenase (XDH) dsRNA reagents of certain modifications of the present invention.

[0153] Some embodiments of the present invention include RNAs having phosphorothioate backbones and oligonucleosides having heteroatom backbones, particularly -CH2-NH-CH2-, -CH2-N(CH3)-O-CH2- [referred to as methylene (methylimino) or MMI backbones], -CH2-ON(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2-, and -N(CH3)-CH2- [where the natural phosphodiester backbone is represented as -OPO-CH2-]. Methods for preparing RNAs having phosphorothioate backbones and oligonucleosides having heteroatom backbones are routinely practiced in the art, and such methods can be used to prepare certain modified xanthine dehydrogenase (XDH) dsRNA agents, certain xanthine dehydrogenase (XDH) antisense polynucleotides, and / or certain xanthine dehydrogenase (XDH) sense polynucleotides of the present invention.

[0154] The modified RNA may also comprise one or more substituted sugar moieties. The xanthine dehydrogenase (XDH) dsRNA, xanthine dehydrogenase (XDH) antisense polynucleotides and / or xanthine dehydrogenase (XDH) sense polynucleotides of the present invention may comprise one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, wherein alkyl, alkenyl, and alkynyl groups may be substituted or unsubstituted C1 to C 10 Alkyl or C2 to C 10 Exemplary suitable modifications include: O[(CH2) n O] m CH3, O(CH2) n OCH3, O(CH2) n NH2, O(CH2) nCH3, O(CH2) n ONH2, and O(CH2) n ON[(CH2) n CH3)]2, wherein n and m are 1 to about 10. In other embodiments, the dsRNA comprises one of the following at the 2' position: C1 to C 10 Lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino; substituted silyl, RNA cleavage group, reporter group, intercalator; a group for improving the pharmacokinetic properties of xanthine dehydrogenase (XDH) dsRNA agent; or a group for improving the pharmacodynamic properties of xanthine dehydrogenase (XDH) dsRNA agent, xanthine dehydrogenase (XDH) antisense polynucleotide and / or xanthine dehydrogenase (XDH) sense polynucleotide, and other substituents with similar properties. In some embodiments, the modification includes 2'-methoxyethoxy (2'-O-CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., alkoxy-alkoxy. Another exemplary modification is 2'-dimethylaminoethoxyethoxy, i.e., O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, as described in the Examples below; and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O-CH2-O-CH2-N(CH2)2. Methods for preparing modified RNAs such as those described are routinely practiced in the art, and such methods can be used to prepare certain modified xanthine dehydrogenase (XDH) dsRNA agents of the invention.

[0155] Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2) and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of the xanthine dehydrogenase (XDH) dsRNA reagents, xanthine dehydrogenase (XDH) antisense polynucleotides of the present invention, xanthine dehydrogenase (XDH) sense polynucleotides and / or xanthine dehydrogenase (XDH) sense polynucleotides, particularly at the 3' position of the sugar on the 3' terminal nucleotide or in the 2'-5' linked xanthine dehydrogenase (XDH) dsRNA, xanthine dehydrogenase (XDH) antisense polynucleotides or xanthine dehydrogenase (XDH) sense polynucleotides, and at the 5' position of the 5' terminal nucleotide. Xanthine dehydrogenase (XDH) dsRNA agents, xanthine dehydrogenase (XDH) antisense polynucleotides and / or xanthine dehydrogenase (XDH) sense polynucleotides can also have sugar mimetics, such as cyclobutyl moieties instead of pentofuranosyl sugars. Methods for preparing modified RNAs such as those described are routinely practiced in the art, and such methods can be used to prepare certain modified xanthine dehydrogenase (XDH) dsRNA agents, xanthine dehydrogenase (XDH) antisense polynucleotides and / or xanthine dehydrogenase (XDH) sense polynucleotides of the present invention.

[0156] In some embodiments, xanthine dehydrogenase (XDH) dsRNA agents, xanthine dehydrogenase (XDH) antisense polynucleotides and / or xanthine dehydrogenase (XDH) sense polynucleotides can include modifications or substitutions of nucleobases (often referred to in the art as "bases"). As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine. Adenine, 5-uracil (pseudouracil), 4-thiouracil; 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenines and guanines; 5-halo, in particular 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines; 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-azaguanine and 7-azaadenine and 3-azaguanine and 3-azaadenine. Additional nucleobases that can be included in certain embodiments of the xanthine dehydrogenase (XDH) dsRNA agents of the invention are known in the art, see, for example: Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. Ed. Wiley-VCH, 2008; The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, JL, Ed. John Wiley & Sons, 1990, English et al., Angewandte Chemie, International Edition, 1991, 30, 613, Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., Ed., CRC Press, 1993.Methods for preparing dsRNA, xanthine dehydrogenase (XDH) antisense polynucleotides and / or xanthine dehydrogenase (XDH) sense polynucleotides comprising nucleobase modifications and / or substitutions, such as those described herein, are routinely practiced in the art, and such methods can be used to prepare certain modified xanthine dehydrogenase (XDH) dsRNA agents, xanthine dehydrogenase (XDH) sense polynucleotides and / or xanthine dehydrogenase (XDH) antisense polynucleotides of the invention.

[0157] Certain embodiments of the xanthine dehydrogenase (XDH) dsRNA agents, xanthine dehydrogenase (XDH) antisense polynucleotides, and / or xanthine dehydrogenase (XDH) sense polynucleotides of the present invention include RNA modified to include one or more locked nucleic acids (LNAs). Locked nucleic acids are nucleotides with modified ribose moieties that contain an additional bridge connecting the 2' and 4' carbons. This structure effectively "locks" the ribose in a 3'-endo conformation. Adding locked nucleic acid to the xanthine dehydrogenase (XDH) dsRNA reagent, xanthine dehydrogenase (XDH) antisense polynucleotide and / or xanthine dehydrogenase (XDH) sense polynucleotide of the present invention can increase stability in serum and reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). Methods for preparing dsRNA agents comprising locked nucleic acids, xanthine dehydrogenase (XDH) antisense polynucleotides, and / or xanthine dehydrogenase (XDH) sense polynucleotides are routinely practiced in the art, and such methods can be used to prepare certain modified xanthine dehydrogenase (XDH) dsRNA agents of the present invention. Certain embodiments of the xanthine dehydrogenase (XDH) dsRNA compounds, sense polynucleotides, and / or antisense polynucleotides of the present invention include at least one modified nucleotide, wherein the at least one modified nucleotide comprises: 2'-methoxy nucleotides, 2'-fluoro nucleotides, 2'-deoxy nucleotides, 2',3'-seco nucleotide mimetics, locked nucleotides, 2'-F-arabino nucleotides, 2'-methoxyethyl nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, and 3'-Ome nucleotides, nucleotides comprising a 5'-phosphorothioate group, or a terminal nucleotide linked to a cholesterol derivative or a dodecanoic acid bisdecylamide group, a 2'-amino modified nucleotide, a phosphoramidate, or an unnatural base comprising a nucleotide. In some embodiments, the xanthine dehydrogenase (XDH) dsRNA compound comprises an E-vinylphosphonate nucleotide at the 5' end of the antisense strand (also referred to herein as the guide strand).

[0158] In certain embodiments of the present invention, at least one modified nucleotide is included at the 3' and 5' ends of the xanthine dehydrogenase (XDH) dsRNA compound, the sense polynucleotide, and / or the 3' end of the antisense polynucleotide, wherein the at least one modified nucleotide comprises: an abasic nucleotide, a ribitol, an inverted nucleotide, an inverted abasic nucleotide, an inverted 2'-OMe nucleotide, or an inverted 2'-deoxynucleotide. It is known to those skilled in the art that including an abasic or inverted abasic nucleotide at the end of an oligonucleotide can enhance stability (Czauderna et al. Structural variations and stabilizing modifications of synthetic siRNAs in mammalian cells. Nucleic Acids Res. 2003; 31(11): 2705-2716. doi: 10.1093 / nar / gkg393).

[0159] In certain embodiments of the present invention, the xanthine dehydrogenase (XDH) dsRNA compound or antisense polynucleotide comprises at least one modified nucleotide, wherein the at least one modified nucleotide comprises an open-ring nucleic acid nucleotide (UNA) or / and a glycol nucleic acid nucleotide (GNA). It is known to those skilled in the art that UNA and GNA are thermally unstable chemical modifications that can significantly improve the off-target spectrum of siRNA compounds (Janas, et al., Selection of GalNAc-conjugated siRNAs with limited off-target-driven rat hepatotoxicity. Nat Commun. 2018; 9(1): 723. doi: 10.1038 / s41467-018-02989-4; Laursen et al., Utilization of unlocked nucleic acid (UNA) to enhance siRNA performance in vitro and in vivo. Mol BioSyst. 2010; 6: 862–70).

[0160] In certain embodiments, the present invention relates to open-ring nucleic acid (UNA) oligomers for therapeutic use. Open-ring nucleic acid (UNA) is an acyclic analog of RNA in which the bond between the C2' and C3' atoms of the ribose ring has been severed. It has been demonstrated that the incorporation of UNA is well tolerated for siRNA gene silencing activity and can even enhance its activity in some cases (Meghan A. et al. "Locked vs. unlocked nucleic acids (LNA vs. UNA): contrasting structures work towards common therapeutic goals". Chem. Soc. Rev., 2011, 40, 5680–5689).

[0161] UNA is a heat-labile modification, and replacing ribonucleotides with UNA reduces base pairing strength and duplex stability. Strategic placement of UNA in the seed region of the siRNA antisense strand can reduce off-target activity in gene silencing mechanisms mediated by microRNA (miRNA). miRNAs primarily recognize target genes for gene suppression through base pairing between the antisense seed region (positions 2-8 from the 5' end) and the target mRNA. Each miRNA has the potential to regulate a large number of genes. The siRNA antisense strand loaded by the RNA-induced silencing complex (RISC) can also potentially regulate a large number of unintended genes through miRNA-mediated mechanisms. Therefore, the addition of thermally unstable nucleotides, such as UNA, to the seed region of siRNA can reduce off-target activity (Lam JK, Chow MY, Zhang Y, Leung SW. siRNA Versus miRNA as Therapeutics for Gene Silencing. Mol Ther Nucleic Acids. 2015 Sep 15; 4(9): e252. doi: 10.1038 / mtna.2015.23. PMID: 26372022; PMCID: PMC4877448.). Specifically, such RNA oligonucleotides or RNA oligonucleotide complexes contain at least one UNA nucleobase monomer in the seed region (Narendra Vaish et al. "Improved specificity of gene silencing by siRNAs containing unlocked nucleobase analog". Nucleic Acids Research, 2011, Vol. 39, No. 5 1823–1832).

[0162] According to the present invention, potential advantages of incorporating UNA into RNA oligonucleotides or RNA oligonucleotide complexes include but are not limited to:

[0163] 1. Reduce off-target activity. Adding UNA to the siRNA seed region reduces the base pairing strength of the seed region, thereby reducing potential off-target activity caused by the micro-RNA mechanism.

[0164] 2. UNA is well tolerated in terms of siRNA activity. In some cases, UNA can lead to enhanced activity.

[0165] Exemplary UNA monomers that can be used in this technology include, but are not limited to:

[0166] Invab is an inverted abasic (deoxyribose) residue. For example, in some embodiments, the sense strand or the antisense strand may include a "terminal cap," which, as used herein, is a non-nucleotide compound or other moiety that can be incorporated at one or more ends of the strand of the RNAi agents disclosed herein and, in some cases, can provide certain advantageous properties to the RNAi agents, such as, for example, protection against exonuclease degradation. In some embodiments, an inverted abasic residue (Invab) is added as a terminal cap (see, e.g.,

[0167] F. Czauderna, Nucleic Acids Res., 2003, 31(11), 2705-16). Terminal caps are generally known in the art, and in some embodiments, the terminal cap is present at the 5' end, the 3' end, or both the 5' and 3' ends of the sense strand.

[0168] In certain embodiments of the present invention, xanthine dehydrogenase (XDH) dsRNA compounds and antisense polynucleotides comprise at least one modified nucleotide, wherein the at least one modified nucleotide comprises an isomannite nucleotide. In certain embodiments, the sense strand independently comprises an isomannite residue and its stereoisomers at the 3' or 5' end. Examples of specific nucleotides comprising isomannite residues and their stereoisomers include, but are not limited to: wherein the phrase "Olig" each independently represents a polynucleotide moiety. Exemplary isomannite residues (imann) include, but are not limited to, the following: In certain embodiments, the isomannol residue (imann) can be replaced with its stereoisomers. In certain embodiments, the sense strand independently comprises an isomannol residue (imann) at either the 3' or 5' terminus, and optionally further comprises a targeting group conjugated to the 5' terminus, such as N-acetyl-galactosamine, preferably the aforementioned GLS-15. An exemplary structure is shown below:

[0169] wherein the phrase "Olig" each independently represents a polynucleotide portion.

[0170] Another modification can be included in the RNA of the xanthine dehydrogenase (XDH) dsRNA agent, xanthine dehydrogenase (XDH) antisense polynucleotide, and / or xanthine dehydrogenase (XDH) sense polynucleotide of certain embodiments of the present invention, comprising one or more ligands, moieties, or conjugates chemically linked to the RNA that enhance one or more characteristics of the xanthine dehydrogenase (XDH) dsRNA agent, xanthine dehydrogenase (XDH) antisense polynucleotide, and / or xanthine dehydrogenase (XDH) sense polynucleotide, respectively. Non-limiting examples of characteristics that can be enhanced are: xanthine dehydrogenase (XDH) dsRNA agent, xanthine dehydrogenase (XDH) antisense polynucleotide, and / or xanthine dehydrogenase (XDH) sense polynucleotide activity, cellular distribution, delivery of the xanthine dehydrogenase (XDH) dsRNA agent, pharmacokinetic properties of the xanthine dehydrogenase (XDH) dsRNA agent, and cellular uptake of the xanthine dehydrogenase (XDH) dsRNA agent. In some embodiments of the present invention, the xanthine dehydrogenase (XDH) dsRNA agent comprises one or more targeting groups or linking groups, which, in certain embodiments of the xanthine dehydrogenase (XDH) dsRNA agent of the present invention, are conjugated to the sense strand. A non-limiting example of a targeting group is a compound comprising N-acetyl-galactosamine (GalNAc). The terms "delivery molecule," "targeting group," "targeting agent," "linking agent," "targeting compound," and "targeting ligand" are used interchangeably herein. In certain embodiments of the present invention, the xanthine dehydrogenase (XDH) dsRNA agent comprises a targeting compound conjugated to the 5'-end of the sense strand. In certain embodiments of the present invention, the xanthine dehydrogenase (XDH) dsRNA agent comprises a targeting compound conjugated to the 3'-end of the sense strand. In some embodiments of the present invention, the xanthine dehydrogenase (XDH) dsRNA agent comprises a targeting group containing GalNAc. In certain embodiments of the invention, the xanthine dehydrogenase (XDH) dsRNA agent does not comprise a targeting compound conjugated to one or both of the 3'-end and the 5'-end of the sense strand. In certain embodiments of the invention, the xanthine dehydrogenase (XDH) dsRNA agent does not comprise a targeting compound containing GalNAc conjugated to one or both of the 5'-end and the 3'-end of the sense strand.

[0171] Additional targeting and linking agents are well known in the art. For example, targeting and linking agents that can be used in certain embodiments of the present invention include, but are not limited to, lipid moieties, such as cholesterol moieties (Letsinger et al., Proc. Natl. Acids. Sci. USA, 1989, 86:6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), thioethers, such as beryl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20: 533-538), fatty chains, such as dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10: 1111-1118; Kabanov et al., FEBS Lett., 1990, 259: 327-330; Svinarchuk et al., Biochimie, 1993, 75: 49-54), phospholipids, such as di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycerol-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36: 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18: 3777-3783), polyamines or polyethylene glycol chains (Manoharan et al., Tetrahedron Lett., 1995, 36: 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18: 3777-3783), al., Nucleosides & Nucleotides, 1995, 14:969-973) or adamantaneacetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), a palmitoyl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237) or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0172] Certain embodiments of compositions comprising xanthine dehydrogenase (XDH) dsRNA agents, xanthine dehydrogenase (XDH) antisense polynucleotides, and / or xanthine dehydrogenase (XDH) sense polynucleotides may include a ligand that alters the distribution, targeting, and other properties of the xanthine dehydrogenase (XDH) dsRNA agent. In some embodiments of compositions comprising xanthine dehydrogenase (XDH) dsRNA agents of the invention, the ligand increases affinity for a selected target (e.g., a molecule, cell or cell type, compartment, such as a cell or organ compartment, tissue, organ, or body region), for example, compared to a species in which such a ligand is absent. Ligands useful in the compositions and / or methods of the invention can be naturally occurring substances, such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulins), carbohydrates (e.g., glucans, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid), or lipids. The ligand can also be a recombinant or synthetic molecule, such as a synthetic polymer, such as a synthetic polyamino acid or polyamine. Examples of polyamino acids are polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly-(L-lactide-co-glycolic acid) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphazene. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendritic polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or alpha helical peptides.

[0173] The ligands included in the compositions and / or methods of the present invention may comprise a targeting group, non-limiting examples of which are cell or tissue targeting agents, e.g., lectins, glycoproteins, lipids, or proteins, e.g., antibodies that bind to specific cell types such as kidney cells or liver cells. The targeting group may be thyrotropin, melanocyte stimulating hormone, lectin, glycoprotein, surfactant protein A, mucin carbohydrates, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, multivalent fucose, glycosylated polyamino acids, multivalent galactose, transferrin, bisphosphonates, polyglutamate, polyaspartic acid, lipids, cholesterol, steroids, bile acids, folic acid, vitamin B12, vitamin A, biotin, or RGD peptide or RGD peptide mimetic.

[0174] Other examples of ligands include dyes, intercalating agents (e.g., acridine), cross-linking agents (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine); artificial endonucleases (e.g., EDTA), lipophilic molecules, such as cholesterol, bile acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl, hexadecylglycerol, borneol, menthol, 1,3-propylene glycol, heptadecanol, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholic acid, dimethoxytrityl or phenoxazine and peptide conjugates (e.g., antennary peptides, Tat peptides), alkylating agents, phosphates, amino groups, sulfhydryl groups, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino groups, alkyl groups, substituted alkyl groups, radiolabels, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazoles, bisimidazoles, histamine, imidazole clusters, acridine-imidazole conjugates, tetraazamacrocyclic Eu 3+ complex), dinitrophenyl, HRP or AP.

[0175] The part included in the composition and / or method of the present invention can be a protein, such as a glycoprotein or a peptide, for example, a molecule with a specific affinity to a co-ligand, or an antibody, for example, an antibody that is combined with a specific cell type such as a cancer cell, endothelial cell, myocardial cell or osteocyte. In the embodiment of the composition and / or method of the present invention, a useful part can be a hormone or hormone receptor. In the embodiment of the composition and / or method of the present invention, a useful part can be a lipid, a lectin, a carbohydrate, a vitamin, a coenzyme, a multivalent lactose, a multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose or multivalent fucose. In the embodiment of the composition and / or method of the present invention, a useful part can be, for example, a substance that increases the uptake of a xanthine dehydrogenase (XDH) dsRNA agent into a cell by destroying the cytoskeleton of the cell (for example, by destroying the microtubules, microfilaments and / or intermediate filaments of the cell). Non-limiting examples of such agents are: taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, and myoservin.

[0176] In some embodiments, the ligands attached to the xanthine dehydrogenase (XDH) dsRNA agents of the present invention are used as pharmacokinetic (PK) modulators. Examples of PK modulators that can be used in the compositions and methods of the present invention include, but are not limited to, lipophiles, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, cholesterol, fatty acids, bile acid, lithocholic acid, dialkyl glycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, serum protein-binding aptamers, and the like. Oligonucleotides containing many phosphorothioate bonds are also known to bind to serum proteins, and thus, short oligonucleotides containing multiple phosphorothioate bonds in the backbone, such as oligonucleotides of about 5 bases, 10 bases, 15 bases, or 20 bases, can also be used as ligands in the compositions and / or methods of the present invention.

[0177] Xanthine dehydrogenase (XDH) dsRNA reagent composition

[0178] In some embodiments of the present invention, the xanthine dehydrogenase (XDH) dsRNA agent is in a composition. The composition of the present invention may include one or more xanthine dehydrogenase (XDH) dsRNA agents and optionally one or more pharmaceutically acceptable carriers, delivery agents, targeting agents, detectable labels, etc. Non-limiting examples of targeting agents useful in some embodiments of the present methods are agents that direct the xanthine dehydrogenase (XDH) dsRNA agent of the present invention to and / or into the cells to be treated. The choice of targeting agent will depend on the following factors: the nature of the xanthine dehydrogenase (XDH)-related disease or condition, and the target cell type. In one non-limiting example, in some embodiments of the present invention, it may be necessary to target the xanthine dehydrogenase (XDH) dsRNA agent to and / or into hepatocytes. It should be understood that in some embodiments of the present methods, the therapeutic agent comprises a xanthine dehydrogenase (XDH) dsRNA agent with only a delivery agent, such as a delivery agent comprising N-acetylgalactosamine (GalNAc), without any additional linking elements. For example, in some aspects of the invention, a xanthine dehydrogenase (XDH) dsRNA agent can be linked to a delivery compound comprising GalNAc and contained in a composition comprising a pharmaceutically acceptable carrier and administered to a cell or subject without any detectable label or targeting agent, etc., linked to the xanthine dehydrogenase (XDH) dsRNA agent.

[0179] In the case where the xanthine dehydrogenase (XDH) dsRNA agent of the present invention is administered together with one or more delivery agents, targeting agents, labeling agents, etc. and / or is attached thereto, those skilled in the art will understand and be able to select and use suitable agents for use in the methods of the present invention. Labeling agents can be used in certain methods of the present invention to determine the location of the xanthine dehydrogenase (XDH) dsRNA agent in cells and tissues, and can be used to determine the cell, tissue or organ location of a therapeutic composition comprising a xanthine dehydrogenase (XDH) dsRNA agent that has been administered in the methods of the present invention. Means for attaching and using labeling agents such as enzyme labels, dyes, radioactive labels, etc. are well known in the art. It should be understood that in some embodiments of the compositions and methods of the present invention, the labeling agent is attached to one or both of the sense polynucleotide and the antisense polynucleotide contained in the xanthine dehydrogenase (XDH) dsRNA agent.

[0180] Delivery of Xanthine Dehydrogenase (XDH) dsRNA Reagents and Xanthine Dehydrogenase (XDH) Antisense Polynucleotide Reagents

[0181] Certain embodiments of the inventive method include delivering a xanthine dehydrogenase (XDH) dsRNA reagent into a cell. As used herein, the term "delivery" refers to promoting or influencing cellular uptake or absorption. The absorption or uptake of a xanthine dehydrogenase (XDH) dsRNA reagent can occur through independent diffusion or active cellular processes, or can be carried out by using a delivery agent, targeting agent, etc. that can be relevant to the xanthine dehydrogenase (XDH) dsRNA reagent of the present invention. Delivery methods suitable for the inventive method include, but are not limited to, in vivo delivery, wherein the xanthine dehydrogenase (XDH) dsRNA reagent is injected into a tissue site or is administered systemically. In some embodiments of the present invention, a xanthine dehydrogenase (XDH) dsRNA reagent is connected to a delivery agent.

[0182] Non-limiting examples of methods that can be used to deliver xanthine dehydrogenase (XDH) dsRNA agents to cells, tissues and / or objects include: xanthine dehydrogenase (XDH) dsRNA-GalNAc conjugates, SAMiRNA technology, LNP-based delivery methods and naked RNA delivery. These and other delivery methods have been successfully used in the art to deliver therapeutic RNAi agents to treat various diseases and conditions, such as, but not limited to: liver disease, acute intermittent porphyria (AIP), hemophilia, pulmonary fibrosis, etc. Detailed information on various delivery methods can be found in publications such as: Nikam, RR & K.R. Gore (2018) Nucleic Acid Ther, 28(4), 209-224 Aug 2018; Springer AD & S.F. Dowdy (2018) Nucleic Acid Ther. Jun 1; 28(3): 109–118; Lee, K. et al., (2018) Arch Pharm Res, 41(9), 867-874; and Nair, JK et al., (2014) J. Am. Chem. Soc. 136: 16958-16961, the contents of which are incorporated herein by reference.

[0183] Some embodiments of the present invention include using lipid nanoparticles (LNP) by xanthine dehydrogenase (XDH) dsRNA reagent of the present invention is delivered to cell, tissue and / or object.LNP is generally used for delivering xanthine dehydrogenase (XDH) dsRNA reagent in vivo, including therapeutic xanthine dehydrogenase (XDH) dsRNA reagent.A benefit of using LNP or other delivery agents is that, when using LNP or other delivery agents to be delivered to object, the stability of xanthine dehydrogenase (XDH) gene RNA agent increases.In some embodiments of the present invention, LNP comprises the cation LNP that has one or more xanthine dehydrogenase (XDH) gene RNAi molecules of the present invention.The LNP comprising xanthine dehydrogenase (XDH) gene RNAi molecules is applied to object, LNP and the xanthine dehydrogenase (XDH) gene RNAi molecules attached thereto are taken up by cells by endocytosis, and their presence causes the release of RNAi triggering molecules, thus mediating RNAi.

[0184] Another non-limiting example of a delivery agent that can be used in embodiments of the present invention to deliver the xanthine dehydrogenase (XDH) dsRNA agent of the present invention to cells, tissues and / or objects is: a reagent comprising GalNAc, which is connected to the xanthine dehydrogenase (XDH) dsRNA agent of the present invention and delivers the xanthine dehydrogenase (XDH) dsRNA agent to cells, tissues and / or objects. PCT application WO2020191183A1 discloses examples of other delivery agents that can be used in certain embodiments of the methods and compositions of the present invention that comprise GalNAc. A non-limiting example of a GalNAc targeting ligand that can be used in the compositions and methods of the present invention to deliver the xanthine dehydrogenase (XDH) dsRNA agent to cells is a targeting ligand cluster. Examples of targeting ligand clusters proposed herein are: a GalNAc ligand (GLO) with a phosphodiester bond and a GalNAc ligand (GLS) with a phosphorothioate bond. The term "GLX-n" may be used herein to represent an example of a GalNAc-containing compound that is linked, but is not limited to, the following compounds: GLS-1, GLS-2, GLS-3, GLS-4, GLS-5, GLS-6, GLS-7, GLS-8, GLS-9, GLS-10, GLS-11, GLS-12, GLS-13, GLS-14, GLS-15, GLS-16, any one of GLO-1, GLO-2, GLO-3, GLO-4, GLO-5, GLO-6, GLO-7, GLO-8, GLO-9, GLO-10, GLO-11, GLO-12, GLO-13, GLO-14, GLO-15 and GLO-16, the structure of each of which is shown below. In the figure below, the connection position of the GalNAc targeting ligand to the RNAi agent of the present invention is on the far right of each targeting ligand. It will be understood that any of the RNAi and dsRNA molecules of the present invention can be linked to GLS-1, GLS-2, GLS-3, GLS-4, GLS-5, GLS-6, GLS-7, GLS-8, GLS-9, GLS-10, GLS-11, GLS-12, GLS-13, GLS-14, GLS-15, GLS-16, GLO-1, GLO-2, GLO-3, GLO-4, GLO-5, GLO-6, GLO-7, GLO-8, GLO-9, GLO-10, GLO-11, GLO-12, GLO-13, GLO-14, GLO-15, and GLO-16, and the structures of GLS-1 to GLS-2 and GLS-3 to GLO-4 are shown below.

[0185] " Heterocycloalkyl " represents a non-aromatic partially saturated or fully saturated ring (for example, 3-10 or 3-7 membered heterocycloalkyl) with a specified number of ring atoms, wherein the ring atoms are composed of one or more heteroatoms (for example, 1, 2, 3 or 4 heteroatoms) selected from N, O and S, and the remaining ring atoms are carbon. A 5-membered heterocycloalkyl is a heterocycloalkyl with 5 ring atoms. A 6-membered heterocycloalkyl is a heterocycloalkyl with 6 ring atoms. Heterocycloalkyl can be monocyclic or polycyclic (for example, bicyclic, tricyclic). The example of heterocycloalkyl includes oxirane, aziridine, azetidinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, piperidinyl, piperazinyl, morpholinyl and thiomorpholinyl. In addition, one ring of polycyclic heterocycloalkyl can be aromatic (for example, aryl or heteroaryl), as long as the polycyclic heterocycloalkyl is attached to the parent structure via non-aromatic carbon or nitrogen atom. For example, 1,2,3,4-tetrahydroquinolin-1-yl (wherein the moiety is attached to the parent structure via a non-aromatic nitrogen atom) is considered a heterocycloalkyl, whereas 1,2,3,4-tetrahydroquinolin-8-yl (wherein the moiety is attached to the parent structure via an aromatic carbon atom) is not considered a heterocycloalkyl. 3-6 Monocyclic, unless otherwise specified, lower heterocycloalkyl groups are generally preferably fully saturated carbocyclic rings. In some embodiments of the present invention, in vivo delivery can also be by β-glucan delivery system, such as those described in U.S. Patent Nos. 5,032,401 and 5,607,677, and U.S. Publication No. 2005 / 0281781, the entire contents of which are incorporated herein by reference. Xanthine dehydrogenase (XDH) gene RNAi reagents can also be introduced into cells in vitro using methods known in the art such as electroporation and lipofection. In certain embodiments of the present method, xanthine dehydrogenase (XDH) dsRNA is delivered in the absence of a targeting agent. These RNAs can be delivered as "naked" RNA molecules. As a non-limiting example, the xanthine dehydrogenase (XDH) dsRNA of the present invention can be administered to a subject in a pharmaceutical composition comprising an RNAi reagent but not comprising a targeting agent (e.g., a GalNAc targeting compound) to treat a xanthine dehydrogenase (XDH) related disease or condition, such as gout, in the subject.

[0186] It will be understood that in addition to certain delivery modes described herein, other RNAi delivery modes can be used in conjunction with the embodiments of the xanthine dehydrogenase (XDH) gene RNAi agents and treatment methods described herein, such as but not limited to those described herein and those used in the art.

[0187] The xanthine dehydrogenase (XDH) dsRNA agents of the present invention can be administered to a subject in an amount and manner effective to reduce the level and activity of a xanthine dehydrogenase (XDH) polypeptide in a cell and / or subject. In some embodiments of the methods of the present invention, one or more xanthine dehydrogenase (XDH) dsRNA agents are administered to a cell and / or subject to treat a disease or condition associated with xanthine dehydrogenase (XDH) expression and activity. In some embodiments, the methods of the present invention comprise administering one or more xanthine dehydrogenase (XDH) dsRNA agents to a subject in need of such treatment to alleviate a disease or condition associated with xanthine dehydrogenase (XDH) expression in the subject. The xanthine dehydrogenase (XDH) dsRNA agents or xanthine dehydrogenase (XDH) antisense polynucleotide agents of the present invention can be administered to reduce xanthine dehydrogenase (XDH) expression and / or activity in one or more cells in vitro, ex vivo, and in vivo.

[0188] In some embodiments of the present invention, the level of xanthine dehydrogenase (XDH) polypeptide in cells is reduced and its activity is thus reduced by delivering (e.g., introducing) xanthine dehydrogenase (XDH) dsRNA agents or xanthine dehydrogenase (XDH) antisense polynucleotide agents into cells. Targeting agents and methods can be used to help deliver xanthine dehydrogenase (XDH) dsRNA agents or xanthine dehydrogenase (XDH) antisense polynucleotide agents to specific cell types, cell subtypes, organs, spatial regions, and / or subcellular regions within a cell within a subject. Xanthine dehydrogenase (XDH) dsRNA agents can be administered alone or in combination with one or more other xanthine dehydrogenase (XDH) dsRNA agents in certain methods of the present invention. In some embodiments, 2, 3, 4 or more independently selected xanthine dehydrogenase (XDH) dsRNA agents are administered to a subject. In certain embodiments of the present invention, a xanthine dehydrogenase (XDH) dsRNA agent is administered to a subject in combination with one or more additional therapeutic regimens for treating a xanthine dehydrogenase (XDH)-related disease or condition to treat a xanthine dehydrogenase (XDH)-related disease or condition. Non-limiting examples of additional therapeutic regimens are: administration of one or more xanthine dehydrogenase (XDH) antisense polynucleotides of the present invention, administration of a non-xanthine dehydrogenase (XDH) dsRNA therapeutic agent, and behavioral changes. Additional therapeutic regimens may be administered at one or more of the following times: before, simultaneously, and after administration of a xanthine dehydrogenase (XDH) dsRNA agent of the present invention. It should be understood that "simultaneously," as used herein, refers to within 5 minutes of time zero, within 10 minutes of time zero, within 30 minutes of time zero, within 45 minutes of time zero, and within 60 minutes of time zero, wherein "time zero" is the time at which a xanthine dehydrogenase (XDH) dsRNA agent of the present invention is administered to a subject. Non-limiting examples of non-xanthine dehydrogenase (XDH) dsRNA therapeutic agents are: additional therapeutic agents, such as allopurinol, hydroxypurine, febuxostat or interleukin-1 beta (IL-1 beta) antagonists, preferably, the interleukin-1 beta antagonist is selected from canakinumab or rilonacept, thereby treating subjects with diseases that will benefit from reducing xanthine dehydrogenase (XDH) expression, or any combination thereof, and therapeutic agents formulated as a combination of agents for the treatment of hyperuricemia, gout, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), metabolic disorders, insulin resistance, cardiovascular disease, type 2 diabetes, Lesch Nyhan syndrome, glycogen storage disease (GSD), and diseases associated with oxidative stress or other XDH-related diseases. Non-limiting examples of behavioral changes are: dietary regimens, counseling, and exercise regimens.These and other therapeutic agents and behavioral changes are known in the art and can be used to treat xanthine dehydrogenase (XDH) diseases or conditions in a subject and can also be combined with one or more xanthine dehydrogenase (XDH) dsRNA agents of the present invention and administered to a subject to treat a xanthine dehydrogenase (XDH) disease or condition. The xanthine dehydrogenase (XDH) dsRNA agents of the present invention administered to a cell or subject to treat a xanthine dehydrogenase (XDH)-related disease or condition can act synergistically with one or more other therapeutic agents or active ingredients, thereby increasing the effectiveness of the one or more therapeutic agents or active ingredients and / or increasing the effectiveness of the xanthine dehydrogenase (XDH) dsRNA agent in treating a xanthine dehydrogenase (XDH)-related disease or condition.

[0189] The treatment methods of the present invention include administering a xanthine dehydrogenase (XDH) dsRNA agent, which can be used before the onset of a xanthine dehydrogenase (XDH)-related disease or condition and / or when a xanthine dehydrogenase (XDH)-related disease or condition is present, including use at the early, middle, and late stages of the disease or condition, and at all times before and after any of these stages. The methods of the present invention can also treat a subject who has previously been treated for a xanthine dehydrogenase (XDH)-related disease or condition with one or more other therapeutic agents and / or therapeutically active ingredients, wherein the one or more other therapeutic agents and / or therapeutically active ingredients were unsuccessful, minimally successful, and / or no longer successful in treating the subject's xanthine dehydrogenase (XDH)-related disease or condition.

[0190] Vector-encoded dsRNA

[0191] In certain embodiments of the present invention, a vector can be used to deliver a xanthine dehydrogenase (XDH) dsRNA agent to a cell. The xanthine dehydrogenase (XDH) dsRNA agent transcription unit can be contained in a DNA or RNA vector. The preparation and use of vectors for delivering sequences to cells and / or objects encoding transgenics are well known in the art. In the methods of the present invention, a vector that causes transient expression of xanthine dehydrogenase (XDH) dsRNA can be used, for example, for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 hours or more, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks or more. The length of transient expression can be determined using conventional methods based on the following elements: for example, but not limited to, selected specific vector constructs and target cells and / or tissues. Such transgenics can be introduced as linear constructs, circular plasmids, or viral vectors, which can be integrating or non-integrating vectors. Transgenes can also be constructed so that they are inherited as extrachromosomal plasmids (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92: 1292).

[0192] One or more single strands of a xanthine dehydrogenase (XDH) dsRNA agent can be transcribed from a promoter on an expression vector. Where two separate strands are to be expressed to produce, for example, dsRNA, the two separate expression vectors can be co-introduced into cells using, for example, transfection or infection. In certain embodiments, each separate strand of a xanthine dehydrogenase (XDH) dsRNA agent of the present invention can be transcribed from a promoter contained on the same expression vector. In certain embodiments of the present invention, the xanthine dehydrogenase (XDH) dsRNA agent is expressed as an inverted repeat polynucleotide linked by a linker polynucleotide sequence, such that the xanthine dehydrogenase (XDH) dsRNA agent has a stem-loop structure.

[0193] The limiting examples of RNA expression vectors are DNA plasmids or viral vectors. Useful expression vectors in embodiments of the present invention can be compatible with eukaryotic cells. Eukaryotic cell expression vectors are conventionally used in this area and can be obtained from many commercial sources. The delivery of xanthine dehydrogenase (XDH) dsRNA expression vectors can be systemic, such as by intravenous or intramuscular administration, by being administered to the target cells removed from the object and then the target cells are reintroduced into the object, or by any other mode that allows the introduction of required target cells to carry out.

[0194] Viral vector systems that may be included in embodiments of the method include, but are not limited to: (a) adenoviral vectors; (b) retroviral vectors, including, but not limited to, lentiviral vectors, Moloney murine leukemia virus, and the like; (c) adeno-associated viral vectors; (d) herpes simplex viral vectors; (e) SV 40 vectors; (f) polyoma viral vectors; (g) papilloma viral vectors; (h) picornaviral vectors; (i) poxviral vectors, such as orthopoxviral vectors, such as vaccinia viral vectors or avian poxviral vectors, such as canarypox or fowlpox viral vectors; (j) helper-dependent or gutless adenoviral vectors. Constructs for recombinant expression of xanthine dehydrogenase (XDH) dsRNA agents may include regulatory elements, such as promoters, enhancers, and the like, which may be selected to provide constitutive or regulated / inducible expression. Viral vector systems and the use of promoters and enhancers, etc., are conventional in the art and may be used in conjunction with the methods and compositions described herein.

[0195] Certain embodiments of the present invention include the use of viral vectors to deliver xanthine dehydrogenase (XDH) dsRNA agents into cells. Many adenovirus-based delivery systems are routinely used in the art for delivery to, for example, the lungs, liver, central nervous system, endothelial cells, and muscle. Non-limiting examples of viral vectors that can be used in the methods of the present invention are: AAV vectors, poxviruses such as vaccinia virus, modified Ankara virus (MVA), NYVAC, avian pox such as fowlpox or canarypox virus.

[0196] Certain embodiments of the present invention include methods for delivering xanthine dehydrogenase (XDH) dsRNA agents into cells using vectors, and such vectors can be in pharmaceutically acceptable carriers, which can, but need not, include a sustained-release matrix in which a gene delivery vector is embedded. In some embodiments, the vectors for delivering xanthine dehydrogenase (XDH) dsRNA can be produced by recombinant cells, and the pharmaceutical compositions of the present invention can include one or more cells that produce the xanthine dehydrogenase (XDH) dsRNA delivery system.

[0197] Pharmaceutical composition containing xanthine dehydrogenase (XDH) dsRNA or ssRNA reagent

[0198] Certain embodiments of the present invention include the purposes of the pharmaceutical composition containing xanthine dehydrogenase (XDH) dsRNA reagent or xanthine dehydrogenase (XDH) antisense polynucleotide reagent and pharmaceutically acceptable carrier. The pharmaceutical composition comprising xanthine dehydrogenase (XDH) dsRNA reagent or xanthine dehydrogenase (XDH) antisense polynucleotide agent can be used in the method of the present invention to reduce xanthine dehydrogenase (XDH) gene expression and xanthine dehydrogenase (XDH) activity in the cell, and can be used to treat xanthine dehydrogenase (XDH) related diseases or illness. Such pharmaceutical composition can be formulated based on delivery mode. Non-limiting examples of the preparation for delivery mode are: compositions formulated for subcutaneous delivery, compositions formulated for systemic administration by parenteral delivery, compositions formulated for intravenous (IV) delivery, compositions formulated for intrathecal delivery, compositions formulated for direct delivery to the brain, etc. The pharmaceutical compositions of the present invention can be administered in one or more ways to deliver xanthine dehydrogenase (XDH) dsRNA agents or xanthine dehydrogenase (XDH) antisense polynucleotide agents to cells, for example: surface (e.g., via a transdermal patch); pulmonary, for example, by inhalation or insufflation of a powder or aerosol, including via a nebulizer; intratracheal, intranasal, epidermal, and transdermal, oral, or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subcutaneous, for example, via an implantable device; or intracranial, for example, via intraparenchymal; intrathecal or intraventricular administration. Xanthine dehydrogenase (XDH) dsRNA agents or xanthine dehydrogenase (XDH) antisense polynucleotide agents can also be delivered directly to target tissues, for example, directly to the liver, etc. It is understood that "delivering a xanthine dehydrogenase (XDH) dsRNA agent" or "delivering a xanthine dehydrogenase (XDH) antisense polynucleotide agent" into a cell includes delivering a xanthine dehydrogenase (XDH) dsRNA agent or a xanthine dehydrogenase (XDH) antisense polynucleotide agent, directly expressing a xanthine dehydrogenase (XDH) dsRNA agent in a cell, and expressing a xanthine dehydrogenase (XDH) dsRNA agent from an encoding vector delivered to the cell, or causing a xanthine dehydrogenase (XDH) dsRNA or a xanthine dehydrogenase (XDH) antisense polynucleotide agent to appear in the cell in any suitable manner. The preparation and use of the formulation and the means for delivering inhibitory RNA are well known and commonly used in the art.

[0199] As used herein, a "pharmaceutical composition" comprises a pharmacologically effective amount of a xanthine dehydrogenase (XDH) dsRNA agent or a xanthine dehydrogenase (XDH) antisense polynucleotide agent of the present invention and a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for administering a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, glucose, water, glycerol, ethanol, and combinations thereof. The term explicitly excludes cell culture media. For orally administered drugs, pharmaceutically acceptable carriers include, but are not limited to, pharmaceutically acceptable excipients such as inert diluents, disintegrants, binders, lubricants, sweeteners, flavorings, colorants, and preservatives. Suitable inert diluents include sodium carbonate and calcium carbonate, sodium phosphate and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrants. Binders may include starch and gelatin, while lubricants (if present) are typically magnesium stearate, stearic acid, or talc. If desired, tablets may be coated with materials such as glyceryl monostearate or glyceryl distearate to delay absorption in the gastrointestinal tract. The agents included in the pharmaceutical formulations are further described below. As used herein, terms such as "pharmacologically effective amount," "therapeutically effective amount," and "effective amount" refer to the amount of a xanthine dehydrogenase (XDH) dsRNA agent or xanthine dehydrogenase (XDH) antisense polynucleotide agent of the present invention that produces the desired pharmacological, therapeutic, or preventive result. For example, a given clinical treatment is considered effective if a measurable parameter associated with a disease or disorder is reduced by at least 10%. The therapeutically effective amount of a drug for treating the disease or disorder is the amount required to reduce the parameter by at least 10%. For example, a therapeutically effective amount of a xanthine dehydrogenase (XDH) dsRNA agent or xanthine dehydrogenase (XDH) antisense polynucleotide agent can reduce xanthine dehydrogenase (XDH) polypeptide levels by at least 10%. Pharmaceutical compositions can include dsRNAi agents including, for example, duplexes AV00206 to AV00174 shown in Table 1. Preferred dsRNAi agents include, for example, duplexes AV00227, AV00260, AV00285, AV00266, AV00280, AV00238, AV00249, AV00289, AV00233, AV00225, AV00273, or AV00283. In certain embodiments, preferred dsRNAi agents in Table 2 include, for example, duplexes AV00227, AV00260, AV00285, AV00266, or AV00280.In certain embodiments, preferred dsRNAi agents of Table 3 include, for example, duplexes AD00353, AD00356, AD00358, AD00364, AD00365, AD00366, AD00368, AD00370, AD00430, AD00431, AD00432, AD00521, AD00521-1, AD00364-1, AD00365-2, AD00366-1, and AD00432-1.

[0200] effective dose

[0201] In some aspects, the methods of the present invention include contacting a cell with an effective amount of a xanthine dehydrogenase (XDH) dsRNA agent or a xanthine dehydrogenase (XDH) antisense polynucleotide agent to reduce xanthine dehydrogenase (XDH) gene expression in the contacted cell. Certain embodiments of the methods of the present invention include administering a xanthine dehydrogenase (XDH) dsRNA agent or a xanthine dehydrogenase (XDH) antisense polynucleotide agent to a subject in an amount effective to reduce xanthine dehydrogenase (XDH) gene expression and treat a xanthine dehydrogenase (XDH)-related disease or condition in the subject. With respect to reducing xanthine dehydrogenase (XDH) expression and / or for treating a xanthine dehydrogenase (XDH)-related disease or condition, an "effective amount" as used herein is an amount necessary or sufficient to achieve the desired biological effect. For example, an effective amount of a xanthine dehydrogenase (XDH) dsRNA agent or a xanthine dehydrogenase (XDH) antisense polynucleotide agent for treating a xanthine dehydrogenase (XDH)-related disease or condition can be: (i) an amount required to slow or stop the progression of the disease or condition; (ii) an amount required to reverse, reduce or eliminate one or more symptoms of the disease or condition. In some aspects of the invention, an effective amount is an amount of a xanthine dehydrogenase (XDH) dsRNA agent or a xanthine dehydrogenase (XDH) antisense polynucleotide agent that, when administered to a subject in need of treatment for a xanthine dehydrogenase (XDH)-related disease or condition, results in a therapeutic response for the prevention and / or treatment of the disease or condition. The amount of a dsRNA agent or a xanthine dehydrogenase (XDH) antisense polynucleotide agent. According to some aspects of the present invention, an effective amount is an amount of a xanthine dehydrogenase (XDH) dsRNA agent or a xanthine dehydrogenase (XDH) antisense polynucleotide agent of the present invention that, when combined or co-administered with another therapeutic treatment for a xanthine dehydrogenase (XDH)-related disease or condition, results in a therapeutic response that prevents and / or treats the disease or condition. In some embodiments of the present invention, the biological effect of treating a subject with a xanthine dehydrogenase (XDH) dsRNA agent or a xanthine dehydrogenase (XDH) antisense polynucleotide agent of the present invention can be caused by xanthine dehydrogenase. In some embodiments of the present invention, the biological effect is the complete elimination of xanthine dehydrogenase (XDH) related diseases or conditions, for example, as demonstrated by a diagnostic test that indicates that the subject does not have a xanthine dehydrogenase (XDH) related disease or condition. Non-limiting examples of detectable physiological symptoms include a decrease in uric acid levels in the subject's clinical blood or urine after administering the reagent of the present invention. Other methods known in the art for assessing xanthine dehydrogenase (XDH) related diseases or condition states can be used to determine the impact of the reagent and / or method of the present invention on xanthine dehydrogenase (XDH) related diseases or conditions.

[0202] The effective amount of a xanthine dehydrogenase (XDH) dsRNA agent or a xanthine dehydrogenase (XDH) antisense polynucleotide agent that reduces xanthine dehydrogenase (XDH) polypeptide activity to a level that treats a xanthine dehydrogenase (XDH)-related disease or condition is typically determined in a clinical trial, where an effective dose is established for a test population versus a control population in a blinded study. In some embodiments, an effective amount is an amount that results in a desired response, such as an amount that reduces a xanthine dehydrogenase (XDH)-related disease or condition in a cell, tissue, and / or subject suffering from a disease or condition. Thus, an effective amount of a xanthine dehydrogenase (XDH) dsRNA agent or xanthine dehydrogenase (XDH) antisense polynucleotide agent for treating a xanthine dehydrogenase (XDH)-related disease or condition that can be treated by reducing xanthine dehydrogenase (XDH) polypeptide activity can be an amount that, when administered, reduces the amount of xanthine dehydrogenase (XDH) polypeptide activity in a subject to a level below that which would be present in a cell, tissue, and / or subject in the absence of administration of the xanthine dehydrogenase (XDH) dsRNA agent or xanthine dehydrogenase (XDH) antisense polynucleotide agent. In certain aspects of the invention, the level of xanthine dehydrogenase (XDH) polypeptide activity and / or xanthine dehydrogenase (XDH) gene expression present in a cell, tissue, and / or subject that has not been contacted with or administered a xanthine dehydrogenase (XDH) dsRNA agent or xanthine dehydrogenase (XDH) antisense polynucleotide agent of the invention is referred to as a "control" amount. In some embodiments of the methods of the present invention, the control amount of the subject is the pre-treatment amount of the subject; in other words, the level of the subject before administering the xanthine dehydrogenase (XDH) agent can be the control level of the subject and is used to compare its xanthine dehydrogenase (XDH) polypeptide activity and / or xanthine dehydrogenase (XDH) gene expression level after administering siRNA to the subject. In the case of treating a xanthine dehydrogenase (XDH)-related disease or condition, the desired response can be to reduce or eliminate one or more symptoms of the disease or condition in cells, tissues and / or subjects. The reduction or elimination can be temporary or permanent. It should be understood that methods such as determining xanthine dehydrogenase (XDH) polypeptide activity, xanthine dehydrogenase (XDH) gene expression, symptom assessment, clinical testing, etc. can be used to monitor the status of a xanthine dehydrogenase (XDH)-related disease or condition. In some aspects of the present invention, the desired response to treating a xanthine dehydrogenase (XDH)-related disease or condition is to delay the onset of the disease or condition or even prevent the onset of the disease or condition.

[0203] The effective amount of a compound that reduces the activity of a xanthine dehydrogenase (XDH) polypeptide can also be determined by evaluating the physiological effects of administering a xanthine dehydrogenase (XDH) dsRNA agent or a xanthine dehydrogenase (XDH) antisense polynucleotide agent on a cell or subject, such as a reduction in xanthine dehydrogenase (XDH)-related diseases or conditions after administration. Assays and / or symptom monitoring of subjects can be used to determine the efficacy of the xanthine dehydrogenase (XDH) dsRNA agent or xanthine dehydrogenase (XDH) antisense polynucleotide agent of the present invention (which can be administered in the pharmaceutical compound of the present invention) and to determine whether there is a response to treatment. Another non-limiting example is that before and after treating a subject with a xanthine dehydrogenase (XDH) dsRNA agent of the present invention, one or more serum uric acid level tests known in the art can be used to determine the status of a xanthine dehydrogenase (XDH)-related disease or condition in the subject. In another non-limiting example, one or more uric acid level tests known in the art are used to determine the status of a xanthine dehydrogenase (XDH)-related disease in the subject. In this embodiment, the diseases include hyperuricemia, gout, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), metabolic disorders, insulin resistance, cardiovascular disease, type 2 diabetes, Lesch Nyhan syndrome, glycogen storage disease (GSD), and diseases associated with oxidative stress or other XDH-related diseases, and the test is used to determine the uric acid level in the subject before and after treatment with the xanthine dehydrogenase (XDH) dsRNA agent of the present invention.

[0204] Some embodiments of the present invention include methods for determining the efficacy of a xanthine dehydrogenase (XDH) dsRNA agent or a xanthine dehydrogenase (XDH) antisense polynucleotide agent of the present invention administered to a subject for treating a xanthine dehydrogenase (XDH)-related disease or condition by assessing and / or monitoring one or more "physiological characteristics" of a xanthine dehydrogenase (XDH)-related disease or condition in the subject. Non-limiting examples of physiological characteristics of a xanthine dehydrogenase (XDH)-related disease or condition are that many patients also experience elevated uric acid levels in the blood and urine, increased pain (e.g., pain caused by gout), and increased risk of inflammatory events. Standard methods for determining such physiological characteristics are known in the art and include, but are not limited to, blood tests, imaging studies, physical examinations, and the like. The syndrome is defined as the presence of a decrease in uric acid levels in the blood and urine, a decrease in pain (e.g., pain caused by gout), and a decrease in inflammation.

[0205] It will be appreciated that the amount of xanthine dehydrogenase (XDH) dsRNA agent or xanthine dehydrogenase (XDH) antisense polynucleotide agent administered to a subject can be modified based, at least in part, on the results of such determinations of the subject's determined disease and / or condition state and / or physiological characteristics. The therapeutic amount can be varied, for example, by increasing or decreasing the amount of xanthine dehydrogenase (XDH) dsRNA agent or xanthine dehydrogenase (XDH) antisense polynucleotide agent by changing the composition of the xanthine dehydrogenase (XDH) dsRNA agent or xanthine dehydrogenase (XDH) antisense polynucleotide agent administered, by changing the route of administration, by changing the time of administration, etc. The effective amount of xanthine dehydrogenase (XDH) dsRNA agent or xanthine dehydrogenase (XDH) antisense polynucleotide agent will vary depending on the specific condition being treated, the age and physical condition of the subject being treated, the severity of the condition, the duration of treatment, the nature of co-treatments (if any), the specific route of administration, and other factors within the knowledge and expertise of a health practitioner. For example, the effective amount can depend on the desired level of xanthine dehydrogenase (XDH) polypeptide activity and / or xanthine dehydrogenase (XDH) gene expression effective for treating xanthine dehydrogenase (XDH) related diseases or conditions. Technicians can empirically determine the effective amount of a specific xanthine dehydrogenase (XDH) dsRNA agent or xanthine dehydrogenase (XDH) antisense polynucleotide agent for the methods of the present invention without excessive experimentation. In conjunction with the teachings provided herein, by selecting from a variety of xanthine dehydrogenase (XDH) dsRNA agents or xanthine dehydrogenase (XDH) antisense polynucleotide agents of the present invention, and weighing factors such as efficacy, relative bioavailability, patient weight, severity of adverse side effects and preferred modes of administration, an effective preventive or therapeutic treatment regimen can be planned to effectively treat a specific subject. As used in embodiments of the present invention, the effective amount of a xanthine dehydrogenase (XDH) dsRNA agent or xanthine dehydrogenase (XDH) antisense polynucleotide agent of the present invention can be the amount that produces a desired biological effect in a cell when in contact with a cell.

[0206] It should be appreciated that xanthine dehydrogenase (XDH) gene silencing can be performed constitutively or by genomic engineering in any cell expressing xanthine dehydrogenase (XDH) and determined by any suitable assay. In some embodiments of the invention, xanthine dehydrogenase (XDH) gene expression is reduced by at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% by administering a xanthine dehydrogenase (XDH) dsRNA agent of the invention. In some embodiments of the invention, xanthine dehydrogenase (XDH) gene expression is reduced by 5% to 10%, 5% to 25%, 10% to 50%, 10% to 75%, 25% to 75%, 25% to 100%, or 50% to 100% by administering a xanthine dehydrogenase (XDH) dsRNA agent of the invention.

[0207] Drug administration

[0208] Xanthine dehydrogenase (XDH) dsRNA agents and xanthine dehydrogenase (XDH) antisense polynucleotide agents are delivered in pharmaceutical compositions at a dosage sufficient to inhibit xanthine dehydrogenase (XDH) gene expression. In certain embodiments of the present invention, the dosage of xanthine dehydrogenase (XDH) dsRNA agents or xanthine dehydrogenase (XDH) antisense polynucleotide agents is 0.01 to 200.0 mg per kilogram of recipient body weight per day, generally 1 to 50 mg / kg body weight, 5 to 40 mg / kg body weight, 10 to 30 mg / kg body weight, 1 to 20 mg / kg body weight, 1 to 10 mg / kg body weight, 4 to 15 mg / kg body weight, including end values. For example, each single administration of a xanthine dehydrogenase (XDH) dsRNA agent or a xanthine dehydrogenase (XDH) antisense polynucleotide agent can be at a dosage of from about 0.01 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 1 mg / kg, 1.1 mg / kg, 1.2 mg / kg, 1.3 mg / kg, 1.4 mg / kg, 1.5 mg / kg, 1.6 mg / kg, 1.7 mg / kg, 1.8 mg / kg, 1.9mg / kg, 2mg / kg, 2.1mg / kg, 2.2mg / kg, 2.3mg / kg, 2.4mg / kg, 2.5mg / kg, 2.6mg / kg, 2.7mg / kg, 2.8mg / kg, 2 .9mg / kg, 3.0mg / kg, 3.1mg / kg, 3.2mg / kg, 3.3mg / kg, 3.4mg / kg, 3.5mg / kg, 3.6mg / kg, 3.7mg / kg, 3.8mg / kg, 3.9mg / k g, 4mg / kg, 4.1mg / kg, 4.2mg / kg, 4.3mg / kg, 4.4mg / kg, 4.5mg / kg, 4.6mg / kg, 4.7mg / kg, 4.8mg / kg, 4.9mg / kg, 5mg / k g, 5.1mg / kg, 5.2mg / kg, 5.3mg / kg, 5.4mg / kg, 5.5mg / kg, 5.6mg / kg, 5.7mg / kg, 5.8mg / kg, 5.9mg / kg, 6mg / kg, 6.1mg / kg, 6.2mg / kg, 6.3mg / kg, 6.4mg / kg, 6.5mg / kg, 6.6mg / kg, 6.7mg / kg, 6.8mg / kg, 6.9mg / kg, 7mg / kg, 7.1mg / kg, 7.2mg / kg, 7.3mg / kg, 7.4mg / kg, 7.5mg / kg, 7.6mg / kg, 7.7mg / kg, 7.8mg / kg, 7.9mg / kg, 8mg / kg, 8.1mg / kg, 8.2mg / kg, 8.3mg / kg, 8.4mg / kg, 8.5mg / kg, 8.6mg / kg, 8.7mg / kg, 8.8mg / kg, 8.9mg / kg, 9mg / kg, 9.1mg / kg, 9.2mg / kg, 9.3mg / kg, 9.4mg / kg, 9.5mg / kg, 9.6mg / kg , 9.7mg / kg, 9.8mg / kg, 9.9mg / kg, 10mg / kg, 11mg / kg, 12mg / kg, 13mg / kg, 14mg / kg, 15mg / kg, 16mg / kg, 17mg / kg, 18mg / kg, 19mg / kg, 20mg / kg, 21mg / kg, 22mg / kg, 23mg / kg, 24mg / kg, 25mg / kg, 26mg / kg, 27mg / kg, 28mg / kg, 29mg / kg, 30mg / kg, 31mg / kg, 32mg / kg, 33mg / kg, 34mg / kg, 35mg / kg, 36mg / k g, 37mg / kg, 38mg / kg, 39mg / kg, 40mg / kg, 41mg / kg, 42mg / kg, 43mg / kg, 44mg / kg, 45mg / kg, 46mg / kg, 47mg / kg, 48mg / kg, 49mg / kg to 50mg / kg body weight. .

[0209] In determining the delivery dosage and time of xanthine dehydrogenase (XDH) dsRNA reagent of the present invention, various factors can be considered. The absolute amount of the xanthine dehydrogenase (XDH) dsRNA reagent or xanthine dehydrogenase (XDH) antisense polynucleotide agent delivered will depend on various factors, including co-treatment, dosage number and individual subject parameters, including age, physical condition, size and body weight. These are factors well known to those of ordinary skill in the art and can be solved by routine experiments. In some embodiments, a maximum dose can be used, i.e., the highest safe dose according to reasonable medical judgment.

[0210] In some embodiments, the methods of the present invention may include administering 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses of a xanthine dehydrogenase (XDH) dsRNA agent or a xanthine dehydrogenase (XDH) antisense polynucleotide agent to a subject. In some cases, a dose of a pharmaceutical compound (e.g., comprising a xanthine dehydrogenase (XDH) dsRNA agent or comprising a xanthine dehydrogenase (XDH) antisense polynucleotide agent) may be administered to a subject at least daily, every other day, weekly, every other week, monthly, etc., may be administered once daily or more than once daily, for example, 2, 3, 4, 5 or more times within a 24-hour period. The pharmaceutical compositions of the present invention may be administered once daily; or the xanthine dehydrogenase (XDH) dsRNA agent or the xanthine dehydrogenase (XDH) antisense polynucleotide agent may be administered as two, three or more sub-doses at appropriate intervals throughout the day, or even delivered using continuous infusion or by a controlled release formulation. In some embodiments of the methods of the present invention, a pharmaceutical composition of the present invention is administered to a subject once per day or more, once per week or more, once per month or more, or once per year or more.

[0211] In certain aspects, the methods of the present invention include administering a pharmaceutical compound alone; in combination with one or more other xanthine dehydrogenase (XDH) dsRNA agents or xanthine dehydrogenase (XDH) antisense polynucleotide agents; and / or in combination with other drug therapies or therapeutic activities or regimens administered to a subject suffering from a xanthine dehydrogenase (XDH)-related disease or condition. The pharmaceutical compound can be administered in the form of a pharmaceutical composition. The pharmaceutical composition used in the methods of the present invention can be sterile and contain an amount of a xanthine dehydrogenase (XDH) dsRNA agent or a xanthine dehydrogenase (XDH) antisense polynucleotide agent that reduces the activity of a xanthine dehydrogenase (XDH) polypeptide to a level sufficient to produce the desired response in a unit of weight or volume suitable for administration to a subject. The dose of the pharmaceutical composition comprising a xanthine dehydrogenase (XDH) dsRNA agent or a xanthine dehydrogenase (XDH) antisense polynucleotide agent administered to a subject to reduce xanthine dehydrogenase (XDH) protein activity can be selected based on various parameters, particularly the mode of administration used and the subject's condition. Other factors include the desired duration of treatment.If a subject has an inadequate response at an initial dose, a higher dose (or effective dose increase by a different, more localized route of delivery) can be employed as patient tolerance permits.

[0212] treat

[0213] As used herein, the terms "prevent" or "preventing," when used in reference to a disease, disorder, or condition that would benefit from reduced expression of a xanthine dehydrogenase (XDH) gene, means that a subject is less likely to develop symptoms associated with such a disease, disorder, or condition, such as symptoms associated with a disease or condition caused by or associated with xanthine dehydrogenase (XDH) activation, such as gout. Effective prevention is considered effective when, for example, an individual with one or more risk factors for gout fails to develop the disease, disorder, or condition, or develops symptoms associated with the disease, disorder, or condition to a lesser extent (e.g., by at least about 10% on a scale that would indicate clinically affected individuals), or delays the onset of symptoms (e.g., by days, weeks, months, or years), relative to a population with the same risk factors and not receiving treatment as described herein.

[0214] For xanthine dehydrogenase (XDH)-related diseases and conditions, where reduction in the level and / or activity of a xanthine dehydrogenase (XDH) polypeptide is effective in treating the disease or condition, the methods and xanthine dehydrogenase (XDH) dsRNA agents of the present invention can be used to treat the disease or condition by inhibiting xanthine dehydrogenase (XDH) expression. Examples of diseases and conditions that can be treated with the xanthine dehydrogenase (XDH) dsRNA agents or xanthine dehydrogenase (XDH) antisense polynucleotide agents of the present invention and the methods of treatment of the present invention include, but are not limited to, hyperuricemia, gout, NAFLD, NASH, metabolic disorders, insulin resistance, cardiovascular disease, type 2 diabetes, Lesch-Nyhan syndrome, glycogen storage diseases (GSD), such as type I GSD, and diseases associated with oxidative stress, such as chronic low-grade inflammation; or other XDH-related diseases. Such diseases and conditions may be referred to herein as "xanthine dehydrogenase (XDH)-related diseases and conditions" and "diseases and conditions caused and / or regulated by xanthine dehydrogenase (XDH)."

[0215] In certain aspects of the invention, xanthine dehydrogenase (XDH) dsRNA reagents or xanthine dehydrogenase (XDH) antisense polynucleotide reagents of the present invention can be applied to a subject at one or more times before or after diagnosis of a xanthine dehydrogenase (XDH) related disease or condition. In certain aspects of the invention, a subject is in the risk of suffering from or developing a xanthine dehydrogenase (XDH) related disease or condition. Compared to a control risk of developing a xanthine dehydrogenase (XDH) related disease or condition, a subject having a risk of developing a xanthine dehydrogenase (XDH) related disease or condition is a subject whose likelihood of developing a xanthine dehydrogenase (XDH) related disease or condition is improved. In certain embodiments of the invention, the risk level is statistically significant compared to a control level of risk. At-risk subjects may include, for example, subjects who are or will be subjects with a pre-existing disease and / or genetic abnormality that makes the subject more susceptible to a xanthine dehydrogenase (XDH)-related disease or condition than a control subject without the pre-existing disease or genetic abnormality; subjects with a family and / or personal history of a xanthine dehydrogenase (XDH)-related disease or condition; and subjects who have previously received treatment for a xanthine dehydrogenase (XDH)-related disease or condition. It should be understood that a pre-existing disease and / or genetic abnormality that makes a subject more susceptible to a xanthine dehydrogenase (XDH)-related disease or condition may be a disease or genetic abnormality that, when present, has previously been determined to be associated with a higher likelihood of developing a xanthine dehydrogenase (XDH)-related disease or condition.

[0216] Should be understood that, can be based on the medical condition of individual object to object use xanthine dehydrogenase (XDH) dsRNA reagent or xanthine dehydrogenase (XDH) antisense polynucleotide reagent.For example, the medical care provided for object can assess the xanthine dehydrogenase (XDH) level measured in the sample obtained from object, and determine that by using xanthine dehydrogenase (XDH) dsRNA reagent of the present invention or xanthine dehydrogenase (XDH) antisense polynucleotide reagent to reduce the xanthine dehydrogenase (XDH) level of object is desirable.In a non-limiting example, biological sample, such as blood or serum sample, can be obtained from object, and the uric acid level of object is determined in sample. Xanthine dehydrogenase (XDH) dsRNA reagent or xanthine dehydrogenase (XDH) antisense polynucleotide reagent is used to object, and blood or serum sample is obtained from object after administration, and uric acid level is measured using this sample, and the result determined in (previously) sample before this result and object administration is compared. Compared to the level before administration, a decrease in the uric acid level of the subject in the subsequent sample indicates the efficacy of the xanthine dehydrogenase (XDH) dsRNA agent or xanthine dehydrogenase (XDH) antisense polynucleotide agent administered in reducing the xanthine dehydrogenase (XDH) level of the subject. In a non-limiting example, an increase in uric acid levels can be considered a physiological characteristic of a xanthine dehydrogenase (XDH)-related condition, even if the subject is not diagnosed as suffering from a xanthine dehydrogenase (XDH)-related disease, such as a disease disclosed herein. A healthcare provider can monitor changes in the uric acid level of the subject as a measure of the efficacy of the xanthine dehydrogenase (XDH) dsRNA agent or xanthine dehydrogenase (XDH) antisense polynucleotide agent administered. In a non-limiting example, the xanthine dehydrogenase (XDH)-associated disease is hyperuricemia, gout, NAFLD, NASH, metabolic disorders, insulin resistance, cardiovascular disease, type 2 diabetes, Lesch Nyhan syndrome, glycogen storage disease (GSD), such as type I GSD, and diseases associated with oxidative stress, such as chronic low-grade inflammation; or other XDH-associated diseases.

[0217] Certain embodiments of the methods of the present invention include adjusting treatment, wherein the treatment includes administering a dsRNA agent or xanthine dehydrogenase (XDH) antisense polynucleotide agent of the present invention to a subject based at least in part on an assessment of changes in one or more physiological characteristics of a xanthine dehydrogenase (XDH)-related disease or condition in the subject caused by the treatment. For example, in some embodiments of the present invention, the effect of a dsRNA agent or xanthine dehydrogenase (XDH) antisense polynucleotide agent of the present invention administered to a subject can be determined and used to help adjust the amount of a dsRNA agent or xanthine dehydrogenase (XDH) antisense polynucleotide agent of the present invention subsequently administered to the subject. In one non-limiting example, a dsRNA agent or xanthine dehydrogenase (XDH) antisense polynucleotide agent of the present invention is administered to a subject, and the subject's uric acid level is measured after administration; and based at least in part on the determined level, it is determined whether a higher amount of the dsRNA agent or xanthine dehydrogenase (XDH) antisense polynucleotide agent is needed to enhance the physiological effect of the administered agent, such as to reduce or further reduce the subject's uric acid level. In another non-limiting example, a dsRNA agent or xanthine dehydrogenase (XDH) antisense polynucleotide agent of the invention is administered to a subject, and the subject's uric acid level is determined after administration, and based at least in part on the determined level, a lower amount of the dsRNA agent or xanthine dehydrogenase (XDH) antisense polynucleotide agent is expected to be administered to the subject.

[0218] Thus, some embodiments of the present invention include assessing changes in one or more physiological characteristics resulting from a previous treatment of a subject, including a decrease in uric acid levels in the blood and urine, a decrease in pain (e.g., pain caused by gout), a decrease in inflammation, and the like, to adjust the amount of the dsRNA agent or xanthine dehydrogenase (XDH) antisense polynucleotide agent of the invention subsequently administered to the subject. Some embodiments of the methods of the present invention include 1, 2, 3, 4, 5, 6, or more measurements of physiological characteristics of a xanthine dehydrogenase (XDH)-related disease or condition; assessing and / or monitoring the efficacy of the administered xanthine dehydrogenase (XDH) dsRNA agent or xanthine dehydrogenase (XDH) antisense polynucleotide agent; and optionally using the results of the measurements to adjust one or more of the following: the dosage, dosing regimen, and / or dosing frequency of the dsRNA agent or xanthine dehydrogenase (XDH) antisense polynucleotide agent of the invention for treating a xanthine dehydrogenase (XDH)-related disease or condition in the subject. In some embodiments of the methods of the invention, the desired outcome of administering an effective amount of a dsRNA agent or a xanthine dehydrogenase (XDH) antisense polynucleotide agent of the invention to a subject is a decrease in xanthine dehydrogenase (XDH) transcript levels, plasma uric acid levels, or xanthine dehydrogenase (XDH) gene expression in the subject as compared to levels previously determined for the subject.

[0219] As used herein, the terms "treat," "therapeutic," or "therapeutic" when applied to a xanthine dehydrogenase (XDH)-related disease or condition can refer to prophylactic treatment, reducing the likelihood that a subject will develop a xanthine dehydrogenase (XDH)-related disease or condition, and can also refer to treatment after a subject has developed a xanthine dehydrogenase (XDH)-related disease or condition in order to eliminate or reduce the level of the xanthine dehydrogenase (XDH)-related disease or condition, prevent the xanthine dehydrogenase (XDH)-related disease or condition from becoming more severe, and / or slow the progression of a xanthine dehydrogenase (XDH)-related disease or condition in a subject compared to a subject in the absence of a therapy that reduces the activity of a xanthine dehydrogenase (XDH) polypeptide in the subject.

[0220] Certain embodiments of the agents, compositions, and methods of the invention are useful for inhibiting xanthine dehydrogenase (XDH) gene expression. As used herein, the terms "inhibit," "silence," "reduce," "downregulate," and "knockdown" with respect to the expression of a xanthine dehydrogenase (XDH) gene refer to altering the expression of a xanthine dehydrogenase (XDH) gene, for example, by one or more of the following: when a cell, cell population, tissue, organ, or subject is contacted with (e.g., treated with) a xanthine dehydrogenase (XDH) dsRNA agent or a xanthine dehydrogenase (XDH) antisense polynucleotide agent of the invention, the level of RNA transcribed from the gene, the level of xanthine dehydrogenase (XDH) activity expressed, and the level of xanthine dehydrogenase (XDH) polypeptide, protein, or protein subunit translated from mRNA in the cell, cell population, tissue, organ, or subject is reduced, compared to a control level of RNA transcribed from the xanthine dehydrogenase (XDH) gene, a control level of xanthine dehydrogenase (XDH) activity expressed, or a control level of xanthine dehydrogenase (XDH) translated from mRNA, respectively. In some embodiments, the control level is the level in a cell, tissue, organ, or subject that has not been contacted with (eg, treated with) a xanthine dehydrogenase (XDH) dsRNA agent or a xanthine dehydrogenase (XDH) antisense polynucleotide agent.

[0221] Application method

[0222] A variety of routes of administration of xanthine dehydrogenase (XDH) dsRNA reagents or xanthine dehydrogenase (XDH) antisense polynucleotide agents can be used for the method of the present invention. The selection of a specific delivery mode will depend at least in part on the specific condition being treated and the dosage required for therapeutic efficacy. Generally speaking, the method of the present invention can be implemented using any medically acceptable mode of administration, which means producing an effective therapeutic level of xanthine dehydrogenase (XDH) related disease or condition without causing any pattern of clinically unacceptable side effects. In some embodiments of the present invention, xanthine dehydrogenase (XDH) dsRNA reagents or xanthine dehydrogenase (XDH) antisense polynucleotide agents can be administered by oral, enteral, mucosal, subcutaneous and / or parenteral routes. The term "parenteral" includes subcutaneous, intravenous, intrathecal, intramuscular, intraperitoneal and intrasternal injection or infusion techniques. Other approaches include, but are not limited to, nasal (e.g., by nasogastric tube), transdermal, vaginal, rectal, sublingual and inhalation. The delivery route of the present invention may include intrathecal, intraventricular or intracranial. In some embodiments of the present invention, xanthine dehydrogenase (XDH) dsRNA reagent or xanthine dehydrogenase (XDH) antisense polynucleotide agent can be placed in a slow-release matrix and administered by placing the matrix in an object. In some aspects of the present invention, xanthine dehydrogenase (XDH) dsRNA reagent or xanthine dehydrogenase (XDH) antisense polynucleotide agent can be delivered to the subject's cells using nanoparticles coated with a delivery agent targeting specific cells or organelles. Various delivery methods, methods, and reagents are known in the art. Non-limiting examples of delivery methods and delivery agents are provided elsewhere herein. In some aspects of the present invention, the term "delivery" with respect to xanthine dehydrogenase (XDH) dsRNA reagent or xanthine dehydrogenase (XDH) antisense polynucleotide agent can refer to: administering one or more "naked" xanthine dehydrogenase (XDH) dsRNA reagents or xanthine dehydrogenase (XDH) antisense polynucleotide agent sequences to a cell or object. In certain aspects of the invention, "delivering" refers to administering to a cell or subject by transfection, delivering a cell comprising a xanthine dehydrogenase (XDH) dsRNA agent or a xanthine dehydrogenase (XDH) antisense polynucleotide agent to a subject, delivering a vector encoding a xanthine dehydrogenase (XDH) dsRNA agent or a xanthine dehydrogenase (XDH) antisense polynucleotide agent to a cell and / or subject, etc. Delivery of a xanthine dehydrogenase (XDH) dsRNA agent or a xanthine dehydrogenase (XDH) antisense polynucleotide agent using transfection can include administering a vector to a cell and / or subject.

[0223] In some methods of the present invention, one or more xanthine dehydrogenase (XDH) dsRNA agents or xanthine dehydrogenase (XDH) antisense polynucleotide agents can be administered in the form of a formulation or in a pharmaceutically acceptable solution, which can generally contain pharmaceutically acceptable concentrations of salt, buffers, preservatives, compatible carriers, adjuvants, and other optional therapeutic ingredients. In some embodiments of the present invention, xanthine dehydrogenase (XDH) dsRNA agents or xanthine dehydrogenase (XDH) antisense polynucleotide agents can be formulated for simultaneous administration together with another therapeutic agent. According to the methods of the present invention, xanthine dehydrogenase (XDH) dsRNA agents or xanthine dehydrogenase (XDH) antisense polynucleotide agents can be administered in the form of a pharmaceutical composition. Typically, the pharmaceutical composition comprises a xanthine dehydrogenase (XDH) dsRNA agent or a xanthine dehydrogenase (XDH) antisense polynucleotide agent and an optional pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known to those of ordinary skill in the art. As used herein, a pharmaceutically acceptable carrier refers to a nontoxic material that does not interfere with the effectiveness of the biological activity of the active ingredient (e.g., the ability of a xanthine dehydrogenase (XDH) dsRNA agent or a xanthine dehydrogenase (XDH) antisense polynucleotide agent to inhibit the expression of a xanthine dehydrogenase (XDH) gene in a cell or subject). Various methods of administering and delivering xanthine dehydrogenase (XDH) dsRNA agents or xanthine dehydrogenase (XDH) antisense polynucleotide agents for therapeutic use are known in the art and can be used in the methods of the present invention.

[0224] Pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials known in the art. Exemplary pharmaceutically acceptable carriers are described in U.S. Patent No. 5,211,657, while other carriers are known to those skilled in the art. Such preparations may typically contain salts, buffers, preservatives, compatible carriers, and optionally other therapeutic agents. When used in medicine, the salt should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts can be conveniently used to prepare pharmaceutically acceptable salts thereof and are not excluded from the scope of the present invention. Such pharmacologically and pharmaceutically acceptable salts include, but are not limited to, salts prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, formic acid, malonic acid, succinic acid, and the like. In addition, pharmaceutically acceptable salts can be prepared as alkali metal salts or alkaline earth metal salts, such as sodium salts, potassium salts, or calcium salts.

[0225] Some embodiments of the methods of the present invention include direct administration of one or more xanthine dehydrogenase (XDH) dsRNA agents or xanthine dehydrogenase (XDH) antisense polynucleotide agents to tissues. In some embodiments, the tissue to which the compound is administered is a tissue in which a xanthine dehydrogenase (XDH) related disease or condition is present or may occur, a non-limiting example of which is the liver or kidney. Direct tissue administration can be achieved by direct injection or other means. Many orally delivered compounds naturally enter and pass through the liver and kidneys, and some embodiments of the methods of the present invention include oral administration of one or more xanthine dehydrogenase (XDH) dsRNA agents to a subject. Xanthine dehydrogenase (XDH) dsRNA agents or xanthine dehydrogenase (XDH) antisense polynucleotide agents, alone or in combination with other therapeutic agents, can be administered once, or they can be administered multiple times. If administered multiple times, xanthine dehydrogenase (XDH) dsRNA agents or xanthine dehydrogenase (XDH) antisense polynucleotide agents can be administered by different routes. For example, although not intended to be limiting, the first (or first few) administrations may be administered subcutaneously, and one or more additional administrations may be administered orally and / or systemically.

[0226] For embodiments of the present invention in which systemic administration of a xanthine dehydrogenase (XDH) dsRNA agent or a xanthine dehydrogenase (XDH) antisense polynucleotide agent is desired, the xanthine dehydrogenase (XDH) dsRNA agent or a xanthine dehydrogenase (XDH) antisense polynucleotide agent can be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion. Injectable formulations can be presented in unit dosage form, such as ampoules or multidose containers, with or without added preservatives. Xanthine dehydrogenase (XDH) dsRNA agent formulations (also referred to as pharmaceutical compositions) can be in the form of suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain preparatants, such as suspending agents, stabilizers, and / or dispersants.

[0227] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcohol / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose solution, glucose and sodium chloride solution, lactated Ringer's solution or fixed oils. Intravenous excipients include fluid and nutritional supplements, electrolyte supplements (such as those based on Ringer's dextrose solution), etc. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents and inert gases, etc., may also be present. Other forms of administration, such as intravenous administration, will result in lower dosages. If the subject's response under the initial dose is insufficient, a higher dosage (or effectively increasing the dosage by different, more localized delivery routes) can be adopted within the scope allowed by patient tolerance. Multiple doses per day can be used as needed to achieve appropriate systemic or local levels of one or more xanthine dehydrogenase (XDH) dsRNA agents or xanthine dehydrogenase (XDH) antisense polynucleotide agents and to achieve appropriate reduction in xanthine dehydrogenase (XDH) activity.

[0228] In other embodiments, the methods of the present invention include the use of a delivery vehicle, such as a biocompatible microparticle, nanoparticle, or implant suitable for implantation into a recipient, such as a subject. Exemplary biodegradable implants that can be used according to this method are described in PCT Publication WO 95 / 24929 (incorporated herein by reference), which describes a biocompatible, biodegradable polymer matrix for containing biomacromolecules.

[0229] Non-biodegradable and biodegradable polymer matrices can be used in the method of the present invention to deliver one or more xanthine dehydrogenase (XDH) dsRNA reagents or xanthine dehydrogenase (XDH) antisense polynucleotide reagents to a subject. In some embodiments, the matrix can be biodegradable. The matrix polymer can be a natural or synthetic polymer. The polymer can be selected based on the time period of the desired release, usually on the order of a few hours to a year or longer. Usually, the release within a period of time between a few hours and three to twelve months can be used. The polymer is optionally in the form of a hydrogel, which can absorb water up to about 90% of its weight, and is also optionally cross-linked with multivalent ions or other polymers.

[0230] Typically, xanthine dehydrogenase (XDH) dsRNA reagent or xanthine dehydrogenase (XDH) antisense polynucleotide reagent can be delivered using biodegradable implants by diffusion or by the degradation of a polymer matrix in some embodiments of the present invention. Exemplary synthetic polymers for such purposes are well known in the art. Using methods known in the art, biodegradable polymers and non-biodegradable polymers can be used for delivering xanthine dehydrogenase (XDH) dsRNA reagent or xanthine dehydrogenase (XDH) antisense polynucleotide reagent. Bioadhesive polymers such as bioerodible hydrogels (HS Sawhney, CP Pathak and JA Hubell in Macromolecules, 1993, 26, 581-587) can also be used for delivering xanthine dehydrogenase (XDH) dsRNA reagent or xanthine dehydrogenase (XDH) antisense polynucleotide reagent, to treat xanthine dehydrogenase (XDH) related diseases or illness. Other suitable delivery systems can include timed release, delayed release or sustained release delivery systems. Such systems can avoid repeated administration of xanthine dehydrogenase (XDH) dsRNA agents or xanthine dehydrogenase (XDH) antisense polynucleotide agents, thereby improving the convenience of subjects and healthcare professionals. Many types of release delivery systems are available and known to those of ordinary skill in the art. See, for example, U.S. Patent Nos. 5,075,109, 4,452,775, 4,675,189, 5,736,152, 3,854,480, 5,133,974, and 5,407,686. In addition, pump-based hardware delivery systems can be used, some of which are also suitable for implantation.

[0231] The use of long-term sustained release implant can be applicable to the preventive treatment of object and the object with the risk of recurrent xanthine dehydrogenase (XDH) related disease or illness.As used herein, long-term release refers to that implant is constructed and arranged to deliver xanthine dehydrogenase (XDH) dsRNA reagent or xanthine dehydrogenase (XDH) antisense polynucleotide reagent of treatment level at least up to 10 days, 20 days, 30 days, 60 days, 90 days, six months, one year or longer time.Long-term sustained release implant is well known to those of ordinary skill in the art and comprises above-mentioned some release systems.

[0232] Therapeutic formulations of xanthine dehydrogenase (XDH) dsRNA agents or xanthine dehydrogenase (XDH) antisense polynucleotide agents can be prepared for storage by mixing the molecule or compound having the desired purity with optional pharmaceutically acceptable carriers, excipients or stabilizers [Remington's Pharmaceutical Sciences 21st edition, (2006)] in the form of a lyophilized formulation or an aqueous solution. Acceptable carriers, excipients or stabilizers are non-toxic to recipients at the doses and concentrations employed and include buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol or benzyl alcohol; parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., zinc-protein complexes); and / or nonionic surfactants, such as or polyethylene glycol (PEG).

[0233] Cells, subjects, and controls

[0234] Method of the present invention can be used in combination with cell, tissue, organ and / or object. In some aspects of the present invention, object is people or vertebrate mammals, includes but not limited to dog, cat, horse, cow, goat, mouse, rat and primate, for example monkey. Therefore, the present invention can be used for treating xanthine dehydrogenase (XDH) related disease or the disease of people and non-human object. In some aspects of the present invention, object can be farm animal, zoo animal, domesticated animal or non-domesticated animal, and method of the present invention can be used for veterinary prevention and treatment scheme. In some embodiments of the present invention, object is people and method of the present invention can be used for people prevention and treatment scheme.

[0235] Non-limiting examples of subjects to which the present invention can be applied are subjects diagnosed with, suspected of having, or at risk of having a disease or condition associated with a higher than desired xanthine dehydrogenase (XDH) expression and / or activity, also referred to as "elevated xanthine dehydrogenase (XDH) expression level." Non-limiting examples of diseases and conditions associated with higher than desired levels of xanthine dehydrogenase (XDH) expression and / or activity are described elsewhere herein. The methods of the present invention can be applied to subjects who have been diagnosed with the disease or condition at the time of treatment, subjects associated with higher than desired xanthine dehydrogenase (XDH) expression and / or activity, or subjects believed to be at risk of having or developing a disease or condition associated with higher than desired xanthine dehydrogenase (XDH) expression and / or activity. In some aspects of the invention, the disease or condition associated with higher than desired xanthine dehydrogenase (XDH) expression and / or activity levels is an acute disease or condition; in certain aspects of the invention, the disease or condition associated with higher than desired xanthine dehydrogenase (XDH) expression and / or activity levels is a chronic disease or condition.

[0236] In one non-limiting example, a xanthine dehydrogenase (XDH) dsRNA agent of the invention is administered to a subject diagnosed with an XDH-related disease, including but not limited to: hyperuricemia, gout, NAFLD, NASH, metabolic disorders, insulin resistance, cardiovascular disease, type 2 diabetes, Lesch-Nyhan syndrome, glycogen storage disease (GSD), such as type I GSD, and diseases associated with oxidative stress, such as chronic low-grade inflammation; or other XDH-related diseases. The methods of the invention can be applied to subjects who have been diagnosed with the disease or condition at the time of treatment, or who are considered to be at risk of developing or developing the disease or condition.

[0237] In another non-limiting example, the xanthine dehydrogenase (XDH) dsRNA agent of the present invention is administered to treat a disease or disorder caused by or associated with the activation of xanthine dehydrogenase (XDH), or a disease or disorder whose symptoms or progression are responsive to the inactivation of xanthine dehydrogenase (XDH). The term "xanthine dehydrogenase (XDH)-related disease" includes diseases, disorders, or conditions that benefit from reduced expression of xanthine dehydrogenase (XDH). Such diseases include, but are not limited to, hyperuricemia, gout, NAFLD, NASH, metabolic disorders, insulin resistance, cardiovascular disease, type 2 diabetes, Lesch Nyhan syndrome, glycogen storage disease (GSD), such as type I GSD, and diseases associated with oxidative stress, such as chronic low-grade inflammation; or other XDH-related diseases.

[0238] The cell that can be applied the method of the present invention includes in vitro, in vivo, in vitro cell.Cell can be in object, in culture and / or in suspension, or in any other suitable state or condition.The cell that can be applied the method of the present invention can be: liver cell (liver cell), liver cell (hepatocyte), heart cell, pancreatic cell, cardiovascular cell, kidney cell or other types of vertebrate cells, including human and non-human mammalian cells.In some aspects of the present invention, the cell that can be applied the method of the present invention is healthy normal cell, which is not known to be disease cell.In some embodiments of the present invention, the method and composition of the present invention are applied to the cell of liver cell, liver cell, heart cell, pancreatic cell, cardiovascular cell and / or kidney cell.In some aspects of the present invention, control cell is normal cell, but it should be understood that the cell with disease or illness can also be used as control cell in specific cases, for example, when comparing the treated cell with disease or illness and the result of untreated cell with disease or illness etc.

[0239] According to the methods of the present invention, the level of xanthine dehydrogenase (XDH) polypeptide activity can be determined and compared to a control level of xanthine dehydrogenase (XDH) polypeptide activity. The control can be a predetermined value, which can take a variety of forms. It can be a single cutoff value, such as a median or mean. It can be established based on a comparison group, for example, a group with normal levels of xanthine dehydrogenase (XDH) polypeptide and / or xanthine dehydrogenase (XDH) polypeptide activity and a group with increased levels of xanthine dehydrogenase (XDH) polypeptide and / or xanthine dehydrogenase (XDH) polypeptide activity. Another non-limiting example of a comparison group can be a group with one or more symptoms or diagnoses of a xanthine dehydrogenase (XDH)-related disease or condition and a group without one or more symptoms or diagnoses of the disease or condition; a group of subjects to whom the siRNA of the present invention has been administered and a group of subjects to whom the siRNA of the present invention has not been administered. Typically, the control can be based on apparently healthy normal individuals or apparently healthy cells in an appropriate age group. It should be understood that, in addition to the predetermined value, the control according to the present invention can be a material sample tested in parallel with the experimental material. Examples include samples from a control population or control samples generated by manufacturing for parallel testing with experimental samples. In some embodiments of the present invention, the control may include cells or subjects that have not been contacted or treated with the xanthine dehydrogenase (XDH) dsRNA reagents of the present invention, in which case the control level of xanthine dehydrogenase (XDH) polypeptide and / or xanthine dehydrogenase (XDH) polypeptide activity can be compared to the level of xanthine dehydrogenase (XDH) polypeptide and / or xanthine dehydrogenase (XDH) polypeptide activity in cells or subjects contacted with the xanthine dehydrogenase (XDH) dsRNA reagents or xanthine dehydrogenase (XDH) antisense polynucleotide reagents of the present invention.

[0240] In some embodiments of the present invention, the control level can be a xanthine dehydrogenase (XDH) polypeptide level determined for a subject, wherein the xanthine dehydrogenase (XDH) polypeptide level determined for the same subject at different times is compared with the control level. In a non-limiting example, the level of xanthine dehydrogenase (XDH) is determined in a biological sample obtained from a subject that has never received xanthine dehydrogenase (XDH) treatment of the present invention. In some embodiments, the biological sample is a serum sample. The uric acid level measured in the sample obtained from the subject can serve as the baseline or control value of the subject. After administering one or more xanthine dehydrogenase (XDH) dsRNA reagents to the subject in the therapeutic methods of the present invention, one or more additional serum samples can be obtained from the subject, and the uric acid level in the subsequent one or more samples can be compared with the control / baseline level of the subject. Such comparisons can be used to assess the onset, progression, or regression of xanthine dehydrogenase (XDH)-related diseases or conditions in the subject. For example, a baseline sample obtained from a subject containing xanthine dehydrogenase (XDH) polypeptide levels that are higher than the levels obtained from the same subject after administration of a xanthine dehydrogenase (XDH) dsRNA agent or a xanthine dehydrogenase (XDH) antisense polynucleotide agent of the invention to the subject indicates regression of a xanthine dehydrogenase (XDH)-related disease or condition and indicates efficacy of the administered xanthine dehydrogenase (XDH) dsRNA agent of the invention in treating a xanthine dehydrogenase (XDH)-related disease or condition.

[0241] In certain aspects of the invention, the biological sample is a hepatocyte sample. One or more values ​​of the xanthine dehydrogenase (XDH) polypeptide and / or xanthine dehydrogenase (XDH) polypeptide activity levels determined for a subject can be used as a control value and used to compare xanthine dehydrogenase (XDH) polypeptide and / or xanthine dehydrogenase (XDH) activity levels in the same subject later, thereby allowing assessment of changes in "baseline" xanthine dehydrogenase (XDH) polypeptide activity in a subject. Therefore, when the initial level is used as the control level for the subject, the initial xanthine dehydrogenase (XDH) polypeptide level and / or the initial xanthine dehydrogenase (XDH) polypeptide activity level can be used to demonstrate and / or determine that the methods and compounds of the present invention can reduce the level of xanthine dehydrogenase (XDH) polypeptide and / or xanthine dehydrogenase (XDH) polypeptide activity in the subject in the subject.

[0242] Using the methods of the present invention, xanthine dehydrogenase (XDH) dsRNA agents and / or xanthine dehydrogenase (XDH) antisense polynucleotide agents of the present invention can be administered to a subject. Such dsRNAi agents include, for example, duplexes AV00206 to AV00292 shown in Table 1. In some embodiments, preferred dsRNAi agents include, for example, duplexes AV00227, AV00260, AV00285, AV00266, AV00280, AV00238, AV00249, AV00289, AV00233, AV00225, AV00273, or AV00283. In other embodiments, preferred dsRNAi agents in Table 2 include, for example, duplexes AV00227, AV00260, AV00285, AV00266, or AV00280. In other embodiments, preferred dsRNAi agents of Table 3 include, for example, duplexes AD00353, AD00356, AD00358, AD00364, AD00365, AD00366, AD00368, AD00370, AD00430, AD00431, AD00521, AD00521-1, AD00364-1, AD00365-2, AD00366-1, and AD00432-1. The efficacy of the administration and treatment of the present invention can be assessed as follows: the level of uric acid in a serum sample obtained from the subject after administration and treatment is reduced by at least 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more compared to the pre-administration level of uric acid in a serum sample obtained from the subject at a previous time point, or compared to the uric acid level in a non-contact control (e.g., the uric acid level in a control serum sample). It should be understood that the level of uric acid is correlated with the level of xanthine dehydrogenase (XDH) gene expression. Certain embodiments of the methods of the present invention include administering a xanthine dehydrogenase (XDH) dsRNA and / or a xanthine dehydrogenase (XDH) antisense agent of the present invention to a subject in an amount effective to inhibit the expression of the xanthine dehydrogenase (XDH) gene, thereby reducing the level of xanthine dehydrogenase (XDH) polypeptide in the subject and reducing the level of xanthine dehydrogenase (XDH) polypeptide activity.In some embodiments of the methods of the invention, contacting a cell with an siRNA agent of the invention (also referred to herein as treatment) results in at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110 %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100%, for example, to below the level of detection in an assay.

[0243] Some embodiments of the present invention include determining the presence, absence, and / or amount (also referred to herein as level) of a xanthine dehydrogenase (XDH) polypeptide in one or more biological samples obtained from one or more subjects. This assay can be used to evaluate the efficacy of the therapeutic methods of the present invention. For example, the methods and compositions of the present invention can be used to determine the level of a xanthine dehydrogenase (XDH) polypeptide in a biological sample obtained from a subject previously treated with a xanthine dehydrogenase (XDH) dsRNA reagent and / or a xanthine dehydrogenase (XDH) antisense agent of the present invention. A reduction in the level of xanthine dehydrogenase (XDH) polypeptide in a hepatocyte sample obtained from the subject by at least 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, when administered and treated, as compared to pre-dose levels of xanthine dehydrogenase (XDH) polypeptide in a hepatocyte sample obtained from the subject at a previous time point, or as compared to uncontacted control levels (e.g., levels of xanthine dehydrogenase (XDH) polypeptide in a control hepatocyte sample), indicates a level of efficacy of the treatment administered to the subject.

[0244] In some embodiments of the present invention, the physiological characteristics of the xanthine dehydrogenase (XDH) related disease or illness determined for the object can be used as a control result, and the determination result of the physiological characteristics of the same object at different times is compared with the control result. In a non-limiting example, the pathological feature uric acid level is measured from an object that has never been treated with the xanthine dehydrogenase (XDH) of the present invention, which can be used as the baseline or control value of the object. After the xanthine dehydrogenase (XDH) dsRNA reagent is applied to the object once or more in the method for treating the present invention, the uric acid level is compared with the control / baseline level of the object respectively. This type of comparison can be used to assess the onset, progression or disappearance of the xanthine dehydrogenase (XDH) related disease or illness in the object. For example, a baseline uric acid level obtained from a subject that is higher than the uric acid level measured from the same subject after administration of a xanthine dehydrogenase (XDH) dsRNA agent or a xanthine dehydrogenase (XDH) antisense polynucleotide agent of the invention to the subject indicates regression of a xanthine dehydrogenase (XDH)-related disease or condition and indicates the efficacy of the administered xanthine dehydrogenase (XDH) dsRNA agent of the invention in treating a xanthine dehydrogenase (XDH)-related disease or condition.

[0245] Some embodiments of the present invention include determining the presence, absence, and / or changes in physiological characteristics of a xanthine dehydrogenase (XDH)-associated disease or condition using, for example, but not limited to, the following methods: (1) measuring uric acid levels in a subject; (2) evaluating physiological characteristics of one or more biological samples obtained from one or more subjects; (3) or performing a physical examination of the subject. This assay can be used to evaluate the efficacy of the treatment methods of the present invention.

[0246] medicine box

[0247] Kits containing one or more xanthine dehydrogenase (XDH) dsRNA agents and / or xanthine dehydrogenase (XDH) antisense polynucleotide agents and instructions for use in the methods of the present invention are also within the scope of the present invention. The kits of the present invention may contain one or more xanthine dehydrogenase (XDH) dsRNA agents, xanthine dehydrogenase (XDH) sense polynucleotides, and xanthine dehydrogenase (XDH) antisense polynucleotide agents that can be used to treat xanthine dehydrogenase (XDH)-related diseases or conditions. Kits containing one or more xanthine dehydrogenase (XDH) dsRNA agents, xanthine dehydrogenase (XDH) sense polynucleotides, and xanthine dehydrogenase (XDH) antisense polynucleotide agents can be prepared for use in the treatment methods of the present invention. The components of the kits of the present invention can be packaged in aqueous media or lyophilized form. The kits of the present invention can include a carrier that is partitioned to enclose one or more container devices or a series of container devices (e.g., test tubes, vials, flasks, bottles, syringes, etc.) therein. The first container means or a series of container means may contain one or more compounds, such as xanthine dehydrogenase (XDH) dsRNA agents and / or xanthine dehydrogenase (XDH) sense or antisense polynucleotide agents. The second container means or a series of container means may contain targeting agents, labeling agents, delivery agents, etc., which may be included as part of the xanthine dehydrogenase (XDH) dsRNA agents and / or xanthine dehydrogenase (XDH) antisense polynucleotides administered in embodiments of the present invention.

[0248] The kit of the present invention may also include instructions. The instructions are typically in written form and will provide guidance for performing the treatment embodied by the kit and for making decisions based on the treatment.

[0249] The following examples are provided to illustrate specific examples of the practice of the present invention and are not intended to limit the scope of the invention. It will be apparent to one of ordinary skill in the art that the present invention can be applied to a variety of compositions and methods.

[0250] Example

[0251] Example 1. Synthesis of RNAi Reagents

[0252] The xanthine dehydrogenase (XDH) gene RNAi reagent duplexes shown in Tables 1-3 above were synthesized according to the following general procedure:

[0253] The sense and antisense strands of siRNA were synthesized on an oligonucleotide synthesizer using a well-established solid-phase synthesis method based on phosphoramidite chemistry. The oligonucleotide chain growth was achieved through a four-step cycle: deprotection, condensation, capping, and oxidation or sulfurization steps for each nucleotide addition. The synthesis was performed on a controlled pore glass (CPG) ). Monomeric phosphoramidites were purchased from commercial sources. Phosphoramidites with GalNAc ligand clusters (GLPA1, GLPA2, and GLPA15 as non-limiting examples) were synthesized according to the procedures of Examples 2-3 herein. For siRNAs used for in vitro screening (Table 2), synthesis was performed at a 2 μmol scale; for siRNAs used for in vivo testing (Table 3), synthesis was performed at a 5 μmol scale or greater. In cases where the GalNAc ligand (GLO-n as a non-limiting example) was attached to the 3'-end of the sense strand, a CPG solid support with a GalNAc ligand attached was used. In cases where the GalNAc ligand (GLS-1 or GLS-2 as non-limiting examples) was attached to the 5'-end of the sense strand, a GalNAc phosphoramidite (GLPA1 or GLPA2 as non-limiting examples) was used for the final coupling reaction. 3% trichloroacetic acid (TCA) in dichloromethane was used for deprotection of the 4,4'-dimethoxytrityl protecting group (DMT). 5-Ethylthio-1H-tetrazole is used as an activator. I2 in THF / Py / H2O and phenylacetyl disulfide (PADS) in pyridine / MeCN are used for oxidation and sulfurization reactions, respectively. After the final solid phase synthesis step, the solid support-bound oligomer is cut and the protecting group is removed by treating with a 1:1 volume of 20wt% methylamine aqueous solution and 28% ammonium hydroxide solution. In order to synthesize siRNA for in vitro screening, the crude mixture is concentrated. The remaining solid is dissolved in 1.0M NaOAc and ice-cold EtOH is added to precipitate a single-stranded product as a sodium salt, which can be used for annealing without further purification. In order to synthesize siRNA for in vivo testing, the crude single-stranded product is further purified by ion pair reversed-phase HPLC (IP-RP-HPLC). By dissolving the purified single-stranded oligonucleotide product from IP-RP-HPLC in 1.0M NaOAc and precipitating it by adding ice-cold EtOH, it is converted into a sodium salt. Sense and antisense oligonucleotides were annealed with equimolar complementarity in water to form a double-stranded siRNA product, which was lyophilized to provide a fluffy white solid.

[0254] Example 2. Preparation of Intermediate-A and Intermediate-B

[0255] As shown in Scheme 1 below, intermediate-A was synthesized by treating commercially available galactosamine pentaacetate with trimethylsilyl trifluoromethanesulfonate (TMSOTf) in dichloromethane (DCM). Glycosylation was then performed with Cbz-protected 2-(2-aminoethoxy)ethan-1-ol to provide compound II. The Cbz protecting group was removed by hydrogenation to provide intermediate-A as a trifluoroacetate (TFA) salt. Intermediate B was synthesized based on the same protocol, except that Cbz-protected 2-(2-(2-aminoethoxy)ethoxy)ethan-1-ol was used as the starting material.

[0256] Solution 1

[0257] To a solution of compound I (20.0 g, 51.4 mmol) in 100 mL of 1,2-dichloroethane (DCE) was added TMSOTf (17.1 g, 77.2 mmol). The resulting reaction solution was stirred at 60°C for 2 hours and then at 25°C for 1 hour; Cbz-protected 2-(2-aminoethoxy)ethan-1-ol (13.5 g, 56.5 mmol) was dissolved in DCE (100 mL) with Powdered molecular sieve (10g) is dried and added dropwise to the above-mentioned reaction solution at 0°C under N2 atmosphere.Under N2 atmosphere, the resulting reaction mixture is stirred at 25°C for 16 hours.The reaction mixture is filtered and washed with saturated NaHCO3 (200mL), water (200mL) and saturated brine (200mL).The organic layer is dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to obtain a crude product, which is ground together with 2-methyltetrahydrofuran / heptane (5 / 3, v / v, 1.80L) for 2 hours.The resulting mixture is filtered and dried to obtain Compound II (15.0g, 50.3% yield) as a white solid.

[0258] 10% Pd / C (1.50 g) was carefully added to a dry and argon-purged hydrogenation bottle, followed by 10 ml of tetrahydrofuran (THF), followed by a solution of compound II (15.0 g, 26.4 mmol) in THF (300 ml) and TFA (trifluoroacetic acid, 3.00 g, 26.4 mmol). The resulting mixture was degassed and purged 3 times with H2 and stirred at 25 ° C for 3 hours under an atmosphere of H2 (45 psi). Thin layer chromatography (TLC, solvent: DCM: MeOH = 10: 1) showed that compound II was completely consumed. The reaction mixture was filtered and concentrated under reduced pressure. The residue was dissolved in anhydrous DCM (500 mL) and concentrated. The process was repeated 3 times to obtain intermediate-A (14.0 g, 96.5% yield) as a foamy white solid. 1H NMR (400MHz DMSO-d6): δppm 7.90 (d, J=9.29Hz, 1H), 7.78 (br s, 3H), 5.23 (d, J = 3.26Hz, 1H), 4.98 (dd, J = 11.29, 3.26Hz, 1H), 4.56 (d, J = 8. 53Hz,1H),3.98-4.07(m,3H),3.79-3.93(m,2H),3.55-3.66(m,5H),2.98(br d,J=4.77Hz,2H),2.11(s,3H),2.00(s,3H),1.90(s,3H),1.76(s,3H).

[0259] Intermediate-B was synthesized using a similar procedure to that used for the synthesis of Intermediate-A. 1H NMR (400 MHz DMSO-d6): δ ppm 7.90 (br d, J = 9.03 Hz, 4H), 5.21 (d, J = 3.51 Hz, 1H), 4.97 (dd, J = 11.1 Hz, 1H), 4.54 (d, J = 8.53 Hz, 1H), 3.98-4.06 (m, 3H), 3.88 (dt, J = 10.9 Hz, 1H), 3.76-3.83 (m, 1H), 3.49-3.61 (m, 9H), 2.97 (br s, 2H), 2.10 (s, 3H), 1.99 (s, 3H), 1.88 (s, 3H), 1.78 (s, 3H). Mass spectrum calculated for C20H34N2O11: 478.22; found: 479.3 (M+H+).

[0260] Example 3. Synthesis of GalNAc ligand cluster phosphoramidites GLPA1, GLPA2, and GLPA15

[0261] GLPA1 and GLPA2 were prepared according to Scheme 2 below. Starting from benzyl-protected propane-1,3-diamine, it was alkylated with tert-butyl 2-bromoacetate to provide the triester compound I. The benzyl protecting group was removed by hydrogenation to provide the secondary amine compound II. The amide was coupled with 6-hydroxyhexanoic acid to provide compound III. The tert-butyl protecting group was then removed upon treatment with HCl in dioxane to generate the triacid compound IV. Amide coupling between triacid compound IV and intermediate-A or intermediate-B was performed to provide compound Va or Vb. The phosphoramidites GLPA1 or GLPA2 were synthesized by phosphitylation of compound Va or Vb with 2-cyanoethyl N,N-diisopropylchlorophosphoramidite and a catalytic amount of 1H-tetrazole.

[0262] Option 2

[0263] To a solution of N-benzyl-1,3-propanediamine (5.00 g, 30.4 mmol) in dimethylformamide (DMF, 100 mL) was added tert-butyl 2-bromoacetate (23.7 g, 121 mmol); diisopropylethylamine (DIEA, 23.61 g, 182 mmol) was then added dropwise. The resulting reaction mixture was stirred at 25-30°C for 16 hours. LCMS indicated complete consumption of N-benzyl-1,3-propanediamine. The reaction mixture was diluted with H₂O (500 mL) and extracted with EtOAc (500 mL x 2). The combined organics were washed with saturated brine (1 L), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to obtain the crude product, which was then purified by silica gel column chromatography (gradient: petroleum ether:ethyl acetate 20:1 to 5:1). Compound I (12.1 g, 78.4% yield) was obtained as a colorless oil. 1H NMR (400MHz, CDCl3): δppm 7.26-7.40(m,5H),3.79(s,2H),3.43(s,4H),3.21(s,2H),2.72(dt,J=16.9,7.34Hz,4H),1.70(quin,J=7.2Hz,2H),1.44-1.50(m,27H).

[0264] The dried hydrogenation bottle was purged with argon 3 times. Pd / C (200 mg, 10%) was added, followed by MeOH (5 mL), followed by a solution of compound I (1.00 g, 1.97 mmol) in MeOH (5 mL). The reaction mixture was degassed under vacuum and refilled with H 2 . This process was repeated 3 times. The mixture was stirred at 25 ° C for 12 hours under an H 2 (15 psi) atmosphere. LCMS showed that compound I was completely consumed. The reaction mixture was filtered under reduced pressure under an N 2 atmosphere. The filtrate was concentrated under reduced pressure to obtain compound II (655 mg, 79.7% yield) as a yellow oil, which can be used in the next step without further purification. 1H NMR (400MHz, CDCl3): δppm 3.44 (s, 4H), 3.31 (s, 2H), 2.78 (t, J = 7.1Hz, 2H), 2.68 (t, J = 6.9Hz, 2H), 1.88 (br s, 1H), 1.69 (quin, J = 7.03Hz, 2H), 1.44-1.50 (s, 27H).

[0265] A mixture of compound II (655 mg, 1.57 mmol), 6-hydroxyhexanoic acid (249 mg, 1.89 mmol), DIEA (1.02 g, 7.86 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 904 mg, 4.72 mmol) and 1-hydroxybenzotriazole (HOBt, 637 mg, 4.72 mmol) in DMF (6 mL) was degassed and purged with N 2 three times; then stirred at 25 ° C. under an N 2 atmosphere for 3 hours. LCMS indicated the desired product. The reaction mixture was diluted with HO (10 mL) and extracted with EtOAc 20 mL (10 mL×2). The organics were combined and washed with saturated brine (20 mL), dried over anhydrous Na2SO4, filtered, and concentrated to give the crude product, which was purified by silica gel column chromatography (gradient: petroleum ether:ethyl acetate from 5:1 to 1:1) to give Compound III (650 mg, 77.8% yield) as a yellow oil. 1H NMR (400 MHz, CDCl3): δ ppm 3.90-3.95 (s, 2H), 3.63 (t, J = 6.40 Hz, 2H), 3.38-3.45 (m, 6H), 2.72 (t, J = 6.65 Hz, 2H), 2.40 (t, J = 7.28 Hz, 2H), 1.55-1.75 (m, 8H), 1.44 (s, 27H). Mass spectrum calculated for C27H50N2O8: 530.36; found: 531.3 (M+H + ).

[0266] A mixture of compound III (5.5 g, 10.3 mmol) in HCl / dioxane (2 M, 55 mL) was stirred at 25 ° C for 3 hours. LCMS showed that compound III was completely consumed. The reaction mixture was filtered, washed with EtOAc (50 mL), and dried under reduced pressure to give a crude product. It was dissolved in CH3CN (50 mL) and volatiles were removed in vacuo. The process was repeated 3 times to obtain compound IV (2.05 g, 54.5% yield) as a white solid. 1 H NMR (400MHz, D2O): δppm 4.21(s,1H),4.07(d,J=4.5Hz,4H),3.99(s,1H),3.45-3.52(m,3H),3. 42(t,J=6.5Hz,1H),3.32-3.38(m,1H),3.24-3.31(m,1H),2.37(t,J=7 .4Hz,1H),2.24(t,J=7.4Hz,1H),1.99(dt,J=15.5,7.53Hz,1H),1.85-1.94(m,1H),1.85-1.94(m,1H),1.39-1.56(m,4H),1.19-1.31(m,2H).

[0267] A mixture of compound IV (500 mg, 1.05 mmol), intermediate-A (2.02 g, 3.67 mmol), DIEA (813 mg, 6.30 mmol), EDCI (704 mg, 3.67 mmol) and HOBt (496 mg, 3.67 mmol) in DMF (10 milliliters) was degassed and purged with N2 three times, and then the mixture was stirred at 25 ° C under N2 atmosphere for 3 hours. LCMS indicates the desired product. The reaction mixture was quenched by adding H2O (10 mL) and extracted with DCM (10 mL × 2). The combined organic matter was extracted with 10% citric acid (20 mL). The aqueous phase was neutralized with saturated NaHCO3 solution and re-extracted with DCM (10 mL x 2). The organic matter was dried over sodium sulfate, filtered and concentrated under reduced pressure to produce compound Va (570 mg, 0.281 mmol, 26.8% yield) as a white solid. 1H NMR:(400MHz,CDCl3)ppmδ7.84-8.12(m,3H),6.85-7.15(m,2H),6.66-6.81(m,1H),5.36(br d,J=2.7Hz,3H),5.11-5.27(m,3H),4.63-4.85(m,3H),3.90-4.25(m,18H),3.37-3.75(m,28H),3.15-3.28(m,4H),2.64(br d,J=6.53Hz,2H),2.30-2.46(m,2H),2.13-2.18(m,9H),2.05(s,9H),1.94-2.03(m,18H),1.68(br s,2H),1.45(br s,2H),1.12(br t,J=7.0Hz,2H)。

[0268] To a solution of compound Va (260 mg, 0.161 mmol) in anhydrous DCM (5 mL) was added diisopropylammonium tetrazolium (30.3 mg, 0.177 mmol); then 3-bis(diisopropylamino)phosphinoyloxypropionitrile (194 mg, 0.645 mmol) was added dropwise at ambient temperature and N2. The reaction mixture was stirred at 20 to 25 ° C for 2 hours. LCMS showed that compound Va was completely consumed. After cooling to -20 ° C, the reaction mixture was added to a stirred brine / saturated NaHCO3 (1:1, 5 mL) solution at 0 ° C. After stirring for 1 minute, DCM (5 mL) was added. Layers separated. The organic layer was washed with brine / saturated NaHCO3 aqueous solution (1:1, 5 mL), dried over Na2SO4, filtered and concentrated to a volume of approximately 1 mL. The residual solution was added dropwise to 20 mL of methyl tert-butyl ether (MTBE) under stirring. This resulted in the precipitation of a white solid. The mixture was centrifuged and the solid was collected. The solid was redissolved in 1 mL of DCM and precipitated by adding MTBE (20 mL). The solid was isolated again by centrifugation. The collected solid was dissolved in anhydrous CH3CN. The volatiles were removed. This process was repeated two more times to obtain the GalNAc ligand phosphoramidite compound GLPA1 (153 mg, 84.4 μmol) as a white solid. 1H NMR (400MHz, CDCl3): ppmδ7.71-8.06(m,2H),6.60-7.06(m,3H),5.37(br d,J=3.0Hz,3H),5.18-5.32(m,3H),4.70-4.86(m,3H),3.92-4.25(m,18H),3.42-3.85(m,30H),3.25(m ,4H),2.59-2.75(m,4H),2.27-2.44(m,2H),2.15-2.20(s,9H)2.07(s,9H),1.96-2.03(m,18H),1.65(br s,4H),1.44(br d,J=7.28Hz,2H),1.14-1.24(m,12H).31P NMR(CDCl3):ppmδ147.15.

[0269] The GalNAc ligand phosphoramidite compound GLPA2 was synthesized using the same procedure except that Intermediate-B was used. 1H NMR (400 MHz, CDCl 3 ): ppm δ 7.94-8.18 (m, 1H), 7.69 (br s,1H),6.66-7.10(m,3H),5.35(d,J=3.5Hz,3H),5.07-5.25(m,3H),4.76-4 .86(m,3H),4.01-4.31(m,10H),3.91-4.01(m,8H),3.74-3.86(m,4H),3.52- 3.71(m,30H),3.42-3.50(m,6H),3.15-3.25(m,4H),2.52-2.70(m,4H),2.2 2-2.45(m,2H),2.15-2.22(s,9H),2.06(s,9H),1.95-2.03(m,18H),1.77(br s,2H),1.58-1.66(m,4H),1.40(m,2H),1.08-1.24(m,12H).31P NMR(CDCl3):ppmδ147.12.

[0270] GLPA15 was prepared according to the following protocol 3.

[0271] To a solution of intermediate compound II (275 g, 660 mmol, 1.00 equivalent (eq)) in dichloromethane (2.75 L) was added triethylamine (133 g, 1.32 mol, 2.00 eq.), followed by the dropwise addition of Cbz-Cl (169 g, 990 mmol, 1.50 eq.). The reaction mixture was stirred at 25°C for 2 hours. LCMS indicated complete conversion of compound II. The reaction mixture was washed sequentially with a saturated solution of NaHCO₃ (800 mL) and then saturated brine (500 mL). The organic phase was dried over anhydrous Na₂SO₄. After filtering to remove the desiccant, the filtrate was concentrated to dryness. The crude product was purified by column chromatography (SiO₂, PE / EA = 100 / 1 to 5 / 1) to afford compound III-1 (290 g, 527 mmol, 75.7% yield) as a colorless oil. 1 H NMR (400MHz in DMSO-d6): δppm 7.23-7.40(m,5H),5.00-5.12(m,2H),3.86-3.95(m,2H),3.23-3.39(m,6H ),2.55-2.67(m,2H),1.56-1.64(m,2H),1.31-1.46(m,27H).MS(ESI)[M+H] + m / z:551.6.

[0272] To compound III-1 (145 g, 263 mmol, 1.00 eq) was added HCOOH (2.9 L), and the solution was stirred at 60°C for 12 hours. LCMS showed complete conversion of compound III-1. 1.5 L of toluene and 1.5 L of acetonitrile were added to the reaction solution, which was concentrated under reduced pressure to approximately 500 mL. Toluene / acetonitrile (1:1, ~750 mL) was then added and concentrated to approximately 500 mL. Acetonitrile (~1000 mL) was then added and concentrated to dryness. The crude product was triturated with 700 mL of acetonitrile at 60°C for 2 hours and filtered. The solid was collected and dried to yield compound IV-1 (105 g, quantitative) as a white solid. 1H NMR (400MHz in DMSO-d6): δppm 7.26-7.40(m,5H),5.02-5.10(m,2H),3.89-4.00(m,2H),3.36-3.45(m,4H),3.24-3.34(m,2H),2.59-2.72(m,2H),1.40(s,2H).MS(ESI)[M+H] + m / z:383.0.

[0273] To a solution of compound IV-1 (100 g, 261 mmol) and intermediate-A (502 g, 915 mmol, 3.50 eq.) in DMF (1.0 L) were added TBTU (327 g, 1.02 mol, 3.90 eq.) and triethylamine (212 g, 2.09 mol, 8.00 eq.). The reaction was allowed to proceed at 25°C for 1 hour. LCMS indicated complete conversion of compound IV-1. The reaction solution was added to 4000 mL of water and extracted with methyl tert-butyl ether (2000 mL in two portions) to remove impurities. The remaining aqueous phase was extracted with dichloromethane (3000 mL in two portions). The dichloromethane phase was washed sequentially with 10% aqueous citric acid (2000 mL in two portions), saturated NaHCO₃ (2.0 L in two portions), and saturated brine (2.0 L), and then dried over anhydrous Na₂SO₄. The filtrate was filtered and concentrated under reduced pressure to give white solid compound V-1 (260 g, 159 mmol, yield 60.9%). 1H NMR (400MHz in DMSO-d6): δppm 7.99-8.08(m,2H),7.93(br d,J=5.50Hz,1H),7.79-7.86(m,3H),7.26-7.39(m,5H),5.22(d,J=3.13Hz,3H),4.95-5.08(m,5H),4.54(br d,J=8.38Hz,3H),4.03(s,9H),3.81-3.93(m,5H),3.76(br d,J=4.88Hz,3H),3.44-3.62(m,10H),3.34-3.43(m,6H),3.24(br d,J=6.13Hz,7H),3.02-3.09(m,4H),2.40-2.47(m,2H),2.10(s,9H),1.99(s,9H),1.89(s,9H),1.77(s,9H),1.57-1.68(m,2H). MS(ESI)[M+H] + m / z:816.4.

[0274] A 2L hydrogenation reactor was inertized with argon and dry Pd / C (9 g) was carefully added. MeOH (50 mL) was then added to moisten the Pd / C. Then, under an argon atmosphere, a solution of compound V-1 (90 g, 55.1 mmol, 1.00 eq.) and trifluoroacetic acid (6.29 g, 55.1 mmol, 1.00 eq.) in MeOH (850 mL) was slowly added. The mixture was degassed and replaced with a hydrogen atmosphere by adding H2 three times, and stirred at 25°C for 10 hours. LCMS indicated complete conversion of compound V-1. The Pd / C was removed by filtration, and the filtrate was concentrated under reduced pressure to afford compound VI-1 (80 g, 90.2% yield). 1H NMR (400MHz in DMSO-d6): δppm 9.12 (br s, 2H), 8.50 (br t, J=5.19Hz, 1H), 8.10 (br t,J=5.50Hz,2H),7.85-7.91(m,3H),5.22(d,J=3.25Hz,3H),4.95-5.01(m,3H),4.52 -4.58(m,3H),4.03(s,9H),3.84-3.93(m,3H),3.75-3.83(m,3H),3.39-3.61(m,16H) ,3.23-3.32(m,6H),3.15-3.18(m,3H),2.97-3.05(m,2H),2.54-2.61(m,2H),2.10(s ,9H),2.00(s,9H),1.89(s,9H),1.77-1.80(m,9H),1.70-1.76(m,2H).MS(ESI)[M+H] + m / z:749.3.

[0275] To a solution of compound VI-1 (270 g, 168 mmol, 1.00 eq.) and glutaric anhydride (28.6 g, 252 mmol, 1.50 eq.) in dichloromethane (2.7 L) was added triethylamine (67.8 g, 672 mmol, 4.00 eq.). The solution was stirred at 25°C for 1 hour. LCMS indicated complete conversion of compound VI-1 to compound VII. 4-Hydroxypiperidine (42.4 g, 420 mmol, 2.50 eq.) and TBTU (107 g, 335 mmol, 2.00 eq.) were added to the reaction mixture, and stirring was continued at 25°C for 1 hour. LCMS indicated complete conversion of compound VII. The reaction was quenched by the slow addition of saturated NH4Cl (3.0 L). The layers were separated, and the aqueous phase was extracted with dichloromethane (2 x 1000 mL) and combined with the previous organic phase. The combined organic phases were washed with a 1:1 mixture of saturated NaHCO₃(aq) and saturated brine (3.0 L), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was dissolved in 1.5 L of dichloromethane and added dropwise to methyl tert-butyl ether (7.5 L). A translucent white precipitate gradually formed during the addition. The precipitate was vacuum filtered, and the collected solid was dried under vacuum to yield Compound VIII as a white solid (207 g, 72.8% yield). 1H NMR (400MHz in DMSO-d6): δppm 8.05(br d,J=2.00Hz,2H),7.82(br d,J=7.38Hz,3H),5.21(br s,3H),4.98(br d,J=10.26Hz,3H),4.72(br s,1H),4.54(br d,J=7.88Hz,3H),4.03(br s,9H),3.74-3.94(m,9H),3.45-3.71(m,12H),3.40(br s,6H),3.24(br s,7H),3.07(br d,J=14.13Hz,5H),2.91-3.01(m,1H),2.24-2.44(m,5H),2.20(br s,1H),2.10(s,9H),1.96-2.04(m,9H),1.89(br s,9H),1.74-1.81(m,9H),1.51-1.73(m,6H),1.07-1.36(m,3H).MS(ESI)[M+H] + m / z:848.0.

[0276] To a solution of compound VIII (200 g, 118 mmol, 1.00 eq.) and diisopropylammonium tetrazolium (8.08 g, 47.2 mmol, 0.40 eq) in dichloromethane (2.0 L) was added 3-bis(diisopropylamino)phosphonooxypropionitrile (53.3 g, 177 mmol, 1.50 eq.). The reaction solution was stirred at 40 ° C for 2 hours. LCMS showed that the conversion of compound VIII was complete. The reaction mixture was washed with a 1:1 mixture of saturated NaHCO₃ and saturated brine (2.0 L) and dried over anhydrous Na₂SO₄. The filtrate was concentrated, and the resulting crude product was dissolved in dichloromethane (1.2 L) and added dropwise to stirred methyl tert-butyl ether (6.0 L). The suspension was filtered, and the filter cake was rinsed with methyl tert-butyl ether. The collected solid was vacuum-dried, dissolved in dichloromethane (1.0 L), and concentrated to dryness. This process was repeated four times to remove any residual tert-butyl ether, yielding GLPA15 (164 g, 73.3% yield). 1H NMR (400MHz in DMSO-d6): δppm 8.05(br d,J=6.50Hz,2H),7.81(br d,J=9.01Hz,3H),5.22(d,J=3.25Hz,3H),4.98(dd,J=11.26,3.25Hz,3H),4.55(br d,J=8.50Hz,3H),4.03(s,9H),3.64-3.97(m,12H),3.55-3.63(m,6H),3.50(br s,5H),3.40(br d,J=6.13Hz,6H),3.17-3.30(m,9H),3.07(br d,J=14.26Hz,4H),2.76(t,J=5.82Hz,2H),2.18-2.47(m,6H),2.10(s,9H),1.9 9(s,9H),1.89(s,9H),1.78(s,9H),1.52-1.74(m,6H),1.12-1.19(m,12H).31P NMR(DMSO-d6):ppmδ145.25.MS(ESI)[M+H] + m / z:1895.7.

[0277] In certain studies, a method is provided for attaching a targeting group comprising GalNAc (also referred to herein as a GalNAc delivery compound) to the 5'-end of the sense strand, which comprises using a GalNAc phosphoramidite (e.g., GLPA1) in the last coupling step of solid phase synthesis, using such a synthesis process, such as that used in oligonucleotide chain extension (i.e., adding nucleotides to the 5' end of the sense strand), to attach it to the 5'-end of the sense strand.

[0278] In some studies, a method for attaching a targeting group comprising GalNAc to the 3'-end of the sense strand includes using a solid support (CPG) comprising GLO-n. In some studies, a method for attaching a targeting group comprising GalNAc to the 3'-end of the sense strand includes attaching the GalNAc targeting group to a CPG solid support via an ester bond, and using the resulting CPG with the attached GalNAc targeting group when synthesizing the sense strand, which results in the GalNAc targeting group being attached to the 3'-end of the sense strand. Other GalNAc phosphoramidite compounds (GLPAn) can also be obtained using similar methods herein or well known in the art after using reasonably corresponding intermediates, and attached as targeting groups to appropriate positions of the siRNA duplex.

[0279] Example 4. Synthesis of isomannide phosphoramidite (Compound 2)

[0280] DMTrCl (232 g, 684 mmol, 1.0 eq) in pyridine (400 ml) was added to a solution of compound A isomannide (100 g, 684 mmol, 1.0 eq) in pyridine (600 ml), and the mixture was stirred at 25°C for 16 hours. LC-MS showed that compound A was completely consumed, and a main peak with the desired mass was detected. The resulting reaction mixture was diluted with water (500 ml), extracted with dichloromethane (500 ml * 2), and the combined organic phases were washed with brine (500 ml), dried over Na2SO4 and concentrated in vacuo to obtain a residue. The residue was purified by column chromatography (DCM / MeOH=100 / 1 to 50 / 1, 0.1% Et3N) to give compound B (150 g, 48.9% yield) as a yellow solid. 1 H NMR: EC4783-404-P1B1_C (400MHz, DMSO-d6) δppm 7.46 (br d, J=7.63Hz, 2H) 7.28-7.37 (m, 6H) 7.19-7.25 (m, 1H) 6.90 (br d,J=7.88Hz,4H)4.70(d,J=6.50Hz,1H)3.99-4.09(m,6H)3.88-3.96(m,2H)3.83(br dd,J=7.82,6.94Hz,1H)3.74(s,6H)3.41(br t,J=8.13Hz,1H)3.05(t,J=8.44Hz,1H)2.85(br t,J=7.50Hz,1H).

[0281] Under N2 atmosphere, to a solution of compound B (80.0 g, 178 mmol, 1.0 eq) in dichloromethane (5.0 mL) was added dropwise 2H-tetrazole (0.45 M, 436 mL, 1.1 eq) at 25°C, followed by the addition of a solution of compound C (2-cyanoethyldiisopropylchlorophosphoramidite, 80.6 g, 267 mmol, 85.0 mL, 1.5 eq) in dichloromethane (200 mL); the reaction mixture was stirred at 25°C for 1.0 h; LC-MS showed that compound B was completely consumed, and a major peak with the desired mass was detected. The resulting reaction mixture was cooled to -20°C and poured into an ice-cold oven; NaHCO3 (500 mL), extracted with dichloromethane (500 mL * 3), and the combined organic layers were washed with NaHCO3 / brine = 1:1 (300 mL / 300 mL), dried over Na2SO4, and concentrated in vacuo (35°C) to obtain a residue (100 mL). The residue was purified by column chromatography (Al2O3, DCM / MeOH = 100 / 1 to 50 / 1, 0.1% Et3N) to obtain compound 2 (77 g, 119 mmol, yield 66.5%) as a white solid.

[0282] 1H NMR:EC4783-423-P1B1_C(400MHz,DMSO-d6)

[0283] δppm 7.22(br d,J=7.50Hz,2H)7.05-7.14(m,6H)6.96-7.02(m,1H)6.67(br dd,J=8.82,1.81Hz,4H)3.95-4.07(m,2H)3.73-3.83(m,1H)3.62-3.72(m,2H)3.48-3.53(m ,6H)3.27-3.37(m,3H)3.11(s,6H)2.82(td,J=8.54,2.31Hz,1H)2.47-2.63(m,3H)2.28(br d,J=1.63Hz,3H)0.82-1.00(m,13H).

[0284] Other phosphoramidite compounds can be prepared according to methods well known to those skilled in the art, such as but not limited to the methods described in the prior art US Pat. No. 426,220 and WO 02 / 36743.

[0285] Example 5. Preparation of a solid support containing isomannide monomer

[0286] Represents the macroporous aminomethyl polyethylene resin support part

[0287] Place a 50L glass kettle under nitrogen protection, add dichloromethane (19.50kg) to the glass kettle, and start stirring. Control the temperature at 20-30°C, add DMTr-imann (1.47kg) to the glass kettle, add triethylamine (1.50kg), 4-dimethylaminopyridine (0.164kg), and add succinic anhydride (1.34kg) to the reactor. Keep the system warm at 20-30°C for 18 hours, then take a sample and terminate the reaction. Add saturated sodium bicarbonate solution (22.50kg) to the reaction system, stir for 10-20 minutes, and then let it stand until the layers are separated. Transfer the lower organic phase to, extract the upper aqueous phase twice with dichloromethane, combine the organic phases, dry with anhydrous sodium sulfate, filter the filtrate, and then transfer to rotary evaporation to concentrate until there is no fraction, forming 1.83kg of solid gray to off-white solid.

[0288] Add N,N-dimethylformamide (23.50 kg) to a 100 L glass kettle and start stirring. Control the temperature at 20-30 ° C. Under nitrogen protection, add the product from the previous step, O-benzotriazole-tetramethyluronium hexafluorophosphate (0.33 kg), and N,N-diisopropylethylamine (0.13 kg) to the 100 L glass kettle through a solid addition funnel. After stirring for 10-30 minutes, discharge it into a 50 L galvanized bucket for use. Add macroporous amine methyl resin (3.25 kg) (purchased from Tianjin Nankai Hecheng Technology Co., Ltd., batch number HA2X1209, loading capacity 0.48 mmol / g) to the 100 L solid phase synthesis reactor through a solid addition funnel. Control the temperature at 20-30 ° C. Add N,N-dimethylformamide (21.00 kg + 21.00 kg) to the solid phase synthesis reactor and the reaction solution from the previous galvanized bucket for use. The system was kept warm and the reaction was monitored until the solid loading reached ≥250 μmol / g. UV loading was used for detection. The system was filtered under nitrogen pressure, and the filter cake was rinsed three times with N,N-dimethylformamide (26.00 kg + 26.10 kg + 26.00 kg), leaving the filter cake in the kettle. CAP.A (50% acetonitrile and 50% acetic anhydride, 4.40 kg + 4.42 kg + 4.30 kg) and CAP.B (20% pyridine and 30% N-methylimidazole and 50% acetonitrile, 4.40 kg + 4.40 kg + 4.47 kg) were added to an 80-L glass kettle and stirred for 3-8 minutes before use. This operation was repeated three times, and the cap was capped. Acetonitrile (18.00 kg + 18.00 kg + 18.00 kg + 17.50 kg + 17.50 kg) was added to the solid-phase synthesis reactor, and nitrogen was bubbled for 10 to 30 minutes before filter pressing. This operation was repeated four times. The filter cake was purged with nitrogen in the solid-phase synthesis reactor for 2 to 4 hours and then transferred to a 50 L filter press. The temperature was controlled at 15 to 30 ° C and continued to dry. The dried yellow to white solid product weighed 3.516 kg.

[0289] An isomannol residue (imann) can be added to the 5' end or 3' end of an oligonucleotide chain by methods well known to those skilled in the art, such as the invab process, and a targeting group can be further added.

[0290] Example 6. In vitro screening of xanthine dehydrogenase (XDH) siRNA duplexes

[0291] CAL27 cells were trypsinized and adjusted to an appropriate density before being seeded into 24-well plates at 1 × 10 5 cells / well. Simultaneously with plating, cells were transfected with 2 μL per well of test or control siRNA using Lipofectamine RNAiMax (Invitrogen-13778-150). Nonsense control-2 (CON) and test siRNA were tested at two concentrations (0.2 nM and 5 nM). Wells transfected with nonsense control-2 (CON) siRNA also served as negative controls.

[0292] 24 hours after transfection, the culture medium was removed and the cells were harvested for RNA extraction. TRIzol was used according to the manufacturer's instructions. TM Total RNA was extracted using 5-mercaptoethanol Reagent (Invitrogen-15596018).

[0293] According to the manual, use PrimeScript TM cDNA was synthesized using an RT kit and gDNA Eraser (Perfect Real Time) (TaKaRa-RR047A). Xanthine dehydrogenase (XDH) cDNA was detected by qPCR. GAPDH cDNA was detected in parallel as an internal control. PCR was performed as follows: 95°C for 30 seconds, followed by 40 cycles of 95°C for 10 seconds and 60°C for 30 seconds.

[0294] Data Analysis

[0295] The expression of the target gene in each sample was determined by relative quantification (RQ) using the comparative Ct (ΔΔCt) method; this method measures the Ct difference (ΔCt) between the target gene and a housekeeping gene (ACTIN).

[0296] The equation is as follows: ΔCT = target gene average Ct - ACTIN average Ct ΔΔCT = ΔCT (sample) - ΔCT (nonsense siRNA control); Relative quantification of target gene mRNA = 2 - ΔΔCT Inhibition % = (relative quantification of control - relative quantification of sample) / relative quantification of control × 100%.

[0297] Table 4 provides experimental results from in vitro studies using various XDH RNAi agents to inhibit XDH expression; the duplex sequences used correspond to the compounds shown in Table 2.

[0298] Table 4 Experimental results of in vitro study on inhibition of XDH expression by XDH RNAi reagent

[0299] Example 7. In vivo testing of xanthine dehydrogenase (XDH) siRNA duplexes

[0300] To evaluate the in vivo activity of xanthine dehydrogenase (XDH) siRNA, mice infected with AAV encoding human xanthine dehydrogenase (XDH) and luciferase genes were used (4 mice per group). 14 days before siRNA administration, female C57BL / 6J mice were infected by intravenous injection of 1x10^11 or 2x10^11 viral particles of adeno-associated virus 8 (AAV8) vector encoding human xanthine dehydrogenase (XDH) and luciferase genes. On day 0, mice were subcutaneously injected with a single dose of 3 mg / kg, 6 mg / kg, or 9 mg / kg of xanthine dehydrogenase (XDH) siRNA reagent or PBS. Blood samples were collected before siRNA administration on day 0 and at the end of days 7 and 14. Luciferase activity was measured. The knockdown percentage was calculated by comparing the luciferase activity of blood samples collected before administration of the siRNA-treated group with that of blood samples collected on days 7 and 14 and normalizing the activity based on the change in serum samples from the PBS-treated group.

[0301] Example 7 used compounds corresponding to the sequences, chemical modifications, and delivery compounds shown in Table 3, where GLO-n is the delivery compound shown as GalNAc3 in Jayaprakash, et al., (2014) J. Am. Chem. Soc., 136, 16958-16961.

[0302] A single 3-mg / kg subcutaneous dose of XDH siRNA was administered to mice transduced with AAV-XDH-luciferase. Luciferase activity in pre-dose blood samples (day 0) and blood samples collected on day 7 were compared in the siRNA-treated group and normalized to the change in luciferase activity in serum samples from the PBS-treated group to calculate percent knockdown. Data are presented in Table 5.

[0303] Table 5 Subcutaneous dose screening of XDH siRNA (3 mpk) in AAV-XDH-luciferase transduced mice

[0304] XDH siRNA single 6mpk subcutaneous dose screening was carried out in AAV-XDH-luciferase transduced mice. Compare the luciferase activity of the blood sample (day 0) before administration of the siRNA treatment group and the luciferase activity of the blood sample collected on day 7 and normalize based on the luciferase activity changes in the serum samples from the PBS treatment group to calculate the percentage of knockdown. The two experimental data are listed in Table 6 and Table 7 respectively.

[0305] Table 6 Subcutaneous dose screening of XDH siRNA (6 mpk) in AAV-XDH-luciferase transduced mice

[0306] Table 7 Subcutaneous dose screening of XDH siRNA single 6 mpk in AAV-XDH-luciferase transduced mice

[0307] A single 9-mpk subcutaneous dose of XDH siRNA was screened in AAV-XDH-luciferase-transduced mice. Luciferase activity in pre-dose blood samples (day 0) and blood samples collected on day 14 were compared in the siRNA-treated group and normalized to the change in luciferase activity in serum samples from the PBS-treated group to calculate percent knockdown. Data are presented in Table 8.

[0308] Table 8 Subcutaneous dose screening of XDH siRNA (9 mpk) in AAV-XDH-luciferase transduced mice

[0309] Example 8. In vivo testing of xanthine dehydrogenase (XDH) siRNA duplexes

[0310] To evaluate the in vivo activity of siRNA, a total of 18 male cynomolgus macaques (13-22 years old, weighing 7-9 kg) were enrolled in this study, with 3 animals in each group. Each animal was subcutaneously injected with 7.5 mg / kg of the test article. The test articles used correspond to the compounds shown in the table below.

[0311] After an overnight fast, animals were anesthetized (10 mg / kg ketamine, subcutaneous) on Days 14, 21, 42, and 78, and ultrasound-guided biopsy was performed to obtain approximately 10 mg of liver samples. The samples were stored in RNAlater (fixed overnight at 4°C and then transferred to -80°C) for subsequent RT-qPCR analysis. The results are shown in Table 9.

[0312] Table 9 Remaining percentage of XDH mRNA in monkey liver measured by qPCR

[0313] equivalent

[0314] Although several embodiments of the present invention have been described and illustrated herein, those skilled in the art will readily appreciate that a variety of other means and / or structures for performing the functions described herein and / or obtaining the results and / or one or more advantages, as well as each of these variations and / or modifications, are considered to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are exemplary, and that actual parameters, dimensions, materials, and / or configurations will depend on the specific application in which the teachings of the present invention are used. Those skilled in the art will recognize or be able to ascertain, using only routine experimentation, many equivalents to the specific embodiments of the present invention described herein. Therefore, it should be understood that the foregoing embodiments are presented by way of example only and are within the scope of the appended claims and their equivalents, and that the present invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, and / or methods, provided that such features, systems, articles, materials, and / or methods are not mutually inconsistent, is also within the scope of the present invention.

[0315] All definitions, as defined and used herein, should be understood against dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0316] Where no quantity limitation is used in the specification and claims, it should be understood as "at least one" unless explicitly stated to the contrary.

[0317] The phrase "and / or" as used in the specification and claims should be understood to mean "one or both" of the elements so conjoined, i.e., such elements are present in combination in some cases and separately in other cases. In addition to the elements specifically identified by "and / or," other elements may optionally be present, whether related or unrelated to those specifically identified elements, unless explicitly stated to the contrary.

[0318] All references, patents and patent applications and publications cited or referenced in this application are hereby incorporated by reference in their entirety.

Claims

1. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting xanthine dehydrogenase (XDH) expression, wherein the dsRNA agent comprises a sense strand and an antisense strand, nucleotides 2 to 18 of the antisense strand comprise a region complementary to a xanthine dehydrogenase (XDH) gene RNA transcript, wherein the complementary region comprises at least 15 consecutive nucleotides that differ from one of the antisense sequences listed in one of Tables 1-3 by 0, 1, 2 or 3 nucleotides, and optionally comprises a targeting ligand.

2. The dsRNA agent of claim 1, wherein the region complementary to the xanthine dehydrogenase (XDH) gene RNA transcript comprises at least 15, 16, 17, 18 or 19 consecutive nucleotides that differ from any of the antisense sequences listed in any one of Tables 1-3 by no more than 3 nucleotides.

3. The dsRNA agent of claim 1 or 2, wherein the antisense strand of the dsRNA is at least substantially complementary to any one of the target regions in SEQ ID NO: 1 and provided in any one of Tables 1-3.

4. The dsRNA agent of claim 3, wherein the antisense strand of the dsRNA is fully complementary to any target region in SEQ ID NO: 1 and provided in any one of Tables 1-3.

5. The dsRNA agent of claim 1, wherein the dsRNA agent comprises a sense strand sequence of any one of Tables 1-3, wherein the sense strand sequence is at least substantially complementary to an antisense strand sequence in the dsRNA agent.

6. The dsRNA agent of claim 1, wherein the dsRNA agent comprises a sense strand sequence of any one of Tables 1-3, wherein the sense strand sequence is fully complementary to the antisense strand sequence in the dsRNA agent.

7. The dsRNA agent of claim 1, wherein the dsRNA agent comprises an antisense strand sequence listed in any one of Tables 1-3.

8. The dsRNA agent of claim 1, wherein the dsRNA agent comprises a sequence listed as a duplex sequence in any one of Tables 1-3.

9. The dsRNA agent of claim 1, wherein the dsRNA agent comprises at least one modified nucleotide.

10. The dsRNA agent of claim 1, wherein all or substantially all nucleotides in the antisense strand are modified nucleotides.

11. The dsRNA agent of claim 9 or 10, wherein the at least one modified nucleotide comprises: 2'-O-methyl nucleotides, 2'-fluoro nucleotides, 2'-deoxy nucleotides, 2'3'-seco nucleotide mimetics, locked nucleotides, open ring nucleic acid nucleotides (UNA), glycol nucleic acid nucleotides (GNA), 2'-F-arabino nucleotides, 2'-methoxyethyl nucleotides, abasic nucleotides, ribitol, inverted nucleotides, inverted abasic nucleotides, inverted 2'-OMe nucleotides, inverted 2'-deoxy nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides and 3'-OMe nucleotides, nucleotides containing a 5'-phosphorothioate group, or a terminal nucleotide linked to a cholesterol derivative or a dodecanoic acid bisdecylamide group, a 2'-amino modified nucleotide, a phosphoramidate, or a non-natural base containing nucleotide.

12. The dsRNA agent of claim 9 or 10, wherein the guide strand comprises an E-vinylphosphonate nucleotide at the 5' end.

13. The dsRNA agent of claim 1, wherein the dsRNA agent comprises at least one phosphorothioate internucleoside linkage.

14. The dsRNA agent of claim 1, wherein the sense strand comprises at least one phosphorothioate internucleoside linkage.

15. The dsRNA agent of claim 1, wherein the antisense strand comprises at least one phosphorothioate internucleoside linkage.

16. The dsRNA agent of claim 1, wherein the sense strand comprises 1, 2, 3, 4, 5, or 6 phosphorothioate internucleoside linkages.

17. The dsRNA agent of claim 1, wherein the antisense strand comprises 1, 2, 3, 4, 5, or 6 phosphorothioate internucleoside linkages.

18. The dsRNA agent of claim 1, wherein all or substantially all nucleotides of the sense and antisense strands are modified nucleotides.

19. The dsRNA agent of claim 1, wherein the modified sense strand is a modified sense strand sequence listed in one of Tables 2-3.

20. The dsRNA agent of claim 1, wherein the modified antisense strand is a modified antisense strand sequence listed in one of Tables 2-3.

21. The dsRNA agent of claim 1, wherein the sense strand is complementary or substantially complementary to the antisense strand, and the complementary region is 16 to 23 nucleotides in length.

22. The dsRNA agent of claim 21, wherein the region of complementarity is 19 to 21 nucleotides in length.

23. The dsRNA agent of claim 1, wherein each strand is no more than 30 nucleotides in length.

24. The dsRNA agent of claim 1, wherein each strand is no more than 25 nucleotides in length.

25. The dsRNA agent of claim 1, wherein each strand is no more than 23 nucleotides in length.

26. The dsRNA agent of claim 1, wherein the dsRNA agent comprises at least one modified nucleotide and further comprises one or more targeting groups or linking groups.

27. The dsRNA agent of claim 26, wherein the one or more targeting groups or linking groups are conjugated to the sense strand.

28. The dsRNA agent of claim 26 or 27, wherein the targeting group or linker group comprises N-acetyl-galactosamine (GalNAc).

29. The dsRNA agent of claim 28, wherein the targeting group or linking group has a structure as shown in formula (X), comprising a targeting moiety, a linker, and a linker W, wherein the targeting moiety is selected from an N-acetyl-galactosamine derivative (GalNAc), which is linked to the linker W via a linker, and the linker W has a structure as shown in formula (XI), wherein X is selected from O, NH2 or S, and Y ﹣ Selected from: O ﹣ 、S ﹣ , methyl or NR a R b , R a and R b are independently selected from hydrogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, or R a and R b Together with the attached atoms, it forms a 3-12 membered heterocycloalkyl group containing 1-3 N, O, and S heteroatoms, preferably a substituted or unsubstituted C1-C6 alkyl group, or a substituted or unsubstituted C3-C6 cycloalkyl group, wherein the substituent is selected from hydroxyl and amino groups; 30. The dsRNA agent of claim 29, wherein the linker in the targeting group is selected from polyethylene glycol, substituted or unsubstituted C2-C 12 Alkyl, substituted or unsubstituted C3-C 12 Cycloalkyl, substituted or unsubstituted C3-C 12 Heterocycloalkyl, substituted or unsubstituted C3-C 12 Amide.

31. The dsRNA agent of claim 30, wherein the substituted or unsubstituted C2-C 12 Alkyl, substituted or unsubstituted C3-C 12 Cycloalkyl, substituted or unsubstituted C3-C 12 Heterocycloalkyl, substituted or unsubstituted C3-C 12 In the amide, the substituent is selected from hydroxyl and carbonyl.

32. The dsRNA agent of claim 30, wherein the linker in the targeting group is preferably selected from the following fragments: Each m is an integer from 1 to 6, each n, o, and p is 0 or 1, and each q is an integer from 1 to 6. 1 With q 2 Each independently is 0, 1 or 2.

33. The dsRNA agent of claim 32, wherein the linker in the targeting group is more preferably selected from the following fragments:

34. The dsRNA agent of claim 29, wherein the targeting portion of the targeting group has the following structural fragment: n' is 1 or 2.

35. The dsRNA agent of claim 29, wherein X is selected from O or S, and Y ﹣ Selected from: O ﹣ or S ﹣ .

36. The dsRNA agent of claim 26 or 27, wherein the targeting group has the following structure:

37. The dsRNA agent of any one of claims 1-36, wherein the dsRNA agent comprises a targeting group conjugated to the 5'-end of the sense strand.

38. The dsRNA agent of any one of claims 1-36, wherein the dsRNA agent comprises a targeting group conjugated to the 3'-end of the sense strand.

39. The dsRNA agent of any one of claims 1-38, wherein the antisense strand comprises an inverted abasic residue at the 3'-end.

40. The dsRNA agent of any one of claims 1 to 38, wherein the sense strand comprises one or two inverted abasic residues at the 3' or / and 5' termini, and / or the sense strand independently comprises one or two isomannol residues, preferably isomannol residues, at the 3' and / or 5' termini.

41. The dsRNA agent of any one of claims 1-40, wherein the dsRNA agent has two blunt ends.

42. The dsRNA agent of any one of claims 1-40, wherein at least one strand comprises a 3' overhang of at least 1 nucleotide.

43. The dsRNA agent of any one of claims 1-40, wherein at least one strand comprises a 3' overhang of at least 2 nucleotides.

44. The dsRNA agent of any one of claims 1-43, wherein the sense strand has an overhang of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides at the 3'-end and / or the 5'-end.

45. A double-stranded ribonucleic acid (dsRNA) agent that inhibits xanthine dehydrogenase (XDH) expression, wherein the dsRNA agent comprises a sense strand and an antisense strand, wherein each strand is 14 to 30 nucleotides in length, and comprises a region complementary to a xanthine dehydrogenase (XDH) gene RNA transcript at nucleotide positions 2 to 18 in the antisense strand, wherein the complementary region comprises at least 15 consecutive nucleotides that differ from one of the double-stranded ribonucleic acid (dsRNA) agent antisense sequences listed in Formula I by 0, 1, 2, or 3 nucleotides, and optionally comprises a targeting ligand, wherein the antisense strand of the double-stranded ribonucleic acid (dsRNA) agent is as described in Formula I: 5'-N a -(A1 A2 A3 A4)iN b –(B1 B2 B3 B4)-N b -(C1 C2 C3 C4 C5)jN a -3' (Formula I) Wherein: i and j are each independently selected from 0 or 1; Each N a and N b Each independently represents 0-17 oligonucleotides, which are optionally chemically modified; Preferably, the chemical modification is selected from 2'-OMe or 2'-fluorine modification; A1 A2 A3 A4 represents a motif of four consecutive nucleotides modified by lowercase 2'-OMe, uppercase 2'-fluorine, lowercase 2'-OMe, and lowercase 2'-OMe, respectively. Preferably, A1 and A2, and A2 and A3 further contain phosphorothioate internucleoside linkages. B1 B2 B3 B4 represents a motif in which four consecutive nucleotides are modified with one uppercase 2'-fluorine and three consecutive lowercase 2'-OMe; C1 C2 C3 C4 C5 represents a motif in which five consecutive nucleotides are modified with lowercase 2'-OMe, preferably further comprising phosphorothioate internucleoside linkages between C3 and C4 and between C4 and C5.

46. ​​The dsRNA agent of claim 45, N a Each independently represents 0 oligonucleotide, N b Each independently represents 2-5 chemically modified oligonucleotides, i and j each independently represent 1; the chemical modification is selected from 2'-OMe or 2'-fluorine.

47. Formula (I) according to any one of claims 45 to 46, characterized in that Further as shown in formula (II): 5'-xYxxxxYxxxxYxYxYxxxxx-3' (Formula II) Wherein, x represents a lowercase 2'-OMe modified oligonucleotide, and Y represents an uppercase 2'-fluorine modified oligonucleotide, preferably, x and Y and / or x and x further contain a phosphorothioate internucleoside linkage.

48. The dsRNA agent of any one of claims 45-47, wherein the antisense strand of the dsRNA is at least substantially complementary to any target region of SEQ ID NO:

1.

49. The dsRNA agent of any one of claims 45 to 47, wherein the antisense strand of the dsRNA is fully complementary to any target region of SEQ ID NO:

1.

50. The dsRNA agent of any one of claims 45-47, wherein the sense strand sequence of any one of the dsRNAs is at least substantially complementary to the antisense strand sequence of the dsRNA agent.

51. The dsRNA agent of any one of claims 45-47, wherein the sense strand sequence of any one of the dsRNAs is completely complementary to the antisense strand sequence of the dsRNA agent.

52. The dsRNA agent of any one of claims 45 to 47, wherein the dsRNA sense strand comprises one or two inverted abasic residues at the 3' or / and 5' termini, and / or the sense strand independently comprises one or two isomannide residues at the 3' and / or 5' termini, preferably the isomannide residue imann.

53. The dsRNA agent of any one of claims 45-47, wherein the dsRNA has two blunt ends.

54. The dsRNA agent of any one of claims 45-47, wherein the dsRNA sense strand has an overhang of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides at the 3'-end and / or the 5'-end.

55. A composition comprising the dsRNA agent of any one of claims 1 to 54.

56. The composition of claim 55, further comprising a pharmaceutically acceptable carrier.

57. The composition of any one of claims 55-56, further comprising one or more additional therapeutic agents.

58. The composition of any one of claims 55-57, wherein the composition is packaged in a kit, container, package, dispenser, prefilled syringe, or vial.

59. The composition of any one of claims 55-57, wherein the composition is formulated for subcutaneous administration or is formulated for intravenous (IV) administration.

60. A cell comprising the dsRNA agent of any one of claims 1 to 54.

61. The cell according to claim 60, characterized in that The cell is a mammalian cell, optionally a human cell.

62. A method for inhibiting xanthine dehydrogenase (XDH) gene expression in a cell, comprising: (i) preparing a cell comprising an effective amount of a double-stranded ribonucleic acid (dsRNA) agent according to any one of claims 1 to 54 or a composition according to any one of claims 55 to 59; (ii) maintaining the cells prepared in (i) for a sufficient period of time to achieve degradation of the mRNA transcript of the xanthine dehydrogenase (XDH) gene, thereby inhibiting the expression of the xanthine dehydrogenase (XDH) gene in the cells.

63. The method of claim 62, wherein The cells are in a subject and the dsRNA agent is administered subcutaneously to the subject.

64. A method of treating a disease or condition associated with a xanthine dehydrogenase (XDH) protein, the method comprising administering to a subject an effective amount of the double-stranded ribonucleic acid (dsRNA) agent of any one of claims 1 to 54 or the composition of any one of claims 55 to 59 to inhibit xanthine dehydrogenase (XDH) gene expression.

65. The method of claim 64, wherein the disease or condition is associated with xanthine dehydrogenase (XDH) expression, and the disease is selected from one or more of the following: hyperuricemia, gout, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), metabolic disorders, insulin resistance, cardiovascular disease, type 2 diabetes, Lesch Nyhan syndrome, glycogen storage disease (GSD), and diseases associated with oxidative stress or other XDH-related diseases; preferably, the glycogen storage disease (GSD) is selected from type I GSD, and the disease associated with oxidative stress is selected from chronic low-grade inflammation.

66. The method of any one of claims 64-65, which reduces serum uric acid levels.

67. The method of any one of claims 64-65, further comprising administering to the subject an additional therapeutic regimen.

68. The method of claim 67, wherein the additional treatment regimen comprises: One or more xanthine dehydrogenase (XDH) antisense polynucleotides are administered to the subject or a non-xanthine dehydrogenase (XDH) dsRNA therapeutic is administered to the subject.

69. The method of claim 68, wherein the non-xanthine dehydrogenase (XDH) dsRNA therapeutic agent is selected from one or more of the following: allopurinol, hydroxypurine, febuxostat or an interleukin-1β (IL-1β) antagonist, preferably, the interleukin-1β antagonist is selected from canakinumab or rilonacept.