WO2023202686A1 - 用于抑制黄嘌呤脱氢酶(xdh)的组合物和方法 - Google Patents

用于抑制黄嘌呤脱氢酶(xdh)的组合物和方法 Download PDF

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WO2023202686A1
WO2023202686A1 PCT/CN2023/089651 CN2023089651W WO2023202686A1 WO 2023202686 A1 WO2023202686 A1 WO 2023202686A1 CN 2023089651 W CN2023089651 W CN 2023089651W WO 2023202686 A1 WO2023202686 A1 WO 2023202686A1
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xdh
dsrna
xanthine dehydrogenase
reagent
nucleotides
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PCT/CN2023/089651
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English (en)
French (fr)
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舒东旭
邵鹏程
夏时伟
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上海舶望制药有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/06—Antigout agents, e.g. antihyperuricemic or uricosuric agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing

Definitions

  • the present invention relates to compositions and methods useful for inhibiting xanthine dehydrogenase (XDH).
  • XDH xanthine dehydrogenase
  • 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) respectively catalyze hypoxanthine Oxidation to xanthine and xanthine to uric acid.
  • XO Xanthine oxidase
  • XDH xanthine dehydrogenase
  • XDH is primarily expressed in the intestine and liver, but can also be expressed in other tissues, including adipose tissue.
  • Chronic elevation of serum uric acid (chronic hyperuricemia), usually defined as a serum urate level 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 attacks of acute inflammatory arthritis, often caused by insufficient renal clearance of uric acid or excessive uric acid production. Caused by an inflammatory response to uric acid crystals in the joints.
  • Fructose-related gout is associated with variants of a transporter protein expressed in the kidneys, intestines 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 conditions related to oxidative stress, chronic low-grade inflammation, and insulin Disease-related (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).
  • NASH nonalcoholic steatohepatitis
  • NAFLD nonalcoholic fatty liver disease
  • metabolic disorders cardiovascular disease
  • type 2 diabetes type 2 diabetes
  • Allopurinol and febuxostat It is an inhibitor of the enzyme in the form of XDH and is commonly used to treat gout.
  • contraindications for use in patients with comorbidities of gout especially in patients with reduced renal function (e.g. due to chronic kidney disease or hepatic insufficiency).
  • Their use may be inappropriate in patients with metabolic syndrome, hypertension, dyslipidemia, nonalcoholic steatohepatitis (NASH) or nonalcoholic fatty liver disease (NAFLD), cardiovascular disease, or diabetes (type 1 or 2) in patients who have limitations in organ function due to a disease or condition or an adverse interaction with drugs used to treat such conditions.
  • NASH nonalcoholic steatohepatitis
  • NAFLD nonalcoholic fatty liver disease
  • diabetes type 1 or 2
  • a double-stranded ribonucleic acid (dsRNA) reagent for inhibiting xanthine dehydrogenase (XDH) gene expression is provided.
  • the dsRNA reagent includes a sense strand and an antisense strand, and the core in the antisense strand
  • the nucleotide positions 2 to 18 contain a region complementary to the xanthine dehydrogenase (XDH) gene RNA transcript, wherein the complementary region contains a difference of 0, 1, and 2 from one of the antisense sequences listed in Table 1-3 or at least 15 consecutive nucleotides of 3 nucleotides, and optionally includes a targeting ligand.
  • the region complementary to the xanthine dehydrogenase (XDH) gene RNA transcript comprises at least 15, 16, 17, 18 or 19 consecutive nucleotides.
  • the antisense strand of the dsRNA is at least substantially complementary to any target region of SEQ ID NO: 1 and is provided in one of Tables 1-3.
  • the antisense strand of the dsRNA is fully complementary to any target region of SEQ ID NO: 1 and is provided in one of Tables 1-3.
  • the dsRNA reagent includes 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 reagent.
  • the dsRNA reagent includes any one of the sense strand sequences listed in Tables 1-3, wherein the sense strand sequence is completely complementary to the antisense strand sequence in the dsRNA reagent.
  • the dsRNA reagent comprises any of the antisense strand sequences listed in Tables 1-3.
  • the dsRNA agent comprises any of the sequences listed as duplex sequences in Tables 1-3.
  • the dsRNA agent contains at least one modified nucleotide.
  • all or substantially all of the nucleotides of the antisense strand are modified nucleotides.
  • all or substantially all nucleotides of the sense and antisense strands are modified nucleotides.
  • At least one modified nucleotide includes: 2'-methoxy nucleotide, 2'-fluoro nucleotide, 2'-deoxy nucleotide, 2',3'-seco core Glycolic acid mimetic, locked nucleotide, unlocked nucleic acid nucleotide (UNA), glycol nucleic acid nucleotide (GNA), 2'-F-arabinose nucleoside acid, 2'-methoxyethyl nucleotide, abasic nucleotide, ribitol, reverse nucleotide, reverse abasic nucleotide, reverse 2'-OMe nucleotide, reverse To 2'-deoxynucleotides, isomannitol nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides and 3'-OMe core Glycosides
  • the dsRNA agent includes an E-vinylphosphonate nucleotide at the 5' end of the guide strand.
  • the dsRNA agent contains at least one phosphorothioate internucleoside linkage.
  • the sense strand contains at least one phosphorothioate internucleoside linkage.
  • the antisense strand contains at least one phosphorothioate internucleoside linkage.
  • the sense strand contains 1, 2, 3, 4, 5, or 6 phosphorothioate internucleoside linkages.
  • the antisense strand contains 1, 2, 3, 4, 5, or 6 phosphorothioate internucleoside linkages.
  • the antisense strand contains at least 15 modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoronucleotides, of which less than 6 are 2'- Fluoronucleotide modified nucleotides. In certain embodiments, the antisense strand contains 3 or 5 2'-fluoronucleotides, preferably the antisense strand contains 5 2'-fluoronucleotides.
  • the sense strand contains at least 15 modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoronucleotides, of which less than 4 are 2'-fluoronucleotides Nucleotide-modified nucleotides, in certain embodiments, the sense strand contains three 2'-fluoronucleotides.
  • the antisense strand is comprised in the direction from the 5' end to the 3' end, counting from the first paired nucleotide at the 5' end of the antisense strand, the 2nd, 7th, and 7th nucleotides of the antisense strand.
  • Nucleotides at positions 12, 14, and 16 are 2'-fluoro modified nucleotides, and each nucleotide at other positions in the antisense strand is independently a non-fluoro modified nucleotide.
  • the sense strand includes cores at positions 9, 11, and 13 of the sense strand in a direction from the 3' end to the 5' end, counting from the first paired nucleotide 3' of the sense strand.
  • the nucleotide is a 2'-fluoro modified nucleotide, and each nucleotide elsewhere in the sense strand is independently a non-fluoro modified nucleotide.
  • the antisense strand contains 15 or more modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoronucleotides, wherein the antisense strand consists of 5 Positions 2, 7, 12, 14 and/or 16 from the ' end are 2'-fluoro nucleotide modified nucleotides, and the rest are 2'-O-methyl nucleotide modified nucleotides.
  • the sense strand comprises 15 or more modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoronucleotides, wherein the sense strand begins at the 3' end Positions 9, 11 and/or 13 from the first matching position are 2'-fluoronucleotide-modified nucleotides, and the remainder are 2'-O-methyl nucleotide-modified nucleotides.
  • the modified sense strand is a modified sense strand sequence listed in Table 2 and/or Table 3.
  • the modified antisense strand is a modified antisense strand sequence listed in Table 2 and/or Table 3.
  • the sense strand is complementary or substantially complementary to the antisense strand
  • the complementary region is between 16 and 23 nucleotides in length.
  • the complementary region is 19-21 nucleotides in length.
  • the complementary region is 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.
  • each strand is no more than 40 nucleotides in length.
  • each strand is no more than 30 nucleotides in length.
  • each strand is no more than 25 nucleotides in length.
  • each strand is no more than 23 nucleotides in length.
  • each strand is no more than 21 nucleotides in length.
  • a dsRNA agent contains at least one modified nucleotide and further contains one or more targeting or linking groups.
  • one or more targeting groups or linking groups are conjugated to the sense strand.
  • the targeting group or linking group includes N-acetyl-galactosamine (GalNAc).
  • the targeting group or linking group is a structure represented by formula (X), including a targeting moiety, a linker bond, and a linker W, wherein the targeting moiety is selected from N-acetyl-galactosamine derived substance (GalNAc), which is linked to the linker W through a linking bond, the linker W has a structure shown in formula (XI), X is selected from O, NH 2 or S, Y - is selected from: O - , S - , methyl Or NR a R b , R a and R b are each independently selected from hydrogen, substituted or unsubstituted C 1 -C 6 alkyl group, substituted or unsubstituted C 3 -C 6 cycloalkyl group, or R a and R b are linked together with the attached atoms to form a 3-12 membered heterocycloalkyl group containing 1-3 N, O, S heteroatoms.
  • formula (X) including a targeting moiety,
  • the substituent is selected from hydroxyl and amino.
  • the linkage in the targeting group is selected from polyethylene glycol, optionally substituted C 2 -C 12 alkyl, substituted or unsubstituted C 3 -C 12 cycloalkyl, substituted or unsubstituted Substituted C 3 -C 12 heterocycloalkyl, substituted or unsubstituted C 3 -C 12 amide.
  • the linkage in the targeting group is preferably from the following fragments:
  • Each m is independently an integer from 1 to 6, each n, o, and p is independently 0 or 1, and each q 1 and q 2 is independently 0, 1, or 2.
  • the linkage in the targeting group is more preferably from the following fragments:
  • the targeting moiety in the targeting group has the following structural fragment,
  • n' is 1 or 2.
  • the targeting group has the following structure:
  • the dsRNA agent comprises a targeting group conjugated to the 5'-end of the sense strand.
  • the dsRNA agent comprises a targeting group conjugated to the 3'-end of the sense strand.
  • the antisense strand contains an inverted abasic residue at the 3'-end.
  • the sense strand contains one or two reverse abasic residues at the 3' or/and 5' end.
  • the sense strand contains one or two imann residues at the 3' or/and 5' end.
  • the 3' and 5' ends of the sense strand each independently comprise an imann residue.
  • the dsRNA agent has two blunt ends.
  • At least one strand includes a 3' overhang that is at least 1 nucleotide long. In some embodiments, at least one strand includes a 3' overhang that is at least 2 nucleotides long. In one embodiment, the sense strand has 1-10 nucleotide overhangs at the 3'-end and/or 5'-end, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or a 10-nucleotide overhang.
  • dsRNA double-stranded ribonucleic acid
  • the dsRNA reagent includes a sense strand and an antisense strand, wherein the length of each strand is 14 to 30 nucleotides, containing a region complementary to the xanthine dehydrogenase (XDH) gene RNA transcript at nucleotide positions 2 to 18 in the antisense strand, wherein the complementary region contains those listed in Formula I
  • dsRNA agent antisense sequences differs by at least 15 consecutive nucleotides by 0, 1, 2, or 3 nucleotides, and optionally includes a targeting ligand.
  • the antisense strand of the double-stranded ribonucleic acid (dsRNA) reagent is as shown in Formula I: 5'-N a -(A 1 A 2 A 3 A 4 ) i -N b –(B 1 B 2 B 3 B 4 )-N b -(C 1 C 2 C 3 C 4 C 5 ) j -N a -3' (Formula I)
  • i and j are each independently selected from 0 or 1;
  • Each Na and N b independently represent 0-17 oligonucleotides, which are optionally modified by the chemical modifications used herein; preferably, the chemical modifications are selected from 2'-OMe or 2'-Fluorine modification;
  • a 1 A 2 A 3 A 4 represents a motif in which four consecutive nucleotides are sequentially modified by lowercase 2'-OMe, uppercase 2'-fluoro, lowercase 2'-OMe, and lowercase 2'-OMe, preferably Further, both A 1 and A 2 and A 2 and A 3 contain phosphorothioate internucleoside linkages;
  • B 1 B 2 B 3 B 4 represents a motif in which four consecutive nucleotides are modified by one uppercase 2'-Fluoro and three consecutive lowercase 2'-OMe;
  • C 1 C 2 C 3 C 4 C 5 represents a motif in which five consecutive nucleotides are all modified with lowercase 2'-OMe, preferably between C 3 and C 4 , and between C 4 and C 5 Contains phosphorothioate internucleoside linkages.
  • N a independently represents 0-2 oligonucleotides
  • N b independently represents 2-5 oligonucleotides with or without chemical modification as used herein.
  • Na each independently represents 0 oligonucleotides
  • N b each independently represents 2-5 oligonucleotides chemically modified as used herein.
  • Na each independently represents 0 oligonucleotides
  • N b each independently represents 2-5 oligonucleotides chemically modified with 2'-OMe or 2'-fluorine.
  • formula (I) is further represented by formula (II): 5'-xYxxxxYxxxxYxYxYxxxxx-3' (Formula II)
  • x represents a lowercase 2'-OMe modified oligonucleotide
  • Y represents an uppercase 2'-fluoro modified oligonucleotide
  • x and Y and/or x and x further include a phosphorothioate internucleoside. Key link.
  • the sense strand of the double-stranded ribonucleic acid (dsRNA) reagent is as shown in 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) 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′
  • N' F represents 2'-fluorine modified nucleotide
  • N' N1 , N' N2 , N' N3 , N' N4 , N' N5 , and N' N6 each independently represent a modified or unmodified nucleotide.
  • Modified nucleotides; N′ N1 N′ N2 N′ N3 and N′ N4 N′ N5 N′ N6 each independently represent a nucleotide fragment containing at least two different modifications;
  • N′ L each independently represents A modified or unmodified nucleotide that is not a 2'-fluoro modified nucleotide;
  • m' and n' each independently represent an integer from 0 to 7.
  • 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 'it's 1.
  • 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 one of the dsRNA is at least substantially complementary to the antisense strand sequence in the dsRNA agent. In some embodiments, the sense strand sequence of any one of the dsRNA is completely complementary to the antisense strand sequence in the dsRNA agent.
  • the region complementary to SEQ ID NO: 1 includes at least 15, 16, 17, 18, or 19 that differs by no more than 3 nucleotides from one of the antisense sequences listed in Tables 1-3 consecutive nucleotides.
  • the antisense strand of the dsRNA is at least substantially complementary to any target region of SEQ ID NO: 1 and is provided in one of Tables 1-3.
  • the antisense strand of the dsRNA is fully complementary to any target region of SEQ ID NO: 1 and is provided in one of Tables 1-3.
  • the dsRNA reagent includes 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 reagent.
  • the dsRNA reagent includes any one of the sense strand sequences listed in Tables 1-3, wherein the sense strand sequence is completely complementary to the antisense strand sequence in the dsRNA reagent.
  • the dsRNA reagent comprises any of the antisense strand sequences listed in Tables 1-3.
  • the dsRNA agent comprises any of the sequences listed as duplex sequences in Tables 1-3.
  • the sense strand contains one or two reverse abasic residues at the 3' or/and 5' end.
  • the sense strand contains one or two imann residues at the 3' or/and 5' end.
  • the 3' and 5' ends of the sense strand each independently comprise an imann residue.
  • the dsRNA agent has two blunt ends.
  • at least one strand includes a 3' overhang that is at least 1 nucleotide long.
  • at least one strand includes a 3' overhang that is at least 2 nucleotides long.
  • the sense strand has a 1-10 nucleotide overhang at the 3'-end and/or 5'-end, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotide overhang.
  • a dsRNA agent contains at least one modified nucleotide and further contains one or more targeting or linking groups.
  • one or more targeting groups or linking groups are conjugated to the sense strand.
  • the targeting group or linking group includes N-acetyl-galactosamine (GalNAc).
  • the targeting group is as used herein, 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. Any one of GLO-7, GLO-8, GLO-9, GLO-10, GLO-11, GLO-12, GLO-13, GLO-14, GLO-15 and GLO-16.
  • the sense strand independently includes an imann residue at the 3' and 5' ends, wherein either of the 3' and 5' end residues is optionally further linked to a targeting group that targets
  • the group is preferably the aforementioned GLS-15.
  • composition comprising any embodiment of the above-described dsRNA agent aspect of the invention.
  • the composition further includes a pharmaceutically acceptable carrier. In some embodiments, the composition further includes one or more additional therapeutic agents.
  • compositions are packaged in a kit, container, wrapper, dispenser, prefilled syringe, or vial.
  • compositions are formulated for subcutaneous administration or are formulated for intravenous (IV) administration.
  • a cell comprising any embodiment of the above-described dsRNA agent aspect of the invention.
  • the cells are mammalian cells, optionally human cells.
  • a method for inhibiting xanthine dehydrogenase (XDH) gene expression in cells comprising: (i) preparing an effective amount of the above-mentioned dsRNA reagent or any of the above-mentioned compositions cells of the embodiment.
  • XDH xanthine dehydrogenase
  • the method further includes: (ii) maintaining the prepared cells for a period of time sufficient to obtain degradation of the mRNA transcript of the xanthine dehydrogenase (XDH) gene, thereby inhibiting xanthine dehydrogenase in the cell (XDH) gene expression.
  • XDH xanthine dehydrogenase
  • the cells are in the subject and the dsRNA agent is administered subcutaneously to the subject.
  • the cells are in the subject and the dsRNA agent is administered to the subject by IV administration.
  • the method further includes assessing the response to xanthine dehydrogenase after administering the dsRNA agent to the subject.
  • Inhibition of the (XDH) gene wherein the means of assessment include: (i) determining one or more physiological characteristics of a xanthine dehydrogenase (XDH)-related disease or condition in the subject, and (ii) converting the determined physiological characteristics Comparison to baseline pre-treatment physiological characteristics of a xanthine dehydrogenase (XDH)-related disease or condition and/or control physiological characteristics of a xanthine dehydrogenase (XDH)-related disease or condition, wherein the comparison is indicative of xanthine in the subject The presence or absence of inhibition of dehydrogenase (XDH) gene expression.
  • the physiological characteristic determined is uric acid levels in the blood
  • a decrease in uric acid levels in the blood indicates a decrease in xanthine dehydrogenase (XDH) gene expression in the subject.
  • XDH xanthine dehydrogenase
  • a method for inhibiting xanthine dehydrogenase (XDH) gene expression in a subject which includes administering to the subject an effective amount of an embodiment of the aforementioned dsRNA agent or an embodiment of the aforementioned composition.
  • 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 includes: assessing inhibition of the xanthine dehydrogenase (XDH) gene after administration of the dsRNA agent, wherein the means for assessing includes: (i) determining a xanthine dehydrogenase (XDH)-related disease in the subject or one or more physiological characteristics of a condition; (ii) comparing the identified physiological characteristics to baseline pre-treatment physiological characteristics of the disease or condition and/or xanthine dehydrogenase (XDH) Control physiological characteristics of a related disease or disorder are compared; wherein the comparison indicates the presence or absence of inhibition of xanthine dehydrogenase (XDH) gene expression in the subject.
  • the physiological characteristic determined is the level of uric acid in the blood. A decrease in uric acid levels in the blood indicates a decrease in x
  • an application for preparing a medicament for treating diseases or conditions related to xanthine dehydrogenase (XDH) protein comprising any embodiment of the aforementioned dsRNA reagent aspect of the invention or in any embodiment of the aforementioned composition of the present invention, the drug can inhibit xanthine dehydrogenase (XDH) gene expression.
  • XDH xanthine dehydrogenase
  • a method of treating a disease or disorder associated with xanthine dehydrogenase (XDH) protein which includes: administering to a subject an effective amount of any of the foregoing dsRNA reagent aspects of the present invention.
  • the xanthine dehydrogenase (XDH)-related disease or disorder is selected from: 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 diseases (GSD), such as GSD type I, and disorders related to oxidative stress, such as chronic low-grade inflammation; or other XDH-related disease.
  • NAFLD non-alcoholic fatty liver disease
  • NASH non-alcoholic steatohepatitis
  • GSD glycogen storage diseases
  • disorders related to oxidative stress such as chronic low-grade inflammation
  • other XDH-related disease is selected from: 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 diseases (GSD), such as GSD type I
  • 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.
  • the method further includes administering an additional treatment regimen to the subject.
  • additional treatment options include treatment of xanthine dehydrogenase (XDH)-related diseases or conditions.
  • additional treatment regimens include: administering to the subject one or more xanthine dehydrogenase (XDH) antisense polynucleotides of the invention; administering to the subject a non-xanthine dehydrogenase (XDH) ) dsRNA therapeutic agent; and effecting behavioral changes in a subject.
  • the non-xanthine dehydrogenase (XDH) dsRNA therapeutic agent is one of the following additional therapeutic agents: for example, allopurinol, hydroxypurine, febuxostat, or interleukin-1 ⁇ (IL-1 ⁇ ) antagonist, preferably, the interleukin-1 ⁇ antagonist is selected from canakinumab or rilonacept, so that the treatment will be from xanthine Subjects with the disease benefited from a reduction in dehydrogenase (XDH).
  • additional therapeutic agents for example, allopurinol, hydroxypurine, febuxostat, or interleukin-1 ⁇ (IL-1 ⁇ ) antagonist, preferably, the interleukin-1 ⁇ antagonist is selected from canakinumab or rilonacept, so that the treatment will be from xanthine Subjects with the disease benefited from a reduction in dehydrogenase (XDH).
  • 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 includes determining the efficacy of the administered double-stranded ribonucleic acid (dsRNA) agent in the subject.
  • dsRNA double-stranded ribonucleic acid
  • means of determining the efficacy of a treatment in a subject includes: (i) determining one or more physiological characteristics of a xanthine dehydrogenase (XDH)-related disease or condition in the subject; (ii) determining The physiological characteristics of the subject are compared to the baseline pre-treatment physiological characteristics of the xanthine dehydrogenase (XDH) related disease or condition, wherein the comparison indicates the presence, absence and level of efficacy of the double-stranded ribonucleic acid (dsRNA) agent administered to the subject of one or more.
  • the physiological characteristic determined is the level of uric acid in the blood. A decrease in uric acid levels in the blood indicates the presence of effectiveness of the double-stranded ribonucleic acid (dsRNA) agent administered to the subject.
  • a method of reducing the level of xanthine dehydrogenase (XDH) protein in a subject as compared to a baseline pre-treatment level of xanthine dehydrogenase (XDH) protein in the subject comprising administering to the subject An effective amount of any embodiment of the foregoing dsRNA agent aspect of the invention or any embodiment of the foregoing composition of the invention to reduce the level of xanthine dehydrogenase (XDH) gene expression.
  • the dsRNA agent is administered to the subject subcutaneously or by IV.
  • a change in the physiology of a xanthine dehydrogenase (XDH)-related disease or disorder in a subject as compared to baseline pre-treatment physiological characteristics of the xanthine dehydrogenase (XDH)-related disease or disorder in the subject.
  • a characteristic method comprising administering to a subject an effective amount of any embodiment of the foregoing dsRNA agent aspect of the invention or any embodiment of the foregoing composition of the invention to alter a xanthine dehydrogenase (XDH)-related disease in the subject or Physiological characteristics of the disease.
  • the dsRNA agent is administered to the subject subcutaneously or by IV.
  • the physiological characteristic determined is a reduction in uric acid.
  • Duplexes AV00206 to AV00292 are shown in Table 1 and their sense strand sequences are shown.
  • Duplexes AV00206 to AV00292 are shown in Table 1 and their antisense strand sequences are shown.
  • SEQ ID NO:1 is human xanthine dehydrogenase (XDH) mRNA [NCBI reference sequence: NM_000379.4].
  • the delivery molecules used in the in vivo studies are designated "GLX-n" at the 3' or 5' end of each sense strand.
  • RNAi agents capable of inhibiting xanthine dehydrogenase (XDH) gene expression such as, but not limited to, double-stranded (ds) RNAi agents.
  • the xanthine dehydrogenase (XDH) dsRNA reagent of the present invention can target xanthine dehydrogenase (XDH) transcripts, resulting in the inhibition of xanthine dehydrogenase (XDH) protein expression.
  • Some embodiments of the invention also include compositions comprising a xanthine dehydrogenase (XDH) gene RNAi agent 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.
  • Pharmaceutical compositions of the present invention may comprise at least one xanthine dehydrogenase (XDH) dsRNA agent and a delivery compound.
  • the delivery compound is a GalNAc-containing delivery compound.
  • the xanthine dehydrogenase (XDH) gene RNAi reagent delivered to cells can inhibit the xanthine dehydrogenase (XDH) gene expression, thereby reducing the activity of the xanthine dehydrogenase (XDH) protein product of the gene in the cell.
  • the dsRNAi agents of the invention can be used to treat xanthine dehydrogenase (XDH)-related diseases and conditions.
  • dsRNAi agents include, for example, duplexes AV00206 to AV00292 shown in Table 1.
  • preferred dsRNAi agents include, for example, duplexes AV00227, AV00260, AV00285, AV00266, AV00280, AV00238, AV00249, AV00289, AV00233, AV00225, AV00273, or AV00283.
  • preferred dsRNAi agents of Table 2 include, for example, duplexes AV00227, AV00260, AV00285, AV00266, or AV00280.
  • preferred dsRNAi agents in 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.
  • reducing xanthine dehydrogenase (XDH) expression in a cell or subject treats a disease or disorder associated with xanthine dehydrogenase (XDH) expression in a cell or subject, respectively.
  • 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 GSD type I, or a disorder related to oxidative stress, such as chronic low-grade inflammation; or other XDH-related disorders.
  • XDH xanthine dehydrogenase
  • ssRNA single-stranded
  • dsRNA double-stranded
  • XDH xanthine dehydrogenase
  • RNAi is also known in the art and may be referred to as "siRNA”.
  • G generally each represent a nucleotide containing guanine, cytosine, adenine and uracil as bases, respectively.
  • ribonucleotide or “nucleotide” may also refer to modified nucleotides, as described in further detail below, or alternative replacement moieties.
  • guanine, cytosine, adenine and uracil can be replaced by other moieties without substantially altering the base pairing properties of the oligonucleotide containing the nucleotides of such replaced moieties.
  • nucleotide containing inosine as its base may be base paired with a nucleotide containing adenine, cytosine, or uracil. Therefore, in the nucleotide sequences of the present invention, nucleotides containing uracil, guanine or adenine may be replaced by nucleotides containing, for example, inosine. Contains a replacement part like this The sequences are embodiments of the invention.
  • RNAi refers to an agent that contains RNA and mediates targeted cleavage of RNA transcripts through the RNA-induced silencing complex (RISC) pathway.
  • RISC RNA-induced silencing complex
  • an RNAi target region refers to a contiguous portion of the nucleotide sequence of an RNA molecule formed during gene transcription, including messenger RNA (mRNA), which is a processing product of the primary transcript RNA. The target portion of the sequence will be at least long enough to serve as a substrate for RNAi-directed cleavage at or near this portion.
  • mRNA messenger RNA
  • the target sequence may be 8-30 nucleotides long (inclusive), 10-30 nucleotides long (inclusive), 12-25 nucleotides long (inclusive), 15-23 nucleotides long (inclusive), 16-23 nucleotides long (inclusive), or 18-23 nucleotides long (inclusive), including all shorter within each stated range length.
  • the target sequence is 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleosides Sour and long.
  • the target sequence is between 9 and 26 nucleotides in length, inclusive, including all subranges and integers therebetween.
  • the target sequences are 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, the sequence is completely or at least substantially complementary to at least a portion of the RNA transcript of the xanthine dehydrogenase (XDH) gene.
  • XDH xanthine dehydrogenase
  • compositions comprising one or more xanthine dehydrogenase (XDH) dsRNA agents and a pharmaceutically acceptable carrier.
  • XDH xanthine dehydrogenase
  • XDH xanthine dehydrogenase gene RNAi as described herein inhibits the expression of xanthine dehydrogenase (XDH) protein.
  • 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 of compounds.
  • two, three, four or more different positions/regions will be capable of targeting one, two, three, four or more different positions/regions of xanthine dehydrogenase (XDH) mRNA, respectively.
  • Multiple xanthine dehydrogenase (XDH) dsRNAs are administered to a cell or subject.
  • a "dsRNA agent” refers to a composition comprising an RNA or RNA-like (eg, chemically modified RNA) oligonucleotide molecule that is capable of degrading or inhibiting translation of a target mRNA transcript. While not wishing to be bound to a particular theory, the dsRNA agents of the present invention may act through an RNA interference mechanism (i.e., induce RNA interference by interacting with the RNA interference pathway machinery of mammalian cells (RNA-induced silencing complex or RISC) ), or act through any alternative mechanism or pathway.
  • RNA interference mechanism i.e., induce RNA interference by interacting with the RNA interference pathway machinery of mammalian cells (RNA-induced silencing complex or RISC)
  • RISC RNA-induced silencing complex
  • the dsRNA reagent disclosed herein consists of a sense strand and an antisense strand, including but not limited to: short interfering RNA (siRNA), RNAi reagents, microRNA (miRNA), short hairpin RNA (shRNA) and Dicer. substrate.
  • the antisense strand of the dsRNA reagents 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, dsRNA 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).
  • RNA duplex structures are also effective in inducing RNA interference.
  • Book The xanthine dehydrogenase (XDH) dsRNA in certain embodiments of the invention may comprise at least one strand of at least 21 nt in length, or the duplex may have a reduced length based on one of the sequences listed in Tables 1-3. 1, 2, 3nt or shorter length. It can also be effective to reduce 4 nucleotides at one or both ends compared to the dsRNA listed in Tables 1-3 respectively.
  • the xanthine dehydrogenase (XDH) dsRNA agent can have at least 15, 16, 17, 18, 19, 20 or more of one or more sequences from Tables 1-3
  • a partial sequence of contiguous nucleotides and their ability to inhibit xanthine dehydrogenase (XDH) gene expression does not differ by more than 5% from the level of inhibition produced by a dsRNA containing the entire sequence (also referred to herein as the "parental" sequence) , 10%, 15%, 20%, 25% or 30%.
  • the sense sequences, antisense sequences and duplexes disclosed in Tables 1-3 may be referred to as "parent" sequences herein, which means that the sequences disclosed in Tables 1-3 may be modified, shortened, extended, include substitutions, etc., As described herein, the resulting sequences retain all or at least part of the efficacy of their parent sequences in the methods and compositions of the invention.
  • compositions and methods of the present invention include single-stranded RNA in the composition and/or administer the single-stranded RNA to the subject.
  • the antisense strand listed in any of Tables 1-3 can be used as or within a composition that, when administered to a subject, reduces xanthine dehydrogenase (XDH) polypeptide activity in the subject and/or expression of the xanthine dehydrogenase (XDH) gene.
  • Tables 1-3 show the antisense and sense strand core extension base sequences of certain xanthine dehydrogenase (XDH) dsRNA reagents.
  • Single-stranded antisense molecules that may be included in certain compositions of the invention and/or administered in certain methods of the invention are referred to herein as “single-stranded antisense agents” or “antisense polynucleotide agents” .
  • Single-stranded sense molecules that may be included in certain compositions and/or administered in certain methods of the invention are referred to herein as “single-stranded sense agents” or “sense polynucleotide agents.”
  • base sequence refers to a polynucleotide sequence without chemical modifications or delivery compounds.
  • the sense strand shown in Table 1 corresponds to the corresponding base sequence in Table 3; however, the corresponding sequence in Table 3 shows the respective chemical modifications and delivery compounds.
  • Sequences disclosed herein may be assigned identifiers. For example, a single-stranded sense sequence may be identified with "sense strand SS#"; a single-stranded antisense sequence may be identified with "antisense strand AS#”; and a duplex containing a sense strand and an antisense strand may be identified with "duplex Chain AD#" to identify.
  • Table 1 includes the sense and antisense strands and provides the identification number of the duplex formed by the sense and antisense strands on the same row in Table 1.
  • the antisense sequence contains nucleobase u or nucleobase a in position 1 thereof.
  • the antisense sequence comprises nucleobase u at position 1 of the antisense sequence.
  • the term "matching position” refers in a sense to a position in each strand that "pairs" with each other when the two strands act as a duplex.
  • nucleobase at position 1 of the sense strand is in a "matching position" with the nucleobase at position 21 of the antisense strand.
  • nucleobase position 2 of the sense strand is in a matching position with position 22 of the antisense strand.
  • the nucleobase at position 1 of the sense strand is in a matching position with the nucleobase at position 18 of the antisense strand; and the sense strand Nucleobase 4 in the strand matches nucleobase 15 in the antisense strand.
  • the skilled person will understand how to identify matching positions between the sense and antisense strands of duplexes and paired strands.
  • the first column in Table 1 represents the duplex AV# of the duplex containing the sense and antisense sequences in the same row of the table.
  • Table 1 discloses a duplex designated "Duplex AV00206" which contains corresponding sense and antisense strand sequences.
  • each row in Table 1 identifies a duplex of the invention, each containing the sense and antisense sequences shown in the same row, and the assigned identifier for each duplex is shown at the end of that row. in one column.
  • the subject is administered an RNAi comprising a polynucleotide sequence set forth in Table 1 Reagents.
  • the RNAi agent administered to the subject includes 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.
  • it is also included in linking an RNAi agent of the polynucleotide sequence shown in Table 1 to a delivery molecule, a non-limiting example of which is a delivery compound comprising GalNAc.
  • Duplex AV# is the number assigned to the duplex of both strands in the same row in 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. .
  • Table 2 shows the antisense and sense strand sequences of certain chemically modified xanthine dehydrogenase (XDH) gene RNAi agents of the present invention.
  • XDH xanthine dehydrogenase
  • an RNAi agent having the polynucleotide sequence set forth in Table 2 is administered to the cell and/or subject.
  • an RNAi agent having the polynucleotide sequence set forth in Table 2 is administered to the subject.
  • the RNAi agent administered to the subject comprises the duplexes noted in the first column of Table 2, and comprises the sense and reverse duplexes shown in the third and sixth columns of the same row in Table 2, respectively. Sequence modifications in the sense strand sequence.
  • the sequences shown in Table 2 can be linked to (also referred to herein as "conjugated to") a compound capable of delivering an RNAi agent to cells and/or tissues of a subject.
  • delivery compounds useful in certain embodiments of the invention are GalNAc-containing compounds.
  • the first column represents the duplex AV# of the base sequence and corresponds to Table 1.
  • the base sequence identified by duplex AV# not only the base sequences contained in the sense and antisense strands are shown, but also with the specified chemical modifications shown in the same row of Table 2.
  • the first row of Table 1 shows the sense and antisense base single-stranded sequences, which together form a duplex, identified as: duplex AV#AV00206; and in the duplex AV#AV00206 listed in Table 2, As a duplex, it contains the base sequences of AV00206-SS and AV00206-AS, and contains chemical modifications in the sense and antisense sequences shown in the third and sixth columns, respectively.
  • "Sense Strand SS#" in the second column of Table 2 is the designated identifier for the sense sequence (including modifications) shown in the third column of the same row.
  • Antisense Strand AS# in the fifth column of Table 2 is the designated identifier for the antisense sequence (including modifications) shown in the sixth column.
  • SEQ ID NO in the fourth column of Table 2 is the sequence number of the sense sequence (including modifications) shown in the third column of the same row.
  • 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.
  • Table 3 shows the antisense and sense strand sequences of certain chemically modified xanthine dehydrogenase (XDH) gene RNAi reagents of the present invention.
  • an RNAi agent shown in Table 3 is administered to the cell and/or subject.
  • an RNAi agent having the polynucleotide sequence set forth in Table 3 is administered to the subject.
  • the RNAi agent administered to the subject comprises a duplex identified in the first column of Table 3, and comprises the sense and reverse duplexes, respectively, in the third and sixth columns of the same row of Table 3.
  • the sequence shown in the sense strand sequence modifies and/or delivers the compound.
  • the sequences shown in Table 3 can be linked (also referred to herein as "conjugated") to a compound for delivery, a non-limiting example of which is a GalNAc-containing compound , that is, there is a delivery compound identified as “GLX-n” on the sense strand in the third column of Table 3.
  • a delivery compound identified as “GLX-n” on the sense strand in the third column of Table 3.
  • Certain embodiments of delivery compounds are identified as "GLS-5", “GLS-15” or "GLX-n” on the sense strand in the third column of Table 3.
  • “GLS-5", “GLS-15” and “GLX-n” refer to GalNAc-containing compounds.
  • GLX is used to mean a “GLS” or “GLO” delivery compound
  • X can be “S” or “O”
  • GLX-n can be any GLS and GLO that can be linked during synthesis Delivery compounds to the 3'- or 5'-end of the oligonucleotide.
  • GLX-13 and GLX-14 can be connected to the 3'-end of the oligonucleotide of the present invention during the synthesis process
  • GLX-5 and GLX-15 can be connected to the present invention during the synthesis process. the 5'-end of the oligonucleotide.
  • GLX-n is used to represent an attached GalNAc-containing compound, which is replaced with compounds GLS-1, GLS-2, GLS-3, GLS- 4.
  • GLX-n is represented as an example but not limited to the ones disclosed in the prior art (Jayaprakash, et al., (2014) J. Am. Chem. Soc., 136, 16958-16961) that can be used to connect GalNAc-containing compounds used in certain in vivo testing studies described elsewhere herein.
  • dsRNA compounds of the invention wherein 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.
  • the structure of each of these is provided elsewhere in this article.
  • the first column of Table 3 provides the duplex AD# assigned to the duplexes for the sense and antisense sequences in that row of the table.
  • duplex AD#AD00352 is a duplex composed of sense strand AD00352-SS and antisense strand AD00352-AS.
  • Each row in Table 3 provides one sense strand and one antisense strand, and discloses the duplex consisting of the indicated sense strand and antisense strand.
  • 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.
  • "Antisense Strand AS#" in the fifth column of Table 3 is the designated identifier for the antisense sequence (including modifications) shown in the sixth column.
  • 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 is the antisense strand sequence.
  • delivery molecules are represented in sense strand NO:350 to SEQ ID NO:397, SEQ ID NO:446 to SEQ ID "GLS-5", “GLS-15” or "GLX-n” at the 3' or 5' end of NO:451.
  • Chemical modifications are expressed as: upper case: 2'-fluoro; lower case: 2'-methoxy; phosphorothioate: *; Invab: reverse abasic; imann: at the end of each chain: or when further coupled to a delivery molecule or targeting group:
  • mismatches are tolerated for the efficacy of dsRNA, especially if the mismatch is within the terminal region of the dsRNA.
  • Certain mismatches are better tolerated, such as those with wobble base pairs G:U and A:C (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).
  • the xanthine dehydrogenase (XDH) dsRNA reagent may contain one or more mismatches to the xanthine dehydrogenase (XDH) target sequence.
  • the xanthine dehydrogenase (XDH) dsRNA reagents of the invention do not contain mismatches.
  • the xanthine dehydrogenase (XDH) dsRNA reagents of the invention contain no more than 1 mismatch.
  • the xanthine dehydrogenase (XDH) dsRNA reagents of the invention contain no more than 2 mismatches.
  • the xanthine dehydrogenase (XDH) dsRNA reagents of the invention contain no more than 3 mismatches.
  • the antisense strand of the xanthine dehydrogenase (XDH) dsRNA agent contains a mismatch to the xanthine dehydrogenase (XDH) target sequence that is not centered in the complementary region.
  • the antisense strand of the xanthine dehydrogenase (XDH) dsRNA agent contains 1, 2, 3, 4, or more mismatches located at one or both of the 5' or 3' ends of the complementary region within the last 5, 4, 3, 2 or 1 nucleotides.
  • XDH xanthine dehydrogenase
  • the term "complementary/complementary" is used when describing the first nucleotide sequence (e.g., the xanthine dehydrogenase (XDH) dsRNA agent sense strand or the target xanthine dehydrogenase (XDH) XDH) mRNA) is related to a second nucleotide sequence (e.g., xanthine dehydrogenase (XDH) dsRNA reagent antisense strand or single-stranded antisense polynucleotide), refers to the inclusion of the first nucleotide sequence an oligonucleotide or polynucleotide that hybridizes to an oligonucleotide or polynucleotide comprising a second nucleotide sequence [which forms hydrogen bonds between base pairs under mammalian physiological conditions (or similar conditions in vitro)], and The ability to form a double helix or duplex structure
  • Complementary sequences include Watson-Crick base pairs or non-Watson-Crick base pairs, and include natural or modified nucleotides or nucleotide mimetics, so long as they are at least to the extent required for hybridization as described above. . Sequence identity or complementarity is independent of modification.
  • the complementary sequence within a xanthine dehydrogenase (XDH) dsRNA as described herein includes an oligonucleotide or polynucleotide comprising a first nucleotide sequence and an oligonucleotide comprising a second nucleotide sequence.
  • Such sequences may be referred to herein as being "completely complementary" to each other. It will 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 to be mismatches based on complementarity.
  • the xanthine dehydrogenase (XDH) dsRNA reagent contains one oligonucleotide that is 19 nucleotides in length and another oligonucleotide that is 20 nucleotides in length, with the longer oligonucleotide Contains 19 nucleotides that are fully complementary to the shorter oligonucleotide sequence, which for the purposes described here may be referred to as "perfectly complementary.”
  • "perfect complementarity" means that all (100%) 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.
  • the contiguous sequence may comprise all or part of the first or second nucleotide sequence.
  • the term "substantially complementary” means that in hybrid pairs of nucleobase sequences, at least about 85% (but not all) of the bases in the contiguous sequence of the first polynucleotide will be with the second polynucleotide. The same number of bases in a contiguous sequence of nucleotides hybridize.
  • the term "substantially complementary” may be used to refer to the first
  • the sequence 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 body while retaining the ability to hybridize under conditions most relevant to its end application, e.g., inhibition of xanthine dehydrogenase (XDH) gene expression via the RISC pathway.
  • XDH xanthine dehydrogenase
  • partially complementary may be used herein to refer to a hybrid pair of nucleobase sequences in which at least 75% (but not all) of the bases in the contiguous sequence of a first polynucleotide will be with those of a second polynucleotide. Hybridization of the same number of bases in a contiguous sequence.
  • “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 will match the Two polynucleotides hybridize to the same number of bases in a contiguous sequence.
  • antisense strand of xanthine dehydrogenase (XDH) dsRNA agent may refer to the same sequence as “xanthine dehydrogenase (XDH) antisense polynucleotide agent”.
  • nucleic acid sequence contains a sequence that has at least about 85% or greater sequence identity compared to a reference sequence, 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. Percent sequence identity is determined by comparing the best alignment of two sequences over an alignment window.
  • the percentage is calculated by determining the number of positions where the same nucleic acid base occurs in both sequences to yield the number of matching positions; dividing the number of matching positions by the total number of positions in the alignment window, and multiplying the result by 100, resulting in percent sequence identity.
  • the inventions disclosed herein include nucleotide sequences that are substantially identical to those disclosed herein (eg, in Tables 1-3). In some embodiments, the nucleotide sequence is identical to, or at least about 85%, 86%, 87%, 88%, 89%, 90% identical to, a sequence disclosed herein (e.g., in Tables 1-3). %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.
  • strand comprising a sequence refers to an oligonucleotide comprising a strand of nucleotides described by a sequence referred to using standard nucleotide nomenclature.
  • double-stranded RNA or “dsRNA” refers to a sequence comprising an RNA molecule or a complex of RNAi molecules that has a The hybridized double-stranded regions of two antiparallel and substantially or completely complementary nucleic acid strands are referred to as having "sense” and “antisense” orientations relative to the target xanthine dehydrogenase (XDH) gene RNA, respectively.
  • XDH target xanthine dehydrogenase
  • the double-stranded region can be of any desired length that allows specific degradation of the target xanthine dehydrogenase (XDH) gene RNA by the RISC pathway, but is typically 9 to 30 base pairs in length, e.g. 15-30 bases in length right.
  • XDH xanthine dehydrogenase
  • duplexes can be any length within this range, e.g., 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 subrange 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, 18-23 base pairs, 18-22 base pairs, 18-21 base pairs, 18-20 bases pair, 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 reagents produced in cells by processing with Dicer and similar enzymes is typically in the range of 19-22 base pairs.
  • One strand of the double-stranded region of the xanthine dehydrogenase (XDH) dsDNA agent contains a sequence substantially complementary to a region of the target xanthine dehydrogenase (XDH) gene RNA.
  • the two strands forming a duplex structure can come from a single RNA molecule with at least one self-complementary region, or can be formed from two or more separate RNA molecules.
  • the molecule may have a duplex structure formed by one strand of the single-stranded nucleotide strand at the 3'-end and the corresponding other strand at the 5'-end (herein called a "hairpin loop").
  • the hairpin configuration includes 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.
  • XDH xanthine dehydrogenase
  • dsRNA reagent consist of separate RNA molecules, these molecules need not be covalently linked, but may be covalently linked.
  • the connecting structure is called a "linker.”
  • siRNA is also used herein to refer to a dsRNA agent as described herein.
  • a xanthine dehydrogenase (XDH) dsRNA reagent can comprise sense and antisense sequences with unpaired nucleotides or nucleotide analogs at one or both ends of the dsRNA reagent. Ends without unpaired nucleotides are called “blunt ends" and have no nucleotide overhangs.
  • XDH xanthine dehydrogenase
  • the dsRNA is said to be "blunt-ended.”
  • the first end of the dsRNA agent is blunt-ended
  • the second end of the dsRNA agent is blunt-ended
  • the xanthine Both ends of the XDH dsRNA reagent are blunt-ended.
  • the dsRNA does not have one or two blunt ends.
  • nucleotide overhangs exist when the 3'-end of one strand of dsRNA extends beyond the 5'-end of the other strand, and vice versa.
  • the dsRNA may contain overhangs of at least 1, 2, 3, 4, 5, 6, or more nucleotides. Nucleotide overhangs can Contains or consists of nucleotide/nucleoside analogs, including deoxynucleotides/nucleosides.
  • the nucleotide overhangs are 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 overhangs may be present on the antisense strand of the dsRNA reagent.
  • one or more nucleotides in the overhang are replaced with nucleoside phosphorothioates.
  • antisense strand or "guide strand” refers to the strand of a xanthine dehydrogenase (XDH) dsRNA agent that includes a region that is substantially complementary to a xanthine dehydrogenase (XDH) target sequence.
  • sense strand or “passenger strand” refers to a xanthine dehydrogenase (XDH) dsRNA reagent that includes a region that is substantially complementary to a region of the antisense strand of the xanthine dehydrogenase (XDH) dsRNA reagent. chain.
  • the RNA of the xanthine dehydrogenase (XDH) gene RNAi agent is chemically modified to obtain enhanced stability and/or one or more other beneficial properties.
  • Nucleic acids in certain embodiments of the invention can be synthesized and/or modified by methods well known in the art, for example, see “Current protocols in Nucleic Acid Chemistry,” Beaucage, S.L. et al. (Eds.), John Wiley & Sons, Inc. ., New York, N.Y., USA, which is hereby incorporated by reference.
  • Modifications that may be present in certain embodiments of the xanthine dehydrogenase (XDH) dsRNA reagents of the invention include, for example: (a) terminal modifications, such as 5' end modifications (phosphorylation, conjugation, reverse ligation, etc.) , 3' end modification (conjugation, DNA nucleotides, reverse ligation, etc.); (b) base modification, such as with stabilizing bases, destabilizing bases, or bases for base pairing with an expanded partner library base substitutions, missing bases (abasic nucleotides) or conjugated bases; (c) sugar modifications (e.g., at the 2' position or 4' position) or substitutions of sugars; and (d) backbone modifications, including Modification or replacement of phosphodiester bonds.
  • terminal modifications such as 5' end modifications (phosphorylation, conjugation, reverse ligation, etc.) , 3' end modification (conjugation, DNA nucleotides, reverse ligation, etc.
  • base modification such as
  • RNAs useful in certain embodiments of the xanthine dehydrogenase (XDH) dsRNA reagents, xanthine dehydrogenase (XDH) antisense polynucleotides, and xanthine dehydrogenase (XDH) sense polynucleotides of the invention Specific examples of compounds include, but are not limited to, RNAs containing modified backbones or without native internucleoside linkages. As a non-limiting example, RNA with backbone modifications may not have phosphorus atoms in the backbone. RNA that does not have a phosphorus atom in its internucleoside backbone is called an oligonucleotide. In certain embodiments of the invention, the modified RNA has a phosphorus atom in its internucleoside backbone.
  • 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 of the Ribonucleotide/ribonucleoside analogs or derivatives as described or known in the art.
  • ribonucleoside and “ribonucleotide” are used interchangeably herein.
  • RNA molecules may be modified in the nucleobase structure or ribose-phosphate backbone structure (eg, as described below), and molecules containing ribonucleoside analogs or derivatives must retain the ability to form duplexes.
  • the RNA molecule may also comprise at least one modified ribonucleoside, including, but not limited to, 2'-methoxy modified nucleosides, nucleosides containing a 5' phosphorothioate group, and Terminal nucleosides linked to cholesterol derivatives or dodecanoic acid bisdecylamide groups, locked nucleosides, abasic nucleosides, 2'-deoxy-2'-fluoro modified nucleosides, 2'-amino modified nucleosides glycosides, 2'-alkyl modified nucleosides, morpholino nucleosides, phosphoramidates, or non-natural bases containing nucleosides, or any combination thereof.
  • modified ribonucleoside including, but not limited to, 2'-methoxy modified nucleosides, nucleosides containing a 5' phosphorothioate group, and Terminal nucleosides linked to cholesterol derivatives or dodecanoic
  • the RNA molecule contains the following number of modified ribonucleosides: 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 full length of the ribonucleoside of the xanthine dehydrogenase (XDH) dsRNA reagent molecule.
  • the modifications need not be the same for each of the multiple modified ribonucleosides in such an RNA molecule.
  • the dsRNA reagents, xanthine dehydrogenase (XDH) antisense polynucleotides and/or xanthine dehydrogenase (XDH) sense polynucleotides of the invention may comprise one or more independently selected Modify the nucleotide and/or one or more independently selected non-phosphodiester linkages.
  • independently selected is used to refer to selected elements, such as modified nucleotides, non-phosphodiester bonds, etc., meaning that two or more selected elements may be identical to each other but need not be identical to each other.
  • nucleotide base is a heterocyclic pyrimidine or purine compound that is a standard component of all nucleic acids and includes the bases that form nucleotides: adenine Purine (a), guanine (g), cytosine (c), thymine (t) and uracil (u).
  • Nucleobases may be further modified to include, but are not intended to be limited to: universal bases, hydrophobic bases, hybrid bases, size-enlarged bases, and fluorinated bases.
  • ribonucleotide or “nucleotide” may be used herein to refer to unmodified nucleotides, modified nucleotides, or alternative moieties.
  • guanine, cytosine, adenine and uracil can be replaced by other moieties without significantly altering the base pairing of oligonucleotides containing nucleotides with such replaced moieties characteristic.
  • the modified RNA contemplated for use in the methods and compositions described herein is a peptide nucleic acid (PNA) that has the specificity to form the desired duplex structure and allow or mediate the passage of the target RNA via the RISC pathway. ability to degrade.
  • the xanthine dehydrogenase (XDH) gene RNA interference agent includes a target xanthine dehydrogenase (XDH) gene RNA sequence that interacts with the target xanthine dehydrogenase (XDH) gene to direct the target xanthine dehydrogenase (XDH) gene. RNA cleavage of single-stranded RNA.
  • Modified RNA backbones may include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphate triesters, aminoalkyl phosphate triesters, methyl and other alkyl phosphonates (including 3'- Alkylene phosphonates and chiral phosphonates), phosphinates, phosphoramidates (including 3'-aminophosphoramidates and aminoalkyl phosphoramidates), thiophosphates, thioalkyl Phosphonates, thioalkyl phosphate triesters, and boronic acid phosphates (which have the normal 3'-5' linkage, as well as the 2'-5' linkage analogs of these, and those with inverted polarity, in which Adjacent pairs of nucleoside units are linked in a 3'-5' to 5'-3' or 2'-5' to 5'-2' format).
  • XDH modified xanthine dehydrogenase
  • XDH modified xanthine dehydrogenase
  • XDH antisense polynucleotides
  • XDH modified xanthine dehydrogenase
  • Modified RNA backbones that do not contain a phosphorus atom have structures consisting of short-chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chains
  • morpholino bonds formed in part from the sugar moiety of the nucleoside
  • siloxane backbones siloxane backbones
  • sulfide, sulfoxide and sulfone backbones methylacetyl and thiomethylacetyl backbones
  • methylenemethacetyl and thiomethylacetyl backbones Skeletons; olefin-containing skeletons; sulfamate skeletons; methyleneimino and methylenehydrazino skeletons; sulfonate and sulfonamide skeletons; amide skeletons; and other mixtures of N, O, S and CH 2 components part.
  • modified RNA backbones that do not contain phosphorus atoms are routine practice in the art, and such methods can be used to prepare certain modified xanthine dehydrogenase (XDH) dsRNA reagents, certain modified xanthine dehydrogenase (XDH) dsRNA reagents of the invention, Purine dehydrogenase (XDH) antisense polynucleotide and/or Certain modified xanthine dehydrogenase (XDH) sense polynucleotides.
  • XDH modified xanthine dehydrogenase
  • RNA mimics are included in xanthine dehydrogenase (XDH) dsRNA, xanthine dehydrogenase (XDH) antisense polynucleotides, and/or xanthine dehydrogenase (XDH)
  • sense polynucleotides for example, but not limited to, the sugars and internucleoside linkages (ie, backbone) of the nucleotide units are replaced with new groups.
  • base units are maintained for hybridization to the appropriate xanthine dehydrogenase (XDH) nucleic acid target compound.
  • RNA mimetic an RNA mimetic that has been shown to have excellent hybridization properties
  • PNA peptide nucleic acid
  • the sugar backbone of RNA is replaced by an amide-containing backbone, specifically an aminoethylglycine backbone.
  • the nucleobase is retained and bound directly or indirectly to the aza nitrogen atom of the backbone amide moiety.
  • Methods for preparing RNA mimetics are routinely practiced in the art, and such methods can be used to prepare certain modified xanthine dehydrogenase (XDH) dsRNA reagents of the invention.
  • XDH modified xanthine dehydrogenase
  • RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones particularly -CH2 -NH- CH2- , -CH2 -N( CH3 )-O-CH 2- [called methylene (methylimino) or MMI skeleton], -CH 2 -ON(CH 3 )-CH 2 -, -CH 2 -N(CH 3 )-N(CH 3 )-CH 2 - and -N(CH 3 )-CH 2 - [where the natural phosphodiester backbone is represented as -OPO-CH 2 -].
  • RNAs with phosphorothioate backbones and oligonucleotides with heteroatom backbones are routinely practiced in the art, and such methods can be used to prepare certain modified xanthine dehydrogenase (XDH) dsRNAs of the invention Reagents, certain xanthine dehydrogenase (XDH) antisense polynucleotides and/or certain xanthine dehydrogenase (XDH) sense polynucleotides.
  • XDH modified xanthine dehydrogenase
  • Modified RNA may also contain one or more substituted sugar moieties.
  • the xanthine dehydrogenase (XDH) dsRNA, xanthine dehydrogenase (XDH) antisense polynucleotide and/or xanthine dehydrogenase (XDH) sense polynucleotide of the present invention may include 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 the alkyl group, alkenyl group and alkynyl group may be substituted or unsubstituted C 1 to C 10 alkyl group or C 2 to C 10 alkenyl group and alkynyl group.
  • Exemplary suitable modifications include: O[(CH 2 ) n O] m CH 3 , O(CH 2 ) n OCH 3 , O(CH 2 ) n NH 2 , O( CH 2 ) n CH 3 , O( CH 2 ) n ONH 2 , and O(CH 2 ) n ON [(CH 2 ) n CH 3 )] 2 , where n and m range from 1 to about 10.
  • the dsRNA includes one of the following at the 2' position: C 1 to C 10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl, or O-aralkyl , SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , SO 2 CH 3 , ONO 2 , NO 2 , N 3 , NH 2 , heterocycloalkyl, heterocycloalkaryl , aminoalkylamino, polyalkylamino; substituted silyl group, RNA cleavage group, reporter group, intercalator; base for improving the pharmacokinetic properties of xanthine dehydrogenase (XDH) dsRNA reagent group; or used to improve the pharmacodynamic properties of xanthine dehydrogenase (XDH) dsRNA reagents, xanthine dehydrogenase (XDH) anti
  • modifications include 2'-O-CH 2 CH 2 OCH 3 , also known as 2'-O-(2-methoxyethyl) or 2' -MOE) (Martin et al., Helv. Chim. Acta, 1995, 78: 486-504), that is, alkoxy-alkoxy.
  • Another exemplary modification is 2'-dimethylaminoethoxyethoxy, the O( CH2 ) 2ON ( CH3 ) 2 group, also known as 2'-DMAOE, as shown in the examples below as described above; and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e. 2'-O-CH 2 -O-CH 2 -N(CH 2 ) 2 .
  • Methods for preparing modified RNAs such as those described are skill are routinely practiced in the art, and such methods can be used to prepare certain modified xanthine dehydrogenase (XDH) dsRNA reagents of the invention.
  • XDH modified xanthine dehydrogenase
  • modifications include 2'-methoxy (2'-OCH 3 ), 2'-aminopropoxy (2'-OCH 2 CH 2 CH 2 NH 2 ), and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of the xanthine dehydrogenase (XDH) dsRNA reagent, xanthine dehydrogenase (XDH) antisense polynucleotide, and xanthine dehydrogenase (XDH) sense polynucleotide of the present invention.
  • XDH xanthine dehydrogenase
  • XDH xanthine dehydrogenase
  • oligonucleotide and/or other positions of the xanthine dehydrogenase (XDH) sense polynucleotide especially the xanthine dehydrogenase (XDH) dsRNA, xanthine on the 3' terminal nucleotide or 2'-5' connected Dehydrogenase (XDH) antisense polynucleotide or xanthine dehydrogenase (XDH) sense polynucleotide at the 3' position of the sugar and the 5' position of the 5' terminal nucleotide.
  • the xanthine dehydrogenase (XDH) dsRNA reagent, the xanthine dehydrogenase (XDH) antisense polynucleotide and/or the xanthine dehydrogenase (XDH) sense polynucleotide can also have a sugar mimetic, for example instead of pentfuran The cyclobutyl portion of sugar.
  • Methods of 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 reagents, xanthine dehydrogenase (XDH) of the invention.
  • antisense polynucleotide and/or xanthine dehydrogenase (XDH) sense polynucleotide can also have a sugar mimetic, for example instead of pentfuran The cyclobutyl portion of sugar.
  • the xanthine dehydrogenase (XDH) dsRNA reagent, the xanthine dehydrogenase (XDH) antisense polynucleotide, and/or the xanthine dehydrogenase (XDH) sense polynucleotide can include nucleobases (often referred to as "base” in the art) modification or substitution.
  • 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-thiocytosan Pyrimidine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil ; 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenine and guanine; 5-halo, especially 5-bromo, 5- Trifluoromethyl and other
  • nucleobases that may be included in certain embodiments of the xanthine dehydrogenase (XDH) dsRNA reagents 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,J.L,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, S.T.
  • dsRNA, xanthine dehydrogenase (XDH) antisense strand polynucleotides and/or xanthine dehydrogenase (XDH) sense strand polynucleotides (such as those described herein) comprising nucleobase modifications and/or substitutions ) methods are routinely practiced in the art, and such methods can be used to prepare certain modified xanthine dehydrogenase (XDH) dsRNA reagents, xanthine dehydrogenase (XDH) sense polynucleotides and/or Xanthine dehydrogenase (XDH) antisense polynucleotide.
  • XDH modified xanthine dehydrogenase
  • Certain embodiments of glycoside and/or xanthine dehydrogenase (XDH) sense polynucleotides include RNA modified to include one or more locked nucleic acids (LNA). Locked nucleic acids are nucleotides with a modified ribose moiety that contains an additional bridge connecting the 2' and 4' carbons. This structure effectively "locks" ribose in the 3'-endostructural conformation.
  • LNA locked nucleic acids
  • 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 serum stability 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).
  • dsRNA reagents 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 For preparing certain modified xanthine dehydrogenase (XDH) dsRNA reagents of the present invention.
  • XDH xanthine dehydrogenase
  • XDH xanthine dehydrogenase
  • xanthine dehydrogenase (XDH) dsRNA compounds, sense polynucleotides, and/or antisense polynucleotides of the invention include at least one modified nucleotide, wherein the at least one modified core
  • the nucleotides include: 2'-methoxy nucleotide, 2'-fluoro nucleotide, 2'-deoxy nucleotide, 2',3'-seco nucleotide mimic, locked nucleotide, 2'- F-arabinose nucleotides, 2'-methoxyethyl nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides and 3' - Ome nucleotides, nucleotides containing a 5'-phosphorothioate group, or terminal nucleotides linked to cholesterol derivatives or dodecanoic acid bisdec
  • the xanthine dehydrogenase (XDH) dsRNA compound contains an E-vinylphosphonate nucleotide at the 5' end of the antisense strand (also referred to herein as the guide strand).
  • At least one modified Nucleotides wherein at least one modified nucleotide includes: abasic nucleotide, ribitol, reverse nucleotide, reverse abasic nucleotide, reverse 2'-OMe nucleotide, Reverse 2'-deoxynucleotide. It is known to those skilled in the art that the inclusion of abasic or reverse abasic nucleotides at the termini of oligonucleotides 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).
  • the xanthine dehydrogenase (XDH) dsRNA compound, antisense polynucleotide comprises at least one modified nucleotide, wherein the at least one modified nucleotide comprises a closed-loop nucleic acid Nucleotide (UNA) or/and glycol nucleic acid nucleotide (GNA).
  • UNA and GNA are thermally unstable chemical modifications that can significantly improve the off-target profile of siRNA compounds (Janas, et al., Selection of GalNAc-conjugated siRNAs with limited off-target-driven rat hepatotoxicity.
  • the invention relates to open nucleic acid (UNA) oligomers for use in therapy.
  • Unlocked 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. Incorporation of UNA has been shown to be well tolerated against siRNA gene silencing activity, in In some cases, its activity can even be enhanced (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).
  • UNA is a heat-labile modification, and replacing ribonucleotides with UNA will reduce 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 the gene silencing mechanism mediated by microRNA (miRNA).
  • miRNA mainly recognizes the target gene through base pairing between the antisense seed region (positions 2-8 starting from the 5' end) and the target mRNA for gene suppression. 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 a miRNA-mediated mechanism.
  • RISC RNA-induced silencing complex
  • RNA oligonucleotides or complexes of RNA oligonucleotides contain at least one UNA nucleotide 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).
  • RNA oligonucleotides or complexes of RNA oligonucleotides include, but are not limited to:
  • UNA is well tolerated in terms of siRNA activity. In some cases, UNA can lead to increased activity.
  • Exemplary UNA monomers that can be used in this technical solution include, but are not limited to:
  • Invab is the reverse abasic (deoxyribose) residue.
  • the sense or antisense strand can include a "terminal cap," which as used herein is a non-nucleotide compound that can be incorporated at one or more ends of a strand of an RNAi agent disclosed herein or other moieties, and in some cases may provide certain advantageous properties to the RNAi agent, such as, for example, protection against exonuclease degradation.
  • an inverted abasic residue is added as a terminal cap (see, e.g.,
  • End caps are generally known in the art In some embodiments, a terminal cap is present at the 5' end, the 3' end, or both the 5' and 3' ends of the sense strand.
  • the xanthine dehydrogenase (XDH) dsRNA compound, antisense polynucleotide comprises at least one modified nucleotide, wherein the at least one modified nucleotide comprises isomannose Alcohol nucleotides.
  • the sense strand contains an isomannitol residue and its stereoisomers at the 3' or 5' ends, respectively. Examples of specific nucleotides containing isomannitol residues and their stereoisomers include, but are not limited to: The phrase "Olig" each independently represents a polynucleotide moiety.
  • Exemplary isomannitol residues include, but are not limited to, the following:
  • the isomannitol residue (imann) can also be replaced by its stereoisomer.
  • the sense strand includes an isomannitol residue (imann) at the 3' or 5' ends, respectively, and optionally further includes a 5'-end conjugated targeting group, e.g. , targeting group N-acetyl-galactosamine, preferably the above-mentioned GLS-15, the exemplary structure is as follows:.
  • RNA contains another modification that includes enhancing xanthine dehydrogenase (XDH) dsRNA reagents, xanthine dehydrogenase (XDH) antisense polynucleotides, and/or xanthine dehydrogenase (XDH) sense polynucleotides, respectively One or more ligands, moieties, or conjugates of one or more features of an acid chemically linked to RNA.
  • Non-limiting examples of characteristics that can be enhanced are: xanthine dehydrogenase (XDH) dsRNA reagents, xanthine dehydrogenase (XDH) antisense polynucleotides, and/or xanthine dehydrogenase (XDH) sense polynucleotides Activity, cellular distribution, delivery of xanthine dehydrogenase (XDH) dsRNA reagent, pharmacokinetic properties of xanthine dehydrogenase (XDH) dsRNA reagent, and cellular uptake of xanthine dehydrogenase (XDH) dsRNA reagent.
  • XDH xanthine dehydrogenase
  • XDH xanthine dehydrogenase
  • XDH xanthine dehydrogenase
  • XDH xanthine dehydrogenase
  • XDH xanthine dehydrogen
  • the xanthine dehydrogenase (XDH) dsRNA reagents comprise one or more targeting groups or linking groups, in certain aspects of the xanthine dehydrogenase (XDH) dsRNA reagents of the invention. In some embodiments, it is conjugated to the sense strand.
  • targeting groups are compounds containing N-acetyl-galactosamine (GalNAc).
  • GalNAc N-acetyl-galactosamine
  • the xanthine dehydrogenase (XDH) dsRNA reagent comprises a targeting compound conjugated to the 5'-end of the sense strand. In certain embodiments of the invention, the xanthine dehydrogenase (XDH) dsRNA reagent comprises a targeting compound conjugated to the 3'-end of the sense strand. In some embodiments of the invention, the xanthine dehydrogenase (XDH) dsRNA reagent comprises a GalNAc-containing targeting group.
  • the xanthine dehydrogenase (XDH) dsRNA reagent does not comprise a targeting compound conjugated to one or both of the 3'-end and 5'-end of the sense strand. In certain embodiments of the invention, the xanthine dehydrogenase (XDH) dsRNA reagent does not comprise a GalNAc-containing targeting compound conjugated to one or both of the 5'-end and 3'-end of the sense strand.
  • lipid moieties such as cholesterol moieties (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4: 1053-1060),
  • 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.
  • compositions comprising a xanthine dehydrogenase (XDH) dsRNA agent, a xanthine dehydrogenase (XDH) antisense polynucleotide, and/or a xanthine dehydrogenase (XDH) sense polynucleotide can comprise Ligands that change the distribution, targeting and other properties of xanthine dehydrogenase (XDH) dsRNA reagents.
  • compositions comprising a xanthine dehydrogenase (XDH) dsRNA agent of the invention
  • a selected target e.g., molecule, cell, or Affinity of a cell type, compartment, such as a cell or organ compartment, tissue, organ or body region.
  • Ligands useful in the compositions and/or methods of the invention may be naturally occurring substances such as proteins (e.g. human serum albumin (HSA), low density lipoprotein (LDL) or globulin), carbohydrates (e.g. , dextran, amylopectin, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid) or lipids.
  • proteins e.g. human serum albumin (HSA), low density lipoprotein (LDL) or globulin
  • carbohydrates e.g. , dextran, amylopectin, chitin, chitosan, inulin, cyclodextr
  • Ligands may also be recombinant or synthetic molecules, such as synthetic polymers, such as synthetic polyamino acids or polyamines.
  • polyamino acids are polylysine (PLL), polyL-aspartic acid, polyL-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 polyphosphazine.
  • PLL polylysine
  • polyL-aspartic acid polyL-glutamic acid
  • styrene-maleic anhydride copolymer poly(L-lactide-co-glycolic acid)
  • polyamines examples include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendritic polyamines, arginine, amidine , protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines or ⁇ -helical peptides.
  • Ligands included in the compositions and/or methods of the invention may comprise targeting groups, non-limiting examples of which are cell or tissue targeting agents, for example, lectins, glycoproteins, lipids or proteins, e.g. binding to specific Antibodies to cell types such as kidney cells or liver cells.
  • targeting groups non-limiting examples of which are cell or tissue targeting agents, for example, lectins, glycoproteins, lipids or proteins, e.g. binding to specific Antibodies to cell types such as kidney cells or liver cells.
  • Targeting groups can be thyrotropin, melanogen, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl -Glucosamine polyvalent mannose, polyvalent fucose, glycosylated polyamino acids, polyvalent galactose, transferrin, bisphosphonates, polyglutamates, polyaspartic acid, lipids, Cholesterol, steroids, bile acids, folic acid, vitamin B12, vitamin A, biotin or RGD peptide or RGD peptide mimetics.
  • ligands include dyes, intercalators (e.g., acridines), cross-linkers (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazines) , dihydrophenazine); artificial endonucleases (such as EDTA), lipophilic molecules such as cholesterol, cholic acid, adamantane acetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O (ten Hexadecylglycerin, geranyloxyhexyl, cetylglycerin, borneol, peppermint Alcohol, 1,3-propanediol, heptadecanyl, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)
  • biotin e.g. aspirin, vitamin E, folic acid
  • transport/absorption enhancers e.g. aspirin, vitamin E, folic acid
  • synthetic ribonucleases e.g. imidazole, bisimidazole, histamine, imidazole cluster, acridine imidazole conjugates, Eu 3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP or AP.
  • Ligands included in the compositions and/or methods of the present invention may be proteins, such as glycoproteins or peptides, such as molecules with specific affinity for the co-ligand, or antibodies, such as with specific cell types such as cancer cells, endothelial cells, Antibodies that bind to cardiomyocytes or bone cells.
  • Ligands useful in embodiments of the compositions and/or methods of the invention may be hormones or hormone receptors.
  • Ligands useful in embodiments of the compositions and/or methods of the invention may be lipids, lectins, carbohydrates, vitamins, coenzymes, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine polyvalent mannose or polyvalent fucose.
  • Ligands useful in embodiments of the compositions and/or methods of the present invention may be those that increase xanthine depletion, for example, by disrupting the cell's cytoskeleton (e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments).
  • Hydrogenase (XDH) uptake of dsRNA reagents into cells.
  • Non-limiting examples of such agents are: taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine and myoservin.
  • ligands linked to the xanthine dehydrogenase (XDH) dsRNA agents of the invention serve as pharmacokinetic (PK) modulators.
  • PK modulators useful in the compositions and methods of the present invention include, but are not limited to: lipophilic agents, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, cholesterol, fatty acids, bile acids, Lithocholic acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, aptamers that bind to serum proteins, etc.
  • Oligonucleotides containing many phosphorothioate linkages are also known to bind to serum proteins, therefore short oligonucleotides containing multiple phosphorothioate linkages in the backbone, e.g. about 5 bases, 10 bases Base, 15 base or 20 base oligonucleotides may also be used as ligands in the compositions and/or methods of the invention.
  • XDH Xanthine dehydrogenase
  • a xanthine dehydrogenase (XDH) dsRNA agent is in the composition.
  • the compositions of the invention may comprise one or more xanthine dehydrogenase (XDH) dsRNA reagents and optionally one or more pharmaceutically acceptable carriers, delivery agents, targeting agents, detectable labels Etc.
  • targeting agents useful according to some embodiments of the methods of the invention are agents that direct the xanthine dehydrogenase (XDH) dsRNA agent of the invention to and/or into the cells to be treated.
  • the choice of targeting agent will depend on the nature of the xanthine dehydrogenase (XDH)-related disease or condition, and the target cell type.
  • 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 connecting elements.
  • XDH xanthine dehydrogenase
  • xanthine dehydrogenase XDH
  • the dsRNA agent can be linked to a GalNAc-containing delivery compound and included in a composition containing a pharmaceutically acceptable carrier in the absence of any detectable label or targeting agent linked to the xanthine dehydrogenase (XDH) dsRNA agent etc., administered to cells or subjects.
  • xanthine dehydrogenase (XDH) dsRNA reagent of the invention is administered with and/or linked to one or more delivery agents, targeting agents, labeling agents, etc.
  • suitable reagents for use in the methods of the invention.
  • Labeling reagents can be used in certain methods of the invention to determine the location of xanthine dehydrogenase (XDH) dsRNA reagents in cells and tissues, and can be used to determine the location of xanthine dehydrogenase (XDH) dsRNA reagents that have been administered in the methods of the invention.
  • XDH enzymatic
  • Means for attaching and using labeling reagents such as enzyme labels, dyes, radioactive labels, etc. are well known in the art. It will be appreciated that in some embodiments of the compositions and methods of the invention, the labeling reagent is linked to one or both of the sense and antisense polynucleotides comprised in the xanthine dehydrogenase (XDH) dsRNA reagent. .
  • XDH xanthine dehydrogenase
  • XDH xanthine dehydrogenase
  • Certain embodiments of the methods of the invention include delivering a xanthine dehydrogenase (XDH) dsRNA agent into the cell.
  • delivery means promoting or affecting cellular uptake or absorption. Absorption or uptake of the xanthine dehydrogenase (XDH) dsRNA agent can occur by independent diffusion or active cellular processes, or through the use of delivery agents, targets, or agents that can be associated with the xanthine dehydrogenase (XDH) dsRNA agent of the invention. Proceed to the agent and so on.
  • Modes of delivery suitable for use in the methods of the present invention include, but are not limited to, in vivo delivery, wherein the xanthine dehydrogenase (XDH) dsRNA agent is injected into a tissue site or administered systemically.
  • the xanthine dehydrogenase (XDH) dsRNA agent is linked to the delivery agent.
  • Non-limiting examples of methods that can be used to deliver xanthine dehydrogenase (XDH) dsRNA agents to cells, tissues, and/or subjects include: xanthine dehydrogenase (XDH) dsRNA-GalNAc conjugates, SAMiRNA technology, LNP delivery methods and naked RNA delivery. These and other delivery methods have been used successfully in the art to deliver therapeutic RNAi agents to treat a variety of diseases and conditions, such as, but not limited to: liver disease, acute intermittent porphyria (AIP), hemophilia, pulmonary fibrosis wait. Details of various delivery methods can be found in publications such as: Nikam, R.R. & K.R.
  • LNPs lipid nanoparticles
  • XDH xanthine dehydrogenase
  • XDH xanthine dehydrogenase
  • XDH xanthine dehydrogenase
  • XDH xanthine dehydrogenase
  • XDH xanthine dehydrogenase
  • One benefit of using LNP or other delivery agents is that the stability of the xanthine dehydrogenase (XDH) gene RNA agent is increased when delivered to a subject using LNP or other delivery agents.
  • the LNPs comprise cationic LNPs loaded with one or more xanthine dehydrogenase (XDH) gene RNAi molecules of the invention.
  • XDH xanthine dehydrogenase
  • will pack LNP containing a xanthine dehydrogenase (XDH) gene RNAi molecule is administered to a subject.
  • the LNP and its attached xanthine dehydrogenase (XDH) gene RNAi molecule are taken up by cells through endocytosis, and their presence results in an RNAi triggering molecule. release, thereby mediating RNAi.
  • a delivery agent that may be used in embodiments of the present invention to deliver the xanthine dehydrogenase (XDH) dsRNA agent of the invention to cells, tissues, and/or subjects is an agent comprising GalNAc, with The xanthine dehydrogenase (XDH) dsRNA reagents of the invention ligate and deliver the xanthine dehydrogenase (XDH) dsRNA reagent to cells, tissues and/or subjects.
  • GalNAc GalNAc
  • Non-limiting examples of GalNAc targeting ligands that can be used in the compositions and methods of the invention to deliver xanthine dehydrogenase (XDH) dsRNA agents to cells are targeting ligand clusters.
  • Examples of targeting ligand clusters proposed here are GalNAc ligands with phosphodiester linkages (GLO) and GalNAc ligands with phosphorothioate linkages (GLS).
  • GLO phosphodiester linkages
  • GLS phosphorothioate linkages
  • the term "GLX-n" may be used herein to refer to attached GalNAc-containing compounds.
  • Examples are, but are 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, 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 is shown below. In the figure below, the connection position of the GalNAc targeting ligand and the RNAi agent of the present invention is on the rightmost side of each targeting ligand.
  • any RNAi and dsRNA molecules of the 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, the following are GLO-1 to Structure of GLO-16 and GLS-1 to GLS-16.
  • Heterocycloalkyl means a nonaromatic partially saturated or fully saturated ring (eg, 3-10 or 3-7 membered heterocycloalkyl) having the specified number of ring atoms consisting of one or Composed of a plurality of heteroatoms (eg, 1, 2, 3, or 4 heteroatoms) selected from N, O, and S, and the remaining ring atoms are carbon.
  • a 5-membered heterocycloalkyl group is a heterocycloalkyl group having 5 ring atoms.
  • a 6-membered heterocycloalkyl group is a heterocycloalkyl group having 6 ring atoms.
  • Heterocycloalkyl groups may be monocyclic or polycyclic (eg, bicyclic, tricyclic).
  • heterocycloalkyl groups include oxanyl, aziridyl, azetidinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, piperidinyl, piperazinyl, morpholinyl and Thiomorpholinyl.
  • one ring of a polycyclic heterocycloalkyl group can be aromatic (eg, aryl or heteroaryl) so long as the polycyclic heterocycloalkyl group is bonded to the parent structure via a nonaromatic carbon or nitrogen atom.
  • 1,2,3,4-tetrahydroquinolin-1-yl (where 1,2,3,4-tetrahydroquinolin-8-yl (where the moiety is bonded to the parent structure via an aromatic carbon atom) is considered to be heterocycloalkyl parent structure) is not considered a heterocycloalkyl group.
  • Lower heterocycloalkanes generally refer to C 3-6 monocyclic rings. Unless otherwise specified, lower heterocycloalkyl groups can generally be preferably fully saturated carbocyclic rings.
  • in vivo delivery may also be by beta-glucan delivery systems, such as those described in U.S. Patent Nos. 5,032,401 and 5,607,677, and U.S. Publication No.
  • the xanthine dehydrogenase (XDH) gene RNAi agent can also be introduced into cells in vitro using methods known in the art such as electroporation and lipofection.
  • the xanthine dehydrogenase (XDH) dsRNA is delivered without a targeting agent. These RNAs can be delivered as "naked" RNA molecules.
  • the xanthine dehydrogenase (XDH) dsRNA of the invention can be administered to a subject in a pharmaceutical composition that includes an RNAi agent but does not include a targeting agent (eg, a GalNAc targeting compound) to treat xanthine in the subject.
  • a targeting agent eg, a GalNAc targeting compound
  • Purine dehydrogenase (XDH)-related diseases or conditions, such as gout such as gout.
  • RNAi delivery modalities may be used in conjunction with embodiments of the xanthine dehydrogenase (XDH) gene RNAi agents and therapeutic methods described herein, such as, but not limited to, those described herein. those and those used in the field.
  • XDH xanthine dehydrogenase
  • the xanthine dehydrogenase (XDH) dsRNA agent of the invention can be administered to a subject in an amount and in a manner effective to reduce the level and activity of the xanthine dehydrogenase (XDH) polypeptide in the cell and/or subject.
  • one or more xanthine dehydrogenase (XDH) dsRNA agents are administered to cells and/or subjects to treat conditions associated with xanthine dehydrogenase (XDH) expression and activity. Disease or illness.
  • methods of the invention include administering to a subject in need of such treatment one or more xanthine dehydrogenase (XDH) dsRNA agents to alleviate the symptoms associated with xanthine dehydrogenase (XDH) expression in the subject disease or illness.
  • XDH xanthine dehydrogenase
  • the xanthine dehydrogenase (XDH) dsRNA reagents or xanthine dehydrogenase (XDH) antisense polynucleotide reagents of the invention can be administered to reduce xanthine in one or more of cells in vitro, ex vivo, and in vivo. Purine dehydrogenase (XDH) expression and/or activity.
  • xanthine dehydrogenation in the cell is reduced by delivering (eg, introducing) a xanthine dehydrogenase (XDH) dsRNA agent or a xanthine dehydrogenase (XDH) antisense polynucleotide agent into the cell.
  • XDH xanthine dehydrogenase
  • XDH xanthine dehydrogenase
  • XDH xanthine dehydrogenase
  • XDH xanthine dehydrogenase
  • Targeting agents and methods can be used to facilitate the delivery of xanthine dehydrogenase (XDH) dsRNA agents or xanthine dehydrogenase (XDH) antisense polynucleotide agents to specific cell types, cell subtypes, organs, spaces within a subject regions, and/or subcellular regions within cells.
  • the xanthine dehydrogenase (XDH) dsRNA agent can be administered in certain methods of the invention alone or in combination with one or more additional xanthine dehydrogenase (XDH) dsRNA agents.
  • 2, 3, 4, or more independently selected xanthine dehydrogenase (XDH) dsRNA agents are administered to the subject.
  • the xanthine dehydrogenase (XDH) dsRNA agent is administered in combination with one or more additional treatment regimens for the treatment of xanthine dehydrogenase (XDH)-related diseases or conditions.
  • the subject is used to treat xanthine dehydrogenase (XDH) related diseases or conditions.
  • additional treatment regimens are: administration of one or more xanthine dehydrogenase (XDH) antisense polynucleotides of the invention, administration of non-xanthine dehydrogenase (XDH) dsRNA therapeutics, and actions Change.
  • Additional treatment regimens may be administered at one or more of the following times: before, simultaneously with, and after administration of the xanthine dehydrogenase (XDH) dsRNA agent of the invention.
  • XDH xanthine dehydrogenase
  • "at the same time” used in this article refers to zero time Within 5 minutes of time, 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, where “time zero” is when the xanthine dehydrogenation agent of the present invention is administered to the subject Enzyme (XDH) dsRNA reagent time.
  • Non-limiting examples of non-xanthine dehydrogenase (XDH) dsRNA therapeutics are: additional therapeutics such as allopurinol, hydroxypurine, febuxostat or interleukin-1 ⁇ (IL-1 ⁇ ) antagonists, preferably , the interleukin-1 ⁇ antagonist is selected from canakinumab or rilonacept, thereby treating a subject with a disease that would benefit from a reduction in xanthine dehydrogenase (XDH) expression, or any of the above Combinations, and pharmaceutical combinations formulated to treat 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 related to oxidative stress or other XDH-related diseases.
  • additional therapeutics such as allopurinol, hydroxypurine, febuxostat or
  • Non-limiting examples of behavior modification are: dietary programs, counseling, and exercise programs. These and other therapeutic agents and behavior modification agents are known in the art and may be used to treat xanthine dehydrogenase (XDH) diseases or conditions in a subject and may also be combined with one or more xanthine dehydrogenases of the present invention Enzyme (XDH) dsRNA agent combinations are administered to a subject to treat xanthine dehydrogenase (XDH) diseases or conditions.
  • XDH xanthine dehydrogenase
  • XDH xanthine dehydrogenase
  • a xanthine dehydrogenase (XDH) dsRNA agent of the invention that is administered to a cell or subject to treat a xanthine dehydrogenase (XDH)-related disease or disorder may be administered in a synergistic manner with one or more other therapeutic agents or active ingredients. Act to increase the effectiveness of one or more therapeutic agents or active ingredients and/or to increase the effectiveness of a xanthine dehydrogenase (XDH) dsRNA agent in treating a xanthine dehydrogenase (XDH)-related disease or condition.
  • the treatment methods of the present invention include the administration of a xanthine dehydrogenase (XDH) dsRNA agent, which may precede the onset of a xanthine dehydrogenase (XDH)-related disease or disorder and/or when a xanthine dehydrogenase (XDH)-related disease or condition is present or Use during the early, middle, late stages of a disease or condition, and at all times before and after any of these stages.
  • XDH xanthine dehydrogenase
  • the methods of the invention may also treat subjects who have been previously treated for xanthine dehydrogenase (XDH)-related diseases or conditions with one or more other therapeutic agents and/or therapeutic active ingredients, one or more of which Other therapeutic agents and/or therapeutically active ingredients are unsuccessful, minimally successful, and/or no longer successful in treating the xanthine dehydrogenase (XDH)-related disease or condition in the subject.
  • XDH xanthine dehydrogenase
  • a carrier can be used to deliver the xanthine dehydrogenase (XDH) dsRNA agent into a cell.
  • the xanthine dehydrogenase (XDH) dsRNA reagent transcription unit can be contained in a DNA or RNA vector.
  • the preparation and use of such transgene-encoding vectors for delivering sequences into cells and/or subjects is well known in the art.
  • Vectors that result in transient expression of xanthine dehydrogenase (XDH) dsRNA for example, for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 hours or more can be used in the methods of the invention. hours, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more weeks.
  • transient expression can be determined using conventional methods based on factors such as, but not limited to, the specific vector construct selected and the target cells and/or tissues.
  • transgenes 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).
  • One or more single strands of the xanthine dehydrogenase (XDH) dsRNA reagent can be transcribed from a promoter on an expression vector.
  • the two separate expression vectors can be co-introduced into the cell using, for example, transfection or infection.
  • each individual strand of the xanthine dehydrogenase (XDH) dsRNA agent of the invention can be transcribed from a promoter contained on the same expression vector.
  • 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 Stem-loop structure.
  • RNA expression vectors are DNA plasmids or viral vectors.
  • Expression vectors useful in embodiments of the invention may be compatible with eukaryotic cells. Eukaryotic expression vectors are routinely used in the art and are available from many commercial sources. Delivery of the xanthine dehydrogenase (XDH) dsRNA expression vector may be systemic, such as by intravenous or intramuscular administration, by administration to target cells removed from the subject and then reintroduced to the subject, or by allowing Any other means of introducing the desired target cells is performed.
  • XDH xanthine dehydrogenase
  • 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 virus vector; (d) Herpes simplex virus vector; (e) SV 40 vector; (f) Polyoma virus vector; (g) Papilloma virus vector; (h) Picornavirus vector; (i) ) poxvirus vectors, such as orthopoxvirus vectors, such as vaccinia virus vectors or fowlpox virus vectors, such as canarypox or fowlpox virus vectors; (j) helper-dependent or gutless adenovirus vectors.
  • adenoviral vectors include, but are not limited to: (a) adenoviral vectors; (b) retroviral vectors, including but not limited to lentiviral vector
  • Constructs for recombinant expression of xanthine dehydrogenase (XDH) dsRNA agents may contain regulatory elements such as promoters, enhancers, etc., which may be selected to provide constitutive or regulated/inducible expression.
  • regulatory elements such as promoters, enhancers, etc., which may be selected to provide constitutive or regulated/inducible expression.
  • Viral vector systems and the use of promoters and enhancers, etc. are routine in the art and can be used in conjunction with the methods and compositions described herein.
  • Certain embodiments of the invention include the use of viral vectors to deliver xanthine dehydrogenase (XDH) dsRNA agents into cells.
  • XDH xanthine dehydrogenase
  • a number of 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 may be used in the methods of the invention are: AAV vectors, poxviruses such as vaccinia virus, modified Ankara virus (MVA), NYVAC, fowlpox such as fowlpox or canarypox virus.
  • Certain embodiments of the invention include methods of delivering a xanthine dehydrogenase (XDH) dsRNA agent into a cell using a carrier, and such carrier may be in a pharmaceutically acceptable carrier, which may, but need not include, A sustained-release matrix embedded in a gene delivery vector.
  • a xanthine dehydrogenase (XDH) dsRNA agent into a cell using a carrier, and such carrier may be in a pharmaceutically acceptable carrier, which may, but need not include, A sustained-release matrix embedded in a gene delivery vector.
  • vectors for delivering xanthine dehydrogenase (XDH) dsRNA can be produced by recombinant cells, and pharmaceutical compositions of the invention can include one or more xanthine dehydrogenase (XDH)-producing dsRNA delivery system to cells.
  • Certain embodiments of the invention include the use of pharmaceutical compositions containing a xanthine dehydrogenase (XDH) dsRNA agent or a xanthine dehydrogenase (XDH) antisense polynucleotide agent and a pharmaceutically acceptable carrier.
  • XDH xanthine dehydrogenase
  • XDH xanthine dehydrogenase
  • compositions containing xanthine dehydrogenase (XDH) dsRNA agents or xanthine dehydrogenase (XDH) antisense polynucleotide agents It can be used in the methods of the present invention to reduce xanthine dehydrogenase (XDH) gene expression and xanthine dehydrogenase (XDH) activity in cells, and can be used to treat xanthine dehydrogenase (XDH)-related diseases or disorders.
  • Such pharmaceutical compositions can be formulated based on the mode of delivery.
  • Non-limiting examples of formulations for delivery modes are: compositions formulated for subcutaneous delivery, compositions formulated for systemic administration by parenteral delivery, compositions formulated for intravenous (IV) delivery, compositions formulated for Compositions for intrathecal delivery, compositions formulated for direct delivery into the brain, and the like.
  • compositions of the present invention may be administered using one or more means to deliver a xanthine dehydrogenase (XDH) dsRNA agent or a xanthine dehydrogenase (XDH) antisense polynucleotide agent into a cell, for example: Surface (e.g., via a transdermal patch); pulmonary, e.g., by inhalation or insufflation of a powder or aerosol, including via a nebulizer; intraairway, intranasal, epidermal and transdermal, oral or parenteral.
  • XDH xanthine dehydrogenase
  • XDH xanthine dehydrogenase
  • XDH xanthine dehydrogenase
  • Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subcutaneous, for example by an implanted device; or intracranial, for example by intraparenchymal; intrathecal or intraventricular administration.
  • the xanthine dehydrogenase (XDH) dsRNA agent or the xanthine dehydrogenase (XDH) antisense polynucleotide agent can also be delivered directly to the target tissue, such as directly to the liver and the like.
  • delivering a xanthine dehydrogenase (XDH) dsRNA agent” or "delivering a xanthine dehydrogenase (XDH) antisense polynucleotide agent” into a cell respectively includes delivering xanthine dehydrogenase (XDH) dsRNA reagents or xanthine dehydrogenase (XDH) antisense polynucleotide agents, expressing xanthine dehydrogenase (XDH) dsRNA reagents directly in cells, and expressing xanthine dehydrogenase (XDH) from encoding vectors delivered into cells ) dsRNA reagent, or any suitable means of causing the xanthine dehydrogenase (XDH) dsRNA or xanthine dehydrogenase (XDH) antisense polynucleotide reagent to appear in the cell.
  • 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 invention and a pharmaceutically acceptable carrier .
  • XDH xanthine dehydrogenase
  • pharmaceutically acceptable carrier refers to a carrier used to administer a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, glucose, water, glycerol, ethanol, and combinations thereof. This term specifically excludes cell culture media.
  • pharmaceutically acceptable carriers include, but are not limited to, pharmaceutically acceptable excipients, such as inert diluents, disintegrating agents, binders, lubricants, sweeteners, flavoring agents, and coloring agents. and preservatives.
  • pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binders, lubricants, sweeteners, flavoring agents, and coloring agents. and preservatives.
  • suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate and lactose, while cornstarch and alginic acid are suitable disintegrants.
  • Binders may include starch and gelatin, while the lubricant, if present, is usually magnesium stearate, stearic acid or talc.
  • the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate to delay absorption in the gastrointestinal tract.
  • Agents included in the pharmaceutical formulations are described further below.
  • terms such as “pharmacologically effective amount,””therapeutically effective amount,” and “effective amount” refer to the xanthine dehydrogenase (XDH) dsRNA agent or xanthine dehydrogenase (XDH) reagent of the invention.
  • XDH xanthine dehydrogenase
  • XDH xanthine dehydrogenase
  • a given clinical treatment is considered effective if it reduces a measurable parameter associated with the disease or disorder by at least 10%, then a therapeutically effective amount of a drug used to treat the disease or disorder is the amount that reduces that parameter by at least 10%. required amount.
  • a therapeutically effective amount of a xanthine dehydrogenase (XDH) dsRNA agent or a xanthine dehydrogenase (XDH) antisense polynucleotide agent can reduce xanthine dehydrogenase (XDH) polypeptide levels by at least 10%.
  • Pharmaceutical compositions may comprise 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.
  • preferred dsRNAi agents of Table 2 include, for example, duplexes AV00227, AV00260, AV00285, AV00266, or AV00280.
  • DSRNAI reagents in the preferred Table 3 include such as dual-chain AD00353, AD00356, AD00358, AD00364, AD00365, AD00366, AD00368, AD00370, AD00431, AD00521-1, AD00521-1, AD00521-1, AD00521-1, AD00521-1, AD00521-1, AD00521-1, AD00521-1, AD00521-1, AD00521-1,, AD00521-1, AD00521-1, AD00521-1, AD00521-1, AD00521-1,, AD00521-1, AD00521-1, AD00521-1, AD00521-1, AD00521-1, AD00521-1, AD00521-1, AD00521-1, AD00521-1, AD00521-1, AD00521-1, AD00521-1, AD00521-1, AD00521-1, AD00521-1, AD00521-1, AD00521-1, AD00521-1
  • methods of the 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 in the contacted cell Dehydrogenase (XDH) gene expression.
  • XDH xanthine dehydrogenase
  • XDH xanthine dehydrogenase
  • Certain embodiments of the methods of the present invention include administering xanthine dehydrogenase (XDH) to a subject in an amount effective to reduce xanthine dehydrogenase (XDH) gene expression and treat a xanthine dehydrogenase (XDH)-related disease or disorder in the subject )dsRNA reagent or xanthine dehydrogenase (XDH) antisense polynucleotide reagent.
  • the "effective amount" used is that which is necessary to achieve the desired biological effect or Sufficient amount.
  • an effective amount of a xanthine dehydrogenase (XDH) dsRNA agent or a xanthine dehydrogenase (XDH) antisense polynucleotide agent that treats a xanthine dehydrogenase (XDH)-related disease or disorder can be: (i) An amount required to slow or stop the progression of a disease or condition; (ii) reverse, reduce or eliminate one or more symptoms of a disease or condition.
  • an effective amount is xanthine dehydrogenase (XDH) that, when administered to a subject in need of treatment of a xanthine dehydrogenase (XDH)-related disease or disorder, results in a therapeutic response in the prevention and/or treatment of the disease or disorder.
  • XDH xanthine dehydrogenase
  • dsRNA reagent or xanthine dehydrogenase (XDH) antisense polynucleotide agent is xanthine dehydrogenase (XDH) that, when administered to a subject in need of treatment of a xanthine dehydrogenase (XDH)-related disease or disorder.
  • an effective amount is the xanthine dehydrogenase (XDH) dsRNA reagent or xanthine dehydrogenase of the invention
  • Enzyme (XDH) antisense polynucleotide agents when combined or co-administered with another therapeutic treatment for a disease or condition associated with xanthine dehydrogenase (XDH), result in a treatment for the prevention and/or treatment of the disease or condition The amount of response.
  • the biological effect in a subject treated with the xanthine dehydrogenase (XDH) dsRNA agent or xanthine dehydrogenase (XDH) antisense polynucleotide agent of the invention can be Improvement and/or complete elimination of symptoms caused by xanthine dehydrogenase (XDH)-related diseases or disorders.
  • the biological effect is a symptom of a xanthine dehydrogenase (XDH)-related disease or disorder. Complete elimination, for example, as demonstrated by a diagnostic test indicating that the subject is free of xanthine dehydrogenase (XDH)-related disease or disorder.
  • Non-limiting examples of detectable physiological symptoms include clinical signs of blood or urine changes in the subject following administration of an agent of the invention. Reduction in uric acid levels in the fluid.
  • Other methods known in the art for assessing xanthine dehydrogenase (XDH)-related diseases or condition states may be used to determine the effect of the reagents and/or methods of the present invention on xanthine dehydrogenase (XDH)-related diseases. or the effects of disease.
  • Xanthine dehydrogenase (XDH) dsRNA agents or xanthine dehydrogenase are generally determined in clinical trials to reduce xanthine dehydrogenase (XDH) polypeptide activity to levels that treat xanthine dehydrogenase (XDH)-related diseases or conditions.
  • Enzyme (XDH) antisense polynucleotide reagent such clinical trials establish effective doses for test and control populations in a blinded study.
  • an effective amount is an amount that results in a desired response, such as reduction of cells,
  • the amount of xanthine dehydrogenase (XDH) associated with a disease or disorder in a tissue and/or in a subject suffering from the disease or disorder Accordingly, xanthine dehydrogenase (XDH) dsRNA agents or xanthine dehydrogenase ( An effective amount of an ) the amount that would be present in the cell, tissue and/or subject in the case of a dsRNA agent or a xanthine dehydrogenase (XDH) antisense polynucleotide agent.
  • cells, tissues and/or cells that have not been exposed to or administered the xanthine dehydrogenase (XDH) dsRNA agent or xanthine dehydrogenase (XDH) antisense polynucleotide agent of the invention The level of xanthine dehydrogenase (XDH) polypeptide activity and/or xanthine dehydrogenase (XDH) gene expression in a subject is referred to as the "control" amount.
  • the subject's control amount is the subject's pre-treatment amount; in other words, the subject's level prior to administration of the xanthine dehydrogenase (XDH) agent can be the subject's control level and used to compare it to Comparison of xanthine dehydrogenase (XDH) polypeptide activity and/or xanthine dehydrogenase (XDH) gene expression levels following administration of siRNA to a subject.
  • the desired response may be to reduce or eliminate one or more symptoms of the disease or disorder in the cell, tissue, and/or subject. Reduction or elimination can be temporary or permanent.
  • xanthine dehydrogenase (XDH)-related diseases or conditions may be monitored using methods for determining xanthine dehydrogenase (XDH) polypeptide activity, xanthine dehydrogenase (XDH) gene expression, symptom assessment, clinical testing, etc. status.
  • a 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.
  • the effective amount of a compound that reduces the activity of a xanthine dehydrogenase (XDH) polypeptide can also be determined by evaluating administration of a xanthine dehydrogenase (XDH) dsRNA reagent or a xanthine dehydrogenase (XDH) antisense polynucleotide reagent.
  • XDH xanthine dehydrogenase
  • XDH xanthine dehydrogenase
  • XDH xanthine dehydrogenase
  • Assays and/or symptom monitoring in subjects can be used to determine the efficacy of the xanthine dehydrogenase (XDH) dsRNA agents or xanthine dehydrogenase (XDH) antisense polynucleotide agents of the invention (which can be used in pharmaceutical compounds of the invention administration) and determine whether there is a response to treatment.
  • XDH xanthine dehydrogenase
  • XDH xanthine dehydrogenase
  • XDH xanthine dehydrogenase
  • one or more uric acid level tests known in the art are used to determine the status of xanthine dehydrogenase (XDH)-related disease in a subject.
  • 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 related to oxidative stress or other XDH-related diseases, and this test is used to determine whether subjects are treated with the xanthine dehydrogenase (XDH) dsRNA agent of the invention Uric acid levels in subjects before and after.
  • NAFLD non-alcoholic fatty liver disease
  • NASH non-alcoholic steatohepatitis
  • GSD glycogen storage disease
  • XDH xanthine dehydrogenase
  • Some embodiments of the invention include determining the administration of a xanthine dehydrogenase (XDH) dsRNA agent or xanthine dehydrogenase (XDH) antisense polynucleotide agent of the invention to a subject to treat xanthine dehydrogenase (XDH) Methods of treating a disease or disorder by assessing and/or monitoring one or more "physiological characteristics" of a xanthine dehydrogenase (XDH)-related disease or disorder in a subject.
  • XDH xanthine dehydrogenase
  • XDH xanthine dehydrogenase
  • Xanthine dehydrogenase (XDH) related diseases or Non-limiting examples of physiological features of the condition are that many patients also experience elevated levels of uric acid in the blood and urine, increased pain (such as that caused by gout), and increased risk of inflammatory events. Standard methods for determining this physiological profile are known in the art and include, but are not limited to, blood tests, imaging studies, physical examinations, etc.
  • the syndrome is defined by the presence of decreased uric acid levels in the blood and urine, decreased pain (such as pain caused by gout), reduction of inflammation.
  • the xanthine dehydrogenase (XDH) dsRNA agent or the Amount of hydrogenase (XDH) antisense polynucleotide agent can be varied, for example, by varying the composition of the xanthine dehydrogenase (XDH) dsRNA agent or xanthine dehydrogenase (XDH) antisense polynucleotide agent administered, by varying the route of administration, by varying the administration You can increase or decrease the amount of xanthine dehydrogenase (XDH) dsRNA reagent or xanthine dehydrogenase (XDH) antisense polynucleotide reagent according to the medication time, etc.
  • the effective amount of the xanthine dehydrogenase (XDH) dsRNA agent or the 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, and 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 health practitioner's knowledge and expertise.
  • the effective amount may depend on the desired level of xanthine dehydrogenase (XDH) polypeptide activity and/or xanthine dehydrogenase (XDH) gene expression effective in treating xanthine dehydrogenase (XDH)-related diseases or conditions.
  • XDH xanthine dehydrogenase
  • the skilled artisan can empirically determine the effective amount of a particular xanthine dehydrogenase (XDH) dsRNA reagent or xanthine dehydrogenase (XDH) antisense polynucleotide reagent for use in the methods of the invention without undue experimentation.
  • XDH xanthine dehydrogenase
  • XDH xanthine dehydrogenase
  • an effective amount of the xanthine dehydrogenase (XDH) dsRNA agent or xanthine dehydrogenase (XDH) antisense polynucleotide agent of the invention can be The amount in cells that produces the desired biological effect.
  • xanthine dehydrogenase (XDH) gene silencing can be performed constitutively or by genome engineering in any cell expressing xanthine dehydrogenase (XDH) and determined by any suitable assay.
  • xanthine dehydrogenase (XDH) dsRNA agent of the invention by administering the xanthine dehydrogenase (XDH) dsRNA agent 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%.
  • xanthine dehydrogenase (XDH) dsRNA agent of the invention by administering the xanthine dehydrogenase (XDH) dsRNA agent 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%.
  • xanthine dehydrogenase (XDH) dsRNA agent and the xanthine dehydrogenase (XDH) antisense polynucleotide agent are delivered in a pharmaceutical composition in a dose sufficient to inhibit xanthine dehydrogenase (XDH) gene expression.
  • the xanthine dehydrogenase (XDH) dsRNA agent or the xanthine dehydrogenase (XDH) antisense polynucleoside The dose of acid 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 per day. /kg body weight, 4 to 15 mg/kg body weight, inclusive.
  • each single administration of a xanthine dehydrogenase (XDH) dsRNA agent or a xanthine dehydrogenase (XDH) antisense polynucleotide agent can be administered in a dosage ranging from about 0.01 mg/kg, 0.05 mg/kg, 0.1 mg/ kg, 0.2mg/kg, 0.3mg/kg, 0.4mg/kg, 0.5mg/kg, 1mg/kg, 1.1mg/kg, 1.2mg/kg, 1.3mg/kg, 1.4mg/kg, 1.5mg/kg , 1.6mg/kg, 1.7mg/kg, 1.8mg/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, 3.0m
  • xanthine dehydrogenase (XDH) dsRNA agent of the invention Various factors can be considered in determining the dosage and timing of delivery of the xanthine dehydrogenase (XDH) dsRNA agent of the invention.
  • the absolute amount of xanthine dehydrogenase (XDH) dsRNA agent or xanthine dehydrogenase (XDH) antisense polynucleotide agent delivered will depend on a variety of factors, including co-treatment, number of doses, and individual subject parameters, including age. , physical condition, size and weight. These factors are well known to those of ordinary skill in the art and can be addressed through routine experimentation.
  • the maximum dose may be used, ie, the highest safe dose based on sound medical judgment.
  • methods of the invention may comprise administering to a subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses of a xanthine dehydrogenase (XDH) dsRNA agent or xanthine dehydrogenase (XDH) antisense polynucleotide reagent.
  • a pharmaceutical compound e.g., a dsRNA agent containing xanthine dehydrogenase (XDH) or a dsRNA agent containing xanthine dehydrogenase
  • the dosage of the antisense polynucleotide agent may be administered once per day or more than once per day, for example, 2, 3, 4, 5 or more times in a 24-hour period.
  • the pharmaceutical composition of the present invention can be administered once a day; or the xanthine dehydrogenase (XDH) dsRNA reagent or the xanthine dehydrogenase (XDH) antisense polynucleotide reagent can be administered twice a day at appropriate intervals, Three or more subdoses, or even using consecutive Infused or delivered via controlled release formulation.
  • a pharmaceutical composition of the invention is administered to the subject once or more daily, once or more weekly, once or more monthly, or once or more annually.
  • methods of the 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 pharmacotherapies or therapeutic activities or regimens administered to subjects suffering from xanthine dehydrogenase (XDH)-related diseases or conditions.
  • Pharmaceutical compounds can be administered in the form of pharmaceutical compositions.
  • the pharmaceutical composition used in the method of the present invention can be sterile and contain an amount of xanthine dehydrogenase (XDH) dsRNA reagent or xanthine dehydrogenase (XDH) antisense polynucleotide reagent, which will The activity of the purine dehydrogenase (XDH) polypeptide is reduced to a level sufficient to produce a desired response in a unit of weight or volume suitable for administration to a subject.
  • XDH xanthine dehydrogenase
  • XDH purine dehydrogenase
  • the dosage of a 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) can be selected based on different parameters.
  • the activity of the protein is particularly selected based on the mode of administration used and the state of the subject. Other factors include the length of treatment required. If the subject's response at the initial dose is insufficient, a higher dose may be used (or the dose may be effectively increased via a different, more local delivery route) as tolerated by the patient.
  • a related disease, disorder, or disorder, or a reduction in the development of symptoms associated with such disease, disorder, or disorder (e.g., a decrease of at least about 10% on a scale of clinically having the disease or disorder), or a delay in symptoms (e.g., delay of days, weeks, months, or years) is considered effective prevention.
  • XDH xanthine dehydrogenase
  • XDH xanthine dehydrogenase
  • the methods of the present invention and xanthine dehydrogenation may be used.
  • Hydrogenase (XDH) dsRNA reagent was used to inhibit xanthine dehydrogenase (XDH) expression.
  • diseases and conditions that may be treated with the xanthine dehydrogenase (XDH) dsRNA reagents or xanthine dehydrogenase (XDH) antisense polynucleotide reagents of the invention and the treatment methods of the 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 GSD type I and diseases related to oxidative stress, For example, chronic low-grade inflammation; or other XDH-related diseases.
  • GSD glycogen storage diseases
  • xanthine dehydrogenase (XDH)-related diseases and conditions and “diseases and conditions caused and/or modulated by xanthine dehydrogenase (XDH).
  • the xanthine dehydrogenase (XDH)-related disease or condition can be diagnosed before or The subject is administered a xanthine dehydrogenase (XDH) dsRNA agent or xanthine dehydrogenase (XDH) antisense polynucleotide agent of the invention at one or more times thereafter.
  • the subject is at risk of suffering from or developing a xanthine dehydrogenase (XDH)-related disease or disorder.
  • a subject at risk of developing a xanthine dehydrogenase (XDH)-related disease or condition is a subject who is at risk of developing a xanthine dehydrogenase (XDH)-related disease or condition compared to a control risk of developing a xanthine dehydrogenase (XDH)-related disease or condition.
  • the level of risk is statistically significant compared to a control level of risk.
  • a subject at risk may include, for example, a subject who is or will be a subject with a pre-existing disease and/or genetic abnormality that renders the subject more susceptible to xanthine dehydrogenase ( XDH)-related diseases or conditions; subjects with a family and/or personal history of xanthine dehydrogenase (XDH)-related diseases or conditions; and subjects who have been previously treated for xanthine dehydrogenase (XDH)-related diseases or conditions.
  • XDH xanthine dehydrogenase
  • a pre-existing disease and/or genetic abnormality that predisposes a subject to a xanthine dehydrogenase (XDH)-related disease or disorder may be a disease or genetic abnormality that, when present, has been previously identified as being associated with There is an association with a higher likelihood of developing xanthine dehydrogenase (XDH)-related diseases or conditions.
  • a xanthine dehydrogenase (XDH) dsRNA agent or a xanthine dehydrogenase (XDH) antisense polynucleotide agent can be administered to an individual subject based on the medical condition of the individual subject. For example, health care provided to a subject may assess xanthine dehydrogenase (XDH) levels measured in a sample obtained from the subject and determine whether the xanthine dehydrogenase (XDH) dsRNA reagent or It is desirable to use antisense polynucleotide reagents to reduce xanthine dehydrogenase (XDH) levels in a subject.
  • XDH xanthine dehydrogenase
  • XDH xanthine dehydrogenase
  • a biological sample such as a blood or serum sample
  • a biological sample can be obtained from a subject and the subject's uric acid level determined in the sample.
  • administering a xanthine dehydrogenase (XDH) dsRNA reagent or a xanthine dehydrogenase (XDH) antisense polynucleotide reagent to a subject, and obtaining a blood or serum sample from the subject after administration, and using the sample to determine uric acid levels, The results are compared to those determined in the subject's pre-dose (previous) sample.
  • XDH xanthine dehydrogenase
  • XDH xanthine dehydrogenase
  • a decrease in the subject's uric acid levels in subsequent samples compared to pre-dose levels indicates that the administered xanthine dehydrogenase (XDH) dsRNA reagent or Efficacy in terms of xanthine dehydrogenase (XDH) levels.
  • elevated uric acid levels may be considered a physiological signature of a xanthine dehydrogenase (XDH)-related disorder, even if the subject has not been diagnosed with a xanthine dehydrogenase (XDH)-related disorder, such as herein Public disease.
  • a healthcare provider can monitor changes in a subject's uric acid levels as a measure of the efficacy of an administered xanthine dehydrogenase (XDH) dsRNA agent or xanthine dehydrogenase (XDH) antisense polynucleotide agent of the invention.
  • xanthine dehydrogenase (XDH) related diseases are hyperuricemia, gout, NAFLD, NASH, metabolic disorders, insulin resistance, cardiovascular disease, type 2 diabetes, Lesch Nyhan syndrome, glucose Primary storage diseases (GSD), such as GSD type I and diseases related to oxidative stress, such as chronic low-grade inflammation; or other XDH-related diseases.
  • Certain embodiments of the methods of the present invention include modifying a treatment based, at least in part, on changes in one or more physiological characteristics of a xanthine dehydrogenase (XDH)-related disease or disorder in a subject caused by the treatment Evaluation, to administer to a subject a dsRNA agent or a xanthine dehydrogenase (XDH) antisense polynucleotide agent of the invention.
  • XDH xanthine dehydrogenase
  • purine dehydrogenase (XDH) antisense polynucleotide agent is used to help regulate the amount of a dsRNA agent or xanthine dehydrogenase (XDH) antisense polynucleotide agent of the invention that is subsequently administered to a subject.
  • a dsRNA agent or a 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, Determine whether higher amounts of dsRNA agent or xanthine dehydrogenase (XDH) antisense polynucleotide agent are needed to enhance the physiological effects of the administered agent, such as reducing or further reducing the subject's uric acid levels.
  • XDH xanthine dehydrogenase
  • a dsRNA agent or a xanthine dehydrogenase (XDH) antisense polynucleotide agent of the invention is administered to a subject, and the subject's uric acid level is determined following administration, and is based at least in part on the At the determined levels, it is expected that subjects will be administered lower amounts of dsRNA agent or xanthine dehydrogenase (XDH) antisense polynucleotide agent.
  • some embodiments of the invention include assessment of changes in one or more physiological characteristics resulting from prior treatment of the subject, including reductions in uric acid levels in blood and urine, reduction in pain (e.g., pain caused by gout), reduction of inflammation, etc., to adjust the amount of the dsRNA agent or xanthine dehydrogenase (XDH) antisense polynucleotide agent of the invention subsequently administered to the subject.
  • changes in one or more physiological characteristics resulting from prior treatment of the subject including reductions in uric acid levels in blood and urine, reduction in pain (e.g., pain caused by gout), reduction of inflammation, etc.
  • Some embodiments of the methods of the present invention include 1, 2, 3, 4, 5, 6, or more determinations of physiological characteristics of xanthine dehydrogenase (XDH)-related diseases or disorders; assessing and/or monitoring administered The efficacy of the xanthine dehydrogenase (XDH) dsRNA reagent or xanthine dehydrogenase (XDH) antisense polynucleotide reagent of the invention; and optionally using the results of the assay to adjust one or more of the following: Dosage, dosing regimen and/or dosing frequency of the inventive dsRNA reagent or xanthine dehydrogenase (XDH) antisense polynucleotide reagent for treating xanthine dehydrogenase (XDH) related diseases or disorders in a subject.
  • XDH xanthine dehydrogenase
  • the desired outcome of administering to a subject an effective amount of a dsRNA agent or a xanthine dehydrogenase (XDH) antisense polynucleotide agent of the invention is: compared to previously determined for the subject, Subjects were reduced xanthine dehydrogenase (XDH) transcript levels, plasma uric acid levels, and xanthine dehydrogenase (XDH) gene expression.
  • XDH xanthine dehydrogenase
  • treatment when used in reference to a xanthine dehydrogenase (XDH)-related disease or condition may refer to preventive treatment, reduction of a subject's development of ), and may also refer to the elimination or reduction of the level of a xanthine dehydrogenase (XDH)-related disease or condition after the subject has developed the xanthine dehydrogenase (XDH)-related disease or condition.
  • XDH xanthine dehydrogenase
  • XDH xanthine dehydrogenase
  • Certain embodiments of the agents, compositions, and methods of the invention can be used to inhibit xanthine dehydrogenase (XDH) gene expression.
  • XDH xanthine dehydrogenase
  • the terms “inhibit”, “silence”, “reduce”, “down-regulate” and “knock-down” mean, for example, by one or more of the following Change the expression of the xanthine dehydrogenase (XDH) gene: respectively compared with the control level of RNA transcribed by the xanthine dehydrogenase (XDH) gene, the control level of the expressed xanthine dehydrogenase (XDH) activity, or by the mRNA Control levels of translated xanthine dehydrogenase (XDH) when a cell, cell population, tissue, organ or subject is treated with a The level of RNA transcribed from the gene, the level of xanthine dehydrogenase
  • control level is a cell, tissue, organ or subject that has not been exposed to (e.g., treated with) a xanthine dehydrogenase (XDH) dsRNA agent or a xanthine dehydrogenase (XDH) antisense polynucleotide agent. medium level.
  • XDH xanthine dehydrogenase
  • XDH xanthine dehydrogenase
  • XDH xanthine dehydrogenase
  • XDH xanthine dehydrogenase
  • XDH xanthine dehydrogenase
  • antisense polynucleotide agents can be used in the methods of the invention.
  • the selection of a particular delivery mode will depend, at least in part, on the specific condition being treated and the dosage required for therapeutic efficacy.
  • the methods of the present invention may be practiced using any mode of administration that is medically acceptable, meaning that producing effective therapeutic levels of xanthine dehydrogenase (XDH)-related diseases or conditions without causing clinically unacceptable Any pattern of side effects.
  • the xanthine dehydrogenase (XDH) dsRNA agent or the xanthine dehydrogenase (XDH) antisense polynucleotide agent can be administered by oral, enteral, mucosal, subcutaneous, and/or parenteral routes Apply.
  • parenteral includes subcutaneous, intravenous, intrathecal, intramuscular, intraperitoneal and intrasternal injection or infusion techniques.
  • Other routes include, but are not limited to, nasal (eg, via gastronasal tube), transdermal, vaginal, rectal, sublingual, and inhalation. Delivery routes of the present invention may include intrathecal, intraventricular, or intracranial.
  • a xanthine dehydrogenase (XDH) dsRNA agent or a xanthine dehydrogenase (XDH) antisense polynucleotide agent can be placed in a sustained release matrix and administered by placing the matrix in a subject. Apply.
  • a xanthine dehydrogenase (XDH) dsRNA agent or a xanthine dehydrogenase (XDH) antisense polynucleotide agent can be delivered using nanoparticles coated with a delivery agent that targets a specific cell or organelle. to the target cell.
  • a variety of delivery modes, methods, and reagents are known in the art.
  • delivery methods and delivery agents are provided elsewhere herein.
  • the term "delivery" with respect to a xanthine dehydrogenase (XDH) dsRNA agent or a xanthine dehydrogenase (XDH) antisense polynucleotide agent may refer to administering to a cell or subject a or More "naked” xanthine dehydrogenase (XDH) dsRNA reagents or xanthine dehydrogenase (XDH) antisense polynucleotide agent sequences.
  • delivery refers to administering to a cell or subject by transfection a substance containing a xanthine dehydrogenase (XDH) dsRNA reagent or a xanthine dehydrogenase (XDH) antisense polynucleotide reagent.
  • XDH xanthine dehydrogenase
  • XDH xanthine dehydrogenase
  • XDH xanthine dehydrogenase
  • Delivery of a xanthine dehydrogenase (XDH) dsRNA agent or a xanthine dehydrogenase (XDH) antisense polynucleotide agent using transfection may include administering a vector to a cell and/or subject.
  • XDH xanthine dehydrogenase
  • XDH xanthine dehydrogenase
  • one or more xanthine dehydrogenase (XDH) dsRNA reagents or xanthine dehydrogenase (XDH) antisense polynucleotide reagents can be administered in the form of a preparation, or can be administered in a pharmaceutical It is administered in an acceptable solution, which may generally contain pharmaceutically acceptable concentrations of salts, buffers, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients.
  • a xanthine dehydrogenase (XDH) dsRNA agent or a xanthine dehydrogenase (XDH) antisense polynucleotide agent can be formulated with another therapeutic agent for simultaneous administration.
  • the xanthine dehydrogenase (XDH) dsRNA agent or the xanthine dehydrogenase (XDH) antisense polynucleotide agent can be administered in the form of a pharmaceutical composition.
  • compositions typically contain yellow Purine dehydrogenase (XDH) dsRNA agent or xanthine dehydrogenase (XDH) antisense polynucleotide agent and optional pharmaceutically acceptable carrier.
  • XDH yellow Purine dehydrogenase
  • XDH xanthine dehydrogenase
  • XDH xanthine dehydrogenase
  • pharmaceutically acceptable carriers are well known to those of ordinary skill in the art.
  • a pharmaceutically acceptable carrier refers to a cell or subject that does not interfere with the biological activity of the active ingredient (e.g., a xanthine dehydrogenase (XDH) dsRNA agent or a xanthine dehydrogenase (XDH) antisense polynucleotide agent).
  • XDH xanthine dehydrogenase
  • XDH xanthine dehydrogenase
  • XDH xanthine dehydrogenase
  • 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, and other carriers are known to those skilled in the art. Such preparations may generally 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, which 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, propionic acid. Diacid, succinic acid, etc.
  • pharmaceutically acceptable salts may be prepared as alkali metal salts or alkaline earth metal salts, such as sodium, potassium or calcium salts.
  • Some embodiments of the methods of the invention include administering one or more xanthine dehydrogenase (XDH) dsRNA reagents or xanthine dehydrogenase (XDH) antisense polynucleotide reagents directly to the tissue.
  • the tissue to which the compound is administered is a tissue in which a xanthine dehydrogenase (XDH)-related disease or disorder is present or likely to occur, non-limiting examples of which are the liver or kidneys.
  • Direct tissue drug delivery can be achieved by direct injection or other means.
  • xanthine dehydrogenase (XDH) dsRNA agents include orally administering to a subject one or more xanthine dehydrogenase (XDH) dsRNA agents.
  • the xanthine dehydrogenase (XDH) dsRNA agent or the xanthine dehydrogenase (XDH) antisense polynucleotide agent can be administered by different routes.
  • the first (or first few) administrations may be administered subcutaneously, and one or more additional administrations may be oral and/or systemic.
  • xanthine dehydrogenase (XDH) dsRNA agent or a xanthine dehydrogenase (XDH) antisense polynucleotide agent for embodiments of the invention in which it is desired to systemically administer a xanthine dehydrogenase (XDH) dsRNA agent or a xanthine dehydrogenase (XDH) antisense polynucleotide agent
  • the xanthine dehydrogenase (XDH) dsRNA agent or Xanthine dehydrogenase (XDH) antisense polynucleotide agents are used for parenteral administration by injection, such as by bolus injection or continuous infusion.
  • injectable preparations may be presented in unit dosage form such as ampoules or multi-dose containers, with or without added preservatives.
  • Xanthine dehydrogenase (XDH) dsRNA reagent preparations can take the form of suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulations such as suspensions, stabilizers and/or dispersants.
  • Formulations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions.
  • 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, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Lin Grignard's dextrose solution, glucose and sodium chloride solution, lactated Ringer's solution or fixed oil.
  • Intravenous excipients include fluid and nutritional supplements, electrolyte supplements (such as those based on Ringer's dextrose solution), and the like. Preservatives and other additives such as antimicrobials, antioxidants, chelating agents, inert gases, etc. may also be present. Other forms of administration, such as intravenous administration, will result in lower doses. If the subject's response at the initial dose is insufficient, a higher dose may be used (or the dose may be effectively increased via a different, more local delivery route) as tolerated by the patient.
  • Multiple daily doses may 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 of xanthine dehydrogenase (XDH) activity.
  • XDH xanthine dehydrogenase
  • XDH xanthine dehydrogenase
  • XDH xanthine dehydrogenase
  • the methods of the present invention include the use of a delivery vehicle, such as a biocompatible microparticle, nanoparticle, or implant suitable for implantation in a recipient, such as a subject.
  • a delivery vehicle such as a biocompatible microparticle, nanoparticle, or implant suitable for implantation in a recipient, such as a subject.
  • exemplary biodegradable implants that may be used according to this method are described in PCT Publication WO 95/24929 (incorporated herein by reference), which describes biocompatible, biodegradable implants containing biological macromolecules. Polymer matrix.
  • both non-biodegradable and biodegradable polymeric matrices can be used in the methods of the invention to combine one or more xanthine dehydrogenase (XDH) dsRNA reagents or xanthine dehydrogenase (XDH) antisense Delivery of polynucleotide reagents to subjects.
  • the matrix can be biodegradable.
  • Matrix polymers can be natural or synthetic polymers. The polymer can be selected based on the period of time for which release is desired, typically on the order of a few hours to a year or more. Typically, releases for periods ranging from a few hours to three to twelve months are available.
  • the polymer is optionally in the form of a hydrogel, which can absorb up to about 90% of its weight in water, and is optionally also cross-linked with multivalent ions or other polymers.
  • xanthine dehydrogenase (XDH) dsRNA reagents or xanthine dehydrogenase (XDH) antisense polynucleotide reagents may be used in some embodiments of the invention via diffusion or through polymers using biodegradable implants. delivery through degradation of the matrix. Exemplary synthetic polymers for this purpose are well known in the art. Biodegradable polymers and non-biodegradable polymers can be used to deliver xanthine dehydrogenase (XDH) dsRNA agents or xanthine dehydrogenase (XDH) antisense polynucleotide agents using methods known in the art.
  • Bioadhesive polymers such as bioerodible hydrogels (H.S. Sawhney, C.P. Pathak and J.A. Hubell in Macromolecules, 1993, 26, 581-587) can also be used to deliver xanthine dehydrogenase (XDH) dsRNA reagents or xanthine dehydrogenase (XDH) antisense polynucleotide reagents for the treatment of xanthine dehydrogenase (XDH)-related diseases or conditions.
  • XDH xanthine dehydrogenase
  • Other suitable delivery systems may include timed release, delayed release, or sustained release delivery systems.
  • Such systems may avoid repeated administration of xanthine dehydrogenase (XDH) dsRNA reagents or xanthine dehydrogenase (XDH) antisense polynucleotide agents, thereby improving convenience for subjects and healthcare professionals.
  • XDH xanthine dehydrogenase
  • 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.
  • pump-based hardware delivery systems are available, some of which are also suitable for implantation.
  • long-term sustained release implants may be suitable for prophylactic treatment of subjects and subjects at risk of developing recurrent xanthine dehydrogenase (XDH)-related diseases or conditions.
  • long-term release means that the implant is constructed and arranged to release for at least 10 days, 20 days, 30 days, 60 days, 90 days, six months, one year, or more Deliver therapeutic levels of xanthine dehydrogenase (XDH) dsRNA reagents or xanthine dehydrogenase (XDH) antisense polynucleotide reagents.
  • Long-term sustained release implants are well known to those of ordinary skill in the art and include some of the release systems described above.
  • Therapeutic formulations of xanthine dehydrogenase (XDH) dsRNA reagents or xanthine dehydrogenase (XDH) antisense polynucleotide reagents can be obtained by combining the molecule or compound with the desired purity and optionally a pharmaceutically acceptable carrier, Excipients or stabilizers [Remington's Pharmaceutical Sciences 21st edition, (2006)] are prepared for storage by mixing in the form of lyophilized formulations or aqueous solutions.
  • Acceptable carriers, excipients or stabilizers are non-toxic to the recipient at the doses and concentrations employed and include buffering agents such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and Methionine; preservatives (e.g. stearyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butanol or benzyl alcohol; parabens, e.g.
  • Methyl or propylparaben e.g., catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, Examples include serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; simple sugars, Disaccharides and other carbohydrates, including glucose, mannose or dextrin; 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, e.g. or polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the methods of the invention can be used in conjunction with cells, tissues, organs and/or subjects.
  • the subject is a human or vertebrate mammal, including but not limited to dogs, cats, horses, cattle, goats, mice, rats, and primates, such as monkeys.
  • the present invention may be used to treat xanthine dehydrogenase (XDH)-related diseases or conditions in humans and non-human subjects.
  • the subject may be a farm animal, a zoo animal, a domesticated animal, or a non-domesticated animal, and the methods of the invention may be used in veterinary preventive and therapeutic regimens.
  • the subject is a human and the methods of the invention can be used in human prophylactic and therapeutic regimens.
  • Non-limiting examples of subjects to which the present invention may be applied are subjects diagnosed with, suspected of having, or at risk of having a disease or condition associated with: Greater than desired xanthine dehydrogenase (XDH) Expression and/or activity, also known as “elevated xanthine dehydrogenase (XDH) expression levels.”
  • XDH xanthine dehydrogenase
  • 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 may be applied to subjects who have been diagnosed with the disease or condition at the time of treatment, are associated with higher than expected xanthine dehydrogenase (XDH) expression and/or activity, or are thought to be suffering from the disease or condition.
  • the disease or condition associated with higher than desired xanthine dehydrogenase (XDH) expression and/or activity levels is an acute disease or condition; Diseases or conditions associated with xanthine dehydrogenase (XDH) expression and/or activity levels are chronic diseases or conditions.
  • the xanthine dehydrogenase (XDH) dsRNA reagent of the invention is administered to Diagnosed with XDH-related diseases, 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 GSD type I and diseases related to oxidative stress, such as chronic low-grade inflammation; or other XDH-related diseases.
  • GSD glycogen storage disease
  • the methods of the present invention may be applied to subjects who, at the time of treatment, have been diagnosed as having the disease or condition, or who are considered to be at risk of developing or developing the disease or condition.
  • a xanthine dehydrogenase (XDH) dsRNA agent of the invention is administered to treat a disease or disorder caused by or associated with activation of xanthine dehydrogenase (XDH), or a symptom thereof or a disease or disorder that progresses in response to inactivation of xanthine dehydrogenase (XDH).
  • XDH xanthine dehydrogenase
  • 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 diseases (GSD), such as I Type GSD and diseases related to oxidative stress, such as chronic low-grade inflammation; or other XDH-related diseases.
  • GSD glycogen storage diseases
  • Cells to which the method of the present invention can be applied include in vitro, in vivo, and ex vivo cells.
  • the cells may be in a subject, in culture and/or in suspension, or in any other suitable state or condition.
  • the cells to which the method of the present invention can be applied may be: liver cells, hepatocytes, heart cells, pancreatic cells, cardiovascular cells, kidney cells or other types of vertebrate cells, including human and non-human mammalian cells. animal cells.
  • cells to which the methods of the invention can be applied are healthy normal cells that are not known to be disease cells.
  • control cells are normal cells, but it should be understood that cells having a disease or disorder may also be used as control cells in certain circumstances, such as when comparing treated cells having a disease or disorder to cells having a disease or disorder. In the case of other diseases that result from untreated cells.
  • 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 many forms. It can be a single cutoff value, such as the median or mean.
  • comparison group for example in a group with normal levels of xanthine dehydrogenase (XDH) polypeptide and/or xanthine dehydrogenase (XDH) polypeptide activity and with increased xanthine dehydrogenase (XDH) polypeptide and/or xanthine dehydrogenase (XDH) polypeptide activity level in the group.
  • XDH xanthine dehydrogenase
  • XDH xanthine dehydrogenase
  • Another non-limiting example of a comparison group may be a population with one or more symptoms or diagnosis of a xanthine dehydrogenase (XDH) related disease or disorder vs.
  • controls can be based on apparently healthy normal individuals or apparently healthy cells in an appropriate age group. It will be understood that, in addition to predetermined values, a control according to the present invention may be a material sample tested in parallel with the experimental material. Examples include samples from control populations or control samples generated through manufacturing for parallel testing with experimental samples.
  • controls may include cells or subjects that have not been contacted or treated with the xanthine dehydrogenase (XDH) dsRNA reagents of the invention, in which case the xanthine dehydrogenase (XDH) dsRNA reagents of the invention may be compared ) polypeptide and/or xanthine dehydrogenase (XDH) polypeptide activity control levels and contact with the xanthine dehydrogenase (XDH) dsRNA reagent or xanthine dehydrogenase (XDH) antisense polynucleotide reagent of the invention in a cell or object Levels of xanthine dehydrogenase (XDH) polypeptide and/or xanthine dehydrogenase (XDH) polypeptide activity.
  • XDH xanthine dehydrogenase
  • control level may be a xanthine dehydrogenase (XDH) polypeptide level determined for a subject, wherein a xanthine dehydrogenase (XDH) polypeptide level determined for the same subject at a different time is compared with the xanthine dehydrogenase (XDH) polypeptide level determined for the same subject at a different time. Compare to control levels.
  • XDH xanthine dehydrogenase
  • XDH xanthine dehydrogenase
  • XDH xanthine dehydrogenase
  • the biological sample is a serum sample.
  • the uric acid level measured in the sample obtained from the subject may serve as the subject's baseline or control value.
  • a xanthine dehydrogenase (XDH) dsRNA agent to a subject in a treatment method of the invention
  • one or more additional serum samples can be obtained from the subject, and the subsequent one or more
  • the uric acid levels in each sample are compared to the subject's control/baseline levels. Such comparisons may be used to evaluate the onset, progression, or regression of xanthine dehydrogenase (XDH)-related diseases or conditions in a subject.
  • the level of a xanthine dehydrogenase (XDH) polypeptide in a baseline sample obtained from a subject is higher than when the subject is administered a xanthine dehydrogenase (XDH) dsRNA agent or a xanthine dehydrogenase (XDH) antisense polypeptide of the invention.
  • XDH xanthine dehydrogenase
  • Levels obtained from the same subject after glucuronide reagent are indicative of resolution of the xanthine dehydrogenase (XDH)-related disease or disorder and are indicative of administration of the xanthine dehydrogenase (XDH) dsRNA reagent of the invention to treat xanthine dehydrogenase (XDH) Effects caused by related diseases or conditions.
  • the biological sample is a hepatocyte sample.
  • One or more of the xanthine dehydrogenase (XDH) polypeptide and/or xanthine dehydrogenase (XDH) polypeptide activity levels determined for a subject may serve as a control value and be used to later compare xanthine dehydrogenase (XDH) polypeptide activity in the same subject.
  • Purine dehydrogenase (XDH) polypeptide and/or xanthine dehydrogenase (XDH) activity levels thereby allowing assessment of changes in "baseline” xanthine dehydrogenase (XDH) polypeptide activity in a subject.
  • the initial xanthine dehydrogenase (XDH) polypeptide level and/or the initial xanthine dehydrogenase (XDH) polypeptide activity level may be used to display and/or determine where the initial level is used as a control level for the subject.
  • the methods and compounds of the invention are capable of reducing the level of xanthine dehydrogenase (XDH) polypeptide and/or xanthine dehydrogenase (XDH) polypeptide activity in a subject.
  • a xanthine dehydrogenase (XDH) dsRNA agent and/or a xanthine dehydrogenase (XDH) antisense polynucleotide agent of the invention can be administered to a subject.
  • dsRNAi agents include, for example, duplexes AV00206 to AV00292 shown in Table 1.
  • preferred dsRNAi agents include, for example, duplexes AV00227, AV00260, AV00285, AV00266, AV00280, AV00238, AV00249, AV00289, AV00233, AV00225, AV00273, or AV00283.
  • preferred dsRNAi agents of Table 2 include, for example, duplexes AV00227, AV00260, AV00285, AV00266, or AV00280.
  • preferred dsRNAi agents in Table 3 include, for example, duplexes AD00353, AD00356, AD00358, AD00364, AD00365, AD00366, AD00368, AD00370, AD00430, AD00431, AD00521, AD00521-1, AD00364-1, AD0036 5 -2, AD00366-1 and AD00432-1.
  • the efficacy of administration and treatment of the present invention may be assessed as compared to pre-dose levels of uric acid in serum samples obtained from the subject at previous time points, or compared to non-exposure control levels (e.g., uric acid levels in control serum samples) , when administered and treated, the level of uric acid in serum samples obtained from the subject is reduced by at least 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80 %, 90%, 95% or more. It should be understood that the level of uric acid is related to the level of xanthine dehydrogenase (XDH) gene expression.
  • XDH xanthine dehydrogenase
  • Certain embodiments of the methods of the invention Including administering the xanthine dehydrogenase (XDH) dsRNA and/or xanthine dehydrogenase (XDH) antisense reagent of the present invention to the subject in an amount that effectively inhibits the expression of the xanthine dehydrogenase (XDH) gene, thereby reducing the The level of xanthine dehydrogenase (XDH) polypeptide and decreases the level of xanthine dehydrogenase (XDH) polypeptide activity.
  • XDH xanthine dehydrogenase
  • XDH xanthine dehydrogenase
  • contacting a cell with an siRNA agent of the invention results in an inhibition of xanthine dehydrogenase (XDH) gene expression in the cell by 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%
  • Some embodiments of the invention include determining the presence, absence, and/or amount (also referred to herein as levels) of a xanthine dehydrogenase (XDH) polypeptide in one or more biological samples obtained from one or more subjects. .
  • This assay can be used to assess the efficacy of the treatment methods of the invention.
  • the methods and compositions of the present invention can be used to determine the levels of xanthine dehydrogenase (XDH) polypeptides in biological samples obtained previously by administration of the xanthine dehydrogenase (XDH) dsRNA reagents of the present invention and/or or subjects treated with xanthine dehydrogenase (XDH) antisense agents.
  • the level of the xanthine dehydrogenase (XDH) polypeptide in the hepatocyte sample obtained from the subject is reduced by at least 0.5%, 1%, 5%, 10%, 20%, 30 %, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more indicates the level of efficacy of the treatment administered to the subject.
  • the physiological characteristics of a xanthine dehydrogenase (XDH)-related disease or disorder determined for a subject can be used as a control result, and the determination results of the physiological characteristics of the same subject at different times are compared with the control results. Compare.
  • pathological characteristic uric acid levels are measured from subjects who have never been administered the xanthine dehydrogenase (XDH) treatment of the present invention, which can be used as a baseline or control value for the subject.
  • uric acid levels are compared to the subject's control/baseline levels, respectively. Such comparisons may be used to evaluate the onset, progression, or regression of xanthine dehydrogenase (XDH)-related diseases or conditions in a subject.
  • XDH xanthine dehydrogenase
  • a baseline uric acid level obtained from a subject is higher than uric acid measured from the same subject after administration of a xanthine dehydrogenase (XDH) dsRNA agent or xanthine dehydrogenase (XDH) antisense polynucleotide agent of the invention to the subject level, represents the regression of xanthine dehydrogenase (XDH)-related diseases or disorders and represents the efficacy of the administered xanthine dehydrogenase (XDH) dsRNA agent of the present invention in treating xanthine dehydrogenase (XDH)-related diseases or disorders .
  • XDH xanthine dehydrogenase
  • XDH xanthine dehydrogenase
  • XDH xanthine dehydrogenase
  • Some embodiments of the present invention include determining the presence, absence, and/or changes in physiological characteristics of xanthine dehydrogenase (XDH)-related diseases or conditions using methods such as, but not limited to: (1) measuring a subject's uric acid levels; 2) Evaluate the physiological characteristics of one or more biological samples obtained from one or more subjects; (3) Or identify the subject Physical examination. This assay can be used to assess the efficacy of the treatment methods of the invention.
  • XDH xanthine dehydrogenase
  • Kits containing one or more xanthine dehydrogenase (XDH) dsRNA reagents and/or xanthine dehydrogenase (XDH) antisense polynucleotide reagents and instructions for their use in the methods of the invention are also provided herein. within the scope of the invention.
  • the kit of the present invention may comprise a xanthine dehydrogenase (XDH) dsRNA agent useful for treating xanthine dehydrogenase (XDH)-related diseases or disorders, a xanthine dehydrogenase (XDH) sense polynucleotide and a xanthine dehydrogenase One or more of the hydrogenase (XDH) antisense polynucleotide reagents.
  • XDH xanthine dehydrogenase
  • Kits containing one or more xanthine dehydrogenase (XDH) dsRNA reagents, xanthine dehydrogenase (XDH) sense polynucleotide, and xanthine dehydrogenase (XDH) antisense polynucleotide reagents can be prepared for use in the treatment methods of the present invention.
  • the components of the kit of the invention may be packaged in an aqueous medium or in lyophilized form.
  • Kits of the present invention may comprise a carrier divided to receive therein one or more container means or a series of container means (eg test tubes, vials, flasks, bottles, syringes, etc.).
  • the first container device or series of container devices may contain one or more compounds, such as a xanthine dehydrogenase (XDH) dsRNA reagent and/or a xanthine dehydrogenase (XDH) sense or antisense polynucleotide reagent.
  • the second container device or series of container devices may contain targeting agents, labeling agents, delivery agents, etc., which may serve as xanthine dehydrogenase (XDH) dsRNA agents and/or administered in embodiments of the treatment methods of the invention.
  • a portion of the xanthine dehydrogenase (XDH) antisense polynucleotide is included.
  • the kit of the present invention may also contain instructions. Instructions are usually in written form and will provide guidance for performing the treatment embodied by the kit and for making decisions based on that treatment.
  • XDH xanthine dehydrogenase
  • siRNA sense and antisense strand sequences are synthesized on an oligonucleotide synthesizer using a well-established solid-phase synthesis method based on phosphoramidite chemistry. Oligonucleotide chain growth is achieved through a 4-step cycle: deprotection, condensation, capping, and an oxidation or sulfation step for the addition of each nucleotide. The synthesis is based on controlled porous glass (CPG, ) on a solid support. 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.
  • CPG controlled porous glass
  • siRNA used for in vitro screening (Table 2), synthesis was performed at a 2 ⁇ mol scale; for siRNA used for in vivo testing (Table 3), the synthesis scale was 5 ⁇ mol or larger.
  • a GalNAc ligand (GLO-n as a non-limiting example) is attached to the 3'-end of the sense strand
  • a CPG solid support to which the GalNAc ligand is attached is used.
  • a GalNAc ligand (GLS-1 or GLS-2 as a non-limiting example) is attached to the 5'-end of the sense strand
  • a GalNAc phosphoramidite (GLPA1 or GLPA2 as a non-limiting example) is used for the final I linked reaction.
  • Trichloroacetic acid (TCA) in 3% dichloromethane was used for deprotection of the 4,4'-dimethoxytrityl protecting group (DMT). 5-Ethylthio-1H-tetrazole was used as the activator.
  • I in THF/Py/H 2 O and phenylacetyl disulfide (PADS) in pyridine / MeCN were used for the oxidation and sulfidation reactions, respectively.
  • PADS phenylacetyl disulfide
  • the solid support-bound oligomers were cleaved and protecting groups were removed by treatment with a 1:1 volume of 20 wt% aqueous methylamine and 28% ammonium hydroxide solution.
  • the crude mixture was concentrated. The remaining solid was dissolved in 1.0 M NaOAc and ice-cold EtOH was added to precipitate the single-chain product as a sodium salt, which was used for annealing without further purification.
  • the crude single-stranded product was further purified by ion pair reversed-phase HPLC (IP-RP-HPLC).
  • IP-RP-HPLC ion pair reversed-phase HPLC
  • the purified single-stranded oligonucleotide product from IP-RP-HPLC was converted to the sodium salt by dissolving it in 1.0 M NaOAc and precipitating by adding ice-cold EtOH. Annealing of sense and antisense strand oligonucleotides is performed by equimolar complementation in water to form a double-stranded siRNA product, which is lyophilized to provide a fluffy white solid.
  • Intermediate-A was synthesized by treating commercially available galactosamine pentaacetate with trimethylsilyl triflate (TMSOTf) in dichloromethane (DCM) as shown in Scheme 1 below. Glycosylation with Cbz-protected 2-(2-aminoethoxy)ethan-1-ol then provided compound II. The Cbz protecting group is removed by hydrogenation to provide Intermediate-A as the trifluoroacetate (TFA) salt.
  • Intermediate B was synthesized based on the same protocol except using Cbz protected 2-(2-(2-aminoethoxy)ethoxy)ethan-1-ol as starting material.
  • reaction mixture was filtered and washed with saturated NaHCO3 (200 mL), water (200 mL) and saturated brine (200 mL).
  • the organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to obtain crude product, which was triturated with 2-methyltetrahydrofuran/heptane (5/3, v/v, 1.80L) for 2 hours.
  • the resulting mixture was filtered and dried to obtain compound II as a white solid (15.0 g, yield 50.3%).
  • Phosphoramidites GLPA1 or GLPA2 are synthesized by phosphorylation of compounds Va or Vb with 2-cyanoethyl N,N-diisopropyl chloride phosphoramidite and a catalytic amount of 1H-tetrazole.
  • the reaction was quenched by slowly adding saturated NH 4 Cl (3.0 L), the layers were separated, and the aqueous phase was extracted with dichloromethane (2 ⁇ 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 (3.0L), dried over anhydrous Na 2 SO 4 , filtered and concentrated under reduced pressure.
  • the crude product was dissolved in 1.5L methylene chloride and added dropwise to methyl tert-butyl ether (7.5L). A translucent white precipitate gradually formed during the dropping process. The precipitate was filtered under vacuum, and the solid was collected and dried under vacuum to obtain compound VIII as a white solid (207 g, yield 72.8%).
  • reaction solution was washed with a 1:1 mixture of saturated NaHCO 3 and saturated brine (2.0L), dried over anhydrous Na 2 SO 4 , and the filtrate was concentrated and the crude product obtained was dissolved in dichloromethane (1.2L), and added dropwise to stirring In methyl tert-butyl ether (6.0L), filter the suspension, rinse the filter cake with methyl tert-butyl ether, collect the solid and dry it under vacuum, dissolve the product in dichloromethane (1.0L) and concentrate to dryness , repeat the operation 4 times to remove residual tert-butyl ether to obtain GLPA15 (164g, yield 73.3%).
  • GalNAc delivery compounds In some studies, methods are provided for delivering compounds containing GalNAc (also referred to herein as GalNAc delivery compounds).
  • Methods for attaching a targeting group to the 5'-end of the sense strand which involves the use of GalNAc phosphoramidite (e.g., GLPAl) in the last coupling step of solid-phase synthesis, using such synthetic processes as in oligonucleotides
  • a process used in acid chain extension i.e., adding nucleotides to the 5' end of the sense strand to connect it to the 5'-end of the sense strand.
  • the method of attaching a GalNAc-containing targeting group to the 3'-end of the sense strand involves the use of a GLO-n-containing solid support (CPG).
  • CPG GLO-n-containing solid support
  • methods to connect a GalNAc-containing targeting group to the 3'-end of the sense strand include: connecting the GalNAc targeting group to a CPG solid support via an ester bond and using it when synthesizing the sense strand.
  • the resulting CPG has a GalNAc targeting group attached, 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 methods similar to this article or well-known in the art after using reasonably corresponding intermediates, and can be connected to the appropriate position of the siRNA duplex as a targeting group.
  • phosphoramidite compounds can be prepared according to methods well known to those skilled in the art, such as but not limited to the methods in the prior art US426,220 and WO02/36743.
  • N,N-dimethylformamide 23.50kg
  • N,N-dimethylformamide 23.50kg
  • the isomannitol residue (imann) can be added to the 5' end or 3' end of the oligonucleotide chain through methods well known to those skilled in the art, such as the same process as reverse abasic (invab), and Further targeting groups are added.
  • CAL27 cells were trypsinized and adjusted to an appropriate density, then seeded into a 24-well plate at 1 ⁇ 10 cells/well. While inoculating, use Lipofectamine RNAiMax (Invitrogen-13778-150) to transfect cells with 2 ⁇ L per well of siRNA to be tested or control siRNA. 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 were also used as negative controls.
  • Lipofectamine RNAiMax Invitrogen-13778-150
  • cDNA was synthesized using PrimeScript TM RT kit and gDNA Eraser (Perfect Real Time) (TaKaRa-RR047A) according to the manual. Detection of xanthine dehydrogenase (XDH) cDNA 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.
  • the expression of the target gene in each sample was determined by relative quantification (RQ) using the comparative Ct ( ⁇ Ct) method; this method measures the Ct difference ( ⁇ Ct) between the target gene and a housekeeping gene (ACTIN).
  • ⁇ CT average Ct of target gene – average Ct of ACTIN
  • ⁇ CT ⁇ CT(sample)– ⁇ CT(nonsense siRNA control)
  • Table 4 provides experimental results from in vitro studies using various XDH RNAi reagents to inhibit XDH expression; the double-stranded sequences used correspond to the compounds shown in Table 2.
  • mice infected with AAV encoding human xanthine dehydrogenase (XDH) and luciferase genes were used (4 mice per group).
  • mice infected with AAV encoding human xanthine dehydrogenase (XDH) and luciferase genes were used (4 mice per group).
  • mice infected with AAV encoding human xanthine dehydrogenase (XDH) and luciferase genes 4 mice per group.
  • mice infected with AAV encoding human xanthine dehydrogenase (XDH) and luciferase genes were used (4 mice per group).
  • mice infected with AAV encoding human xanthine dehydrogenase (XDH) and luciferase genes were used (4 mice per group).
  • mice infected with AAV encoding human xanthine dehydrogenase (XDH) and luciferase genes were used (4 mice per group).
  • mice were injected subcutaneously with a single dose of 3 mg/kg, 6 mg/kg, or 9 mg/kg 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. Measure luciferase activity.
  • XDH xanthine dehydrogenase
  • Example 7 uses compounds corresponding to the sequences, chemical modifications and delivery shown in Table 3, GLO-n in Jayaprakash, et al., (2014) J. Am. Chem. Soc., 136, 16958-16961 Delivery compounds represented by GalNAc3.
  • luciferase activity of the pre-dose blood sample (day 0) of the siRNA-treated group was compared with the luciferase activity of the blood sample collected on day 7 and normalized based on the luciferase activity change in the serum sample from the PBS-treated group, to calculate knockdown percentage.
  • the data are listed in Table 5.
  • luciferase activity of the pre-dose blood sample (day 0) of the siRNA-treated group was compared with the luciferase activity of the blood sample collected on day 7 and normalized based on the luciferase activity change in the serum sample from the PBS-treated group, to calculate knockdown percentage.
  • the two experimental data are listed in Table 6 and Table 7 respectively.
  • luciferase activity of the pre-dose blood sample (day 0) of the siRNA-treated group was compared with the luciferase activity of the blood sample collected on day 14 and normalized based on the luciferase activity change in the serum sample from the PBS-treated group, to calculate knockdown percentage.
  • the data are listed in Table 8.
  • siRNA In order to evaluate the in vivo activity of siRNA, a total of 18 male cynomolgus monkeys (13-22 years old, weighing 7-9 kg) were selected in this study, with 3 animals in each group. Each animal was given 7.5 mg/kg test product by subcutaneous injection. , the test samples used correspond to the compounds shown in the table below.

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Abstract

提供了可用于降低细胞和对象中黄嘌呤脱氢酶(XDH)表达的黄嘌呤脱氢酶(XDH)dsRNA试剂、及其与黄嘌呤脱氢酶(XDH)反义多核苷酸试剂和/或非黄嘌呤脱氢酶(XDH)dsRNA治疗剂的组合物，其可用于抑制黄嘌呤脱氢酶(XDH)基因表达和用于治疗黄嘌呤脱氢酶(XDH)相关疾病和病症。

Description

用于抑制黄嘌呤脱氢酶(XDH)的组合物和方法 技术领域
本发明涉及可用于抑制黄嘌呤脱氢酶(XDH)的组合物和方法。
背景技术
尿酸(在本文中也称为尿酸盐)是内源性和饮食性嘌呤代谢的最终代谢产物。黄嘌呤氧化酶(在本文中也称为“XO”)(EC 1.1.3.22)和黄嘌呤脱氢酶(在本文中也称为“XDH”)(EC 1.17.1.4)分别可催化次黄嘌呤氧化为黄嘌呤和黄嘌呤氧化为尿酸。这些酶是含钼蝶呤的黄素蛋白，由约145kDa的两个相同亚基组成。这些酶来自哺乳动物，包括人，以脱氢酶形式合成，但是可以通过巯基残基的氧化或蛋白水解容易地转化为氧化酶形式。XDH主要在肠和肝中表达，但也可以在其他组织(包括脂肪组织)中表达。
慢性血清尿酸升高(慢性高尿酸血症)，通常定义为血清尿酸盐水平高于6.8mg/dl(大于360mmol/dl)，超过该水平时，其生理饱和度阈值被超过(Mandell,Cleve.Clin.Med.75：S5-S8,2008)与许多疾病有关，例如痛风，其特征在于急性炎症性关节炎的反复发作，通常是由于对肾的尿酸清除不足或尿酸产生过多而引起的对关节中尿酸晶体的炎性反应所致。果糖相关的痛风与在肾脏，肠和肝脏中表达的转运蛋白的变体相关。慢性尿酸升高还与非酒精性脂肪性肝炎(NASH)，非酒精性脂肪肝疾病(NAFLD)，代谢紊乱，心血管疾病，2型糖尿病以及与氧化应激，慢性低度炎症和胰岛素相关的疾病有关(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).
别嘌醇和非布索坦是以XDH形式存在的酶的抑制剂，通常用于治疗痛风。但是，在患有痛风的合并症，特别是肾功能下降的患者(例如由于慢性肾脏疾病或肝功能不全)中有使用禁忌。它们的使用可能会在患有代谢综合征，高血压，血脂异常，非酒精性脂肪性肝炎(NASH)或非酒精性脂肪肝疾病(NAFLD)，心血管疾病或糖尿病(1型或2型)的患者中受到限制，这是由于疾病或病状或药物与治疗此类病状的药物发生不良相互作用而使器官功能受到限制导致的。
当前，XDH相关疾病或病症的治疗方案不能完全满足患者的需求。因此，针对将受益于XDH基因表达降低的受试者，例如痛风的受试者，存在对其他疗法的需求。
发明内容
根据本发明的一个方面，提供了用于抑制黄嘌呤脱氢酶(XDH)基因表达的双链核糖核酸(dsRNA)试剂，该dsRNA试剂包含正义链和反义链，在反义链中的核苷酸位置2至18处包含与黄嘌呤脱氢酶(XDH)基因RNA转录物互补的区域，其中互补区域包含与表1-3中所列出的反义序列之一相差0、1、2或3个核苷酸的至少15个连续核苷酸，并且任选地包含靶向配体。
在一些实施方案中，与黄嘌呤脱氢酶(XDH)基因RNA转录物互补的区域包含与表1-3中所列出的反义序列之一相差不超过3个核苷酸的至少15、16、17、18或19个连续核苷酸。
在一些实施方案中，dsRNA的反义链与SEQ ID NO:1的任意靶区域至少基本互补，并且在表1-3之一中提供。
在一些实施方案中，dsRNA的反义链与SEQ ID NO:1的任意靶区域完全互补并且在表1-3之一中提供。
在一些实施方案中，dsRNA试剂包含表1-3中所列出的任一个正义链序列，其中正义链序列与dsRNA试剂中的反义链序列至少基本上互补。
在一些实施方案中，dsRNA试剂包含表1-3中列出的任一个正义链序列，其中正义链序列与dsRNA试剂中的反义链序列完全互补。
在一些实施方案中，dsRNA试剂包含表1-3中列出的任一个反义链序列。
在一些实施方案中，dsRNA试剂包含表1-3中作为双链体序列列出的任一个序列。
在一些实施方案中，dsRNA试剂包含至少一个修饰的核苷酸。
在一些实施方案中，反义链的所有核苷酸或基本上所有核苷酸都是修饰的核苷酸。
在一些实施方案中，正义链和反义链的所有或基本上所有核苷酸都是修饰的核苷酸。
在一些实施方案中，至少一种修饰的核苷酸包括：2'-甲氧基核苷酸、2'-氟核苷酸、2'-脱氧核苷酸、2',3'-seco核苷酸模拟物、锁核苷酸、开环核酸核苷酸(unlocked nucleic acid nucleotide,UNA)、乙二醇核酸核苷酸(glycol nucleic acid nucleotide，GNA)、2'-F-阿拉伯糖核苷酸、2'-甲氧基乙基核苷酸、无碱基核苷酸、核糖醇、反向核苷酸、反向无碱基核苷酸、反向2'-OMe核苷酸、反向2'-脱氧核苷酸、异甘露糖醇核苷酸、2'-氨基修饰的核苷酸、2'-烷基修饰的核苷酸、吗啉代核苷酸和3'-OMe核苷酸、包括5'-硫代磷酸酯基团的核苷酸、或与胆固醇衍生物或十二烷酸双癸酰胺基团连接的末端核苷酸、2'-氨基修饰的核苷酸、氨基磷酸酯或包含核苷酸的非天然碱基。
在一些实施方案中，dsRNA试剂包括在引导链的5'末端处的E-乙烯基膦酸酯核苷酸。
在一些实施方案中，dsRNA试剂包含至少一个硫代磷酸酯核苷间键联。
在一些实施方案中，正义链包含至少一个硫代磷酸酯核苷间键联。
在一些实施方案中，反义链包含至少一个硫代磷酸酯核苷间键联。
在一些实施方案中，正义链包含1、2、3、4、5或6个硫代磷酸酯核苷间键联。
在一些实施方案中，反义链包含1、2、3、4、5或6个硫代磷酸酯核苷间键联。
在一些实施方案中，反义链包含至少15个独立地选自2'-O-甲基核苷酸和2'-氟核苷酸的修饰核苷酸，其中少于6个是2'-氟核苷酸修饰的核苷酸。在某些实施方案中，反义链包含3个或5个2'-氟核苷酸，优选地，反义链包含5个2'-氟核苷酸。
在一些实施方案中，正义链包含至少15个独立地选自2'-O-甲基核苷酸和2'-氟核苷酸的修饰核苷酸，其中少于4个是2'-氟核苷酸修饰的核苷酸，在某些实施方案中，正义链包含3个2'-氟核苷酸。
在一些实施方案中，反义链包含在从5'末端到3'末端的方向上，从反义链5'端第一个配对的核苷酸开始计算，反义链的第2、7、12、14和16位的核苷酸是2'-氟修饰的核苷酸，并且反义链中其他位置的每个核苷酸独立地是非氟修饰的核苷酸。
在一些实施方案中，正义链包含在从3'末端到5'末端的方向上，从正义链3'第一个配对的核苷酸开始计算，在正义链的位置9、11和13的核苷酸是2'-氟修饰的核苷酸，并且每个核苷酸在正义链中的其他位置独立地是非氟修饰的核苷酸。
在一些实施方案中，反义链包含15个或更多个独立地选自2'-O-甲基核苷酸和2'-氟核苷酸的修饰核苷酸，其中由反义链5'端起的第2、7、12、14和/或16位是2'-氟核苷酸修饰的核苷酸，其余为2'-O-甲基核苷酸修饰的核苷酸。
在一些实施方案中，正义链包含独立地选自2'-O-甲基核苷酸和2'-氟核苷酸的15个或更多个修饰核苷酸，其中正义链从3'端第一个匹配位置起的第9、11和/或13位是2'-氟核苷酸修饰的核苷酸，其余为2'-O-甲基核苷酸修饰的核苷酸。
在一些实施方案中，修饰的正义链是表2和/或表3中列出的修饰的正义链序列。
在一些实施方案中，修饰的反义链是表2和/或表3中列出的修饰的反义链序列。
在一些实施方案中，正义链与反义链互补或基本互补，并且互补区域的长度在16至23个核苷酸之间。在一些实施方案中，互补区域的长度为19-21个核苷酸。在一些实施方案中，互补区域的长度为14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29或30个核苷酸。
在一些实施方案中，每条链的长度不超过40个核苷酸。
在一些实施方案中，每条链的长度不超过30个核苷酸。
在一些实施方案中，每条链的长度不超过25个核苷酸。
在一些实施方案中，每条链的长度不超过23个核苷酸。
在一些实施方案中，每条链的长度不超过21个核苷酸。
在一些实施方案中，dsRNA试剂包含至少一种修饰的核苷酸并且还包含一个或更多个靶向基团或连接基团。
在一些实施方案中，一个或更多个靶向基团或连接基团与正义链缀合。在一些实施方案中，靶向基团或连接基团包括N-乙酰基-半乳糖胺(GalNAc)。
在一些实施方案中，靶向基团或连接基团如式(X)所示的结构，包括靶向部分、链接键以及接头W，其中靶向部分选自N-乙酰基-半乳糖胺衍生物(GalNAc)，其通过链接键与接头W链接，接头W具有如式(XI)所示的结构，X选自O、NH2或者S，Y﹣选自：O﹣、S﹣、甲基或者NRaRb，Ra和Rb分别独立的选自氢、取代或者未取代的C1-C6的烷基、取代或者未取代的C3-C6的环烷基，或者Ra和Rb与附着的原子一起链接形成含有1-3个N、O、S杂原子组成的3-12元杂环烷基。在一些优选的实施例中，取代或者未取代的C1-C6的烷基、取代或者未取代的C3-C6的环烷基中，所述的取代基选自羟基、氨基。

在一些实施方案中，靶向基团中的链接键选自聚乙二醇、任选取代的C2-C12烷基,取代或者未取代的C3-C12环烷基、取代或者未取代的C3-C12杂环烷基、取代或者未取代的C3-C12酰胺。在一些实施方案中，取代或者未取代的C2-C12烷基,取代或者未取代的C3-C12环烷基、取代或者未取代的C3-C12杂环烷基、取代或者未取代的C3-C12酰胺中，所述的取代基选自羟基、羰基。
在一些实施方案中，靶向基团中的链接键优选自以下片段：
其中每个m独立的为1-6的整数，每个n、o、p独立的为0或者1，每个q1与q2分别独立的为0、1或者2。
在一些实施方案中，靶向基团中的链接键更优选自以下片段：
在一些实施方案中，靶向基团中的靶向部分具有为以下结构片段，
n’为1或者2。
在一些实施方案中，靶向基团具有以下结构：



在一些实施方案中，dsRNA试剂包含与正义链的5'-末端缀合的靶向基团。
在一些实施方案中，dsRNA试剂包含与正义链的3'-末端缀合的靶向基团。
在一些实施方案中，反义链在3'-末端包含一个反向无碱基残基。
在一些实施方案中，正义链在3'或/和5'末端包含一个或两个反向无碱基残基。
在一些实施方案中，正义链在3'或/和5'末端包含一个或两个imann残基。
在一些实施方案中，正义链的3’和5’末端各自独立地包含一个imann残基。
在一些实施方案中，dsRNA试剂具有两个平末端。
在一些实施方案中，至少一条链包含至少1个核苷酸长的3'突出端。在一些实施方案中，至少一条链包含至少2个核苷酸长的3'突出端。在一个实施例中，正义链在3’-末端和/或5’-末端具有1-10个核苷酸突出端，如1、2、3、4、5、6、7、8、9、或10个核苷酸的突出端。
本发明的另一个方面，提供了抑制黄嘌呤脱氢酶(XDH)基因表达的双链核糖核酸(dsRNA)试剂，该dsRNA试剂包含正义链和反义链，其中每条链的长度是14个至30个核苷酸，在反义链中的核苷酸位置2至18处包含与黄嘌呤脱氢酶(XDH)基因RNA转录物互补的区域，其中互补区域包含与式I中所列出的双链核糖核酸(dsRNA)试剂反义序列之一相差0、1、2或3个核苷酸的至少15个连续核苷酸，并且任选地包含靶向配体。
所述双链核糖核酸(dsRNA)试剂反义链如式I所示:
5’-Na-(A1A2A3A4)i-Nb–(B1B2B3B4)-Nb-(C1C2C3C4C5)j-Na-3’
(式I)
其中:i和j各自独立地选自0或1；
每个Na和Nb分别独立的代表0-17寡核苷酸，所述寡核苷酸可选地被本文所用化学修饰进行修饰；优选的，所述化学修饰选自2'-OMe或2'-氟修饰；
A1A2A3A4表示四个连续的核苷酸依次被代表小写2'-OMe、大写2'-氟、小写2'-OMe、小写2'-OMe修饰的一个基序，优选的A1与A2之间、A2与A3之间进一步均包含硫代磷酸核苷间键联；
B1B2B3B4表示四个连续的核苷酸依次被1个大写2'-氟，和连续3个小写2'-OMe修饰的一个基序；
C1C2C3C4C5表示五个连续的核苷酸均被小写2'-OMe修饰的的一个基序，优选的C3与C4之间、C4与C5之间进一步包含硫代磷酸酯核苷间键联。
在一些实施例中，Na分别独立的代表0-2个寡核苷酸，Nb分别独立的代表2-5个经过或未经过本文所用化学修饰的寡核苷酸。
在一些实施例中，Na分别独立的代表0个寡核苷酸，Nb分别独立的代表2-5个经本文所用化学修饰的寡核苷酸。
在一些实施例中，Na分别独立的代表0个寡核苷酸，Nb分别独立的代表2-5个经2'-OMe或2'-氟化学修饰的寡核苷酸。
进一步地，在一些实施例中，式(I)进一步如式(II)所示：
5’-xYxxxxYxxxxYxYxYxxxxx-3’
(式II)
x代表小写2'-OMe修饰的寡核苷酸，Y代表大写2'-氟修饰的寡核苷酸，优选的x与Y和/或x与x之间进一步包含硫代磷酸酯核苷间键联。
进一步地，在一些实施例中，所述双链核糖核酸(dsRNA)试剂正义链如式(III)所示:
5′-(N′L)n′N′LN′LN′LN′N1N′N2N′N3N′LN′FN′LN′N4N′N5N′N6N′LN′LN′L(N′L)m′-3′  式(III)
其中，N’F代表2'-氟修饰的核苷酸；N′N1,N′N2,N′N3,N′N4,N′N5,and N′N6各自分别独立地代表一种修饰或未修饰的核苷酸；N′N1N′N2N′N3和N′N4N′N5N′N6各自独立地代表一种包含至少两种不同修饰的核苷酸片段；N′L各自独立地代表一种修饰或未修饰的核苷酸，所述修饰的核苷酸不是2’-氟修饰的核苷酸；m′和n′各自独立地代表0到7整数。
在一些实施例中，n′是1并且m′是1,或者n′是1并且m′是2,或者n′是1并且m′是3,或者n′是1并且m′是4,或者n′是1并且m′是5,或者n′是3并且m′是1,或者n′是3并且m′是2,或者n′是3并且m′是3,或者n′是5并且m′是1。
在一些实施方案中，dsRNA的反义链与SEQ ID NO:1的任意靶区域至少基本互补。在一些实施方案中，dsRNA的反义链与SEQ ID NO:1的任意靶区域完全互补。在一些实施方案中，dsRNA任一项所述的正义链序列与所述dsRNA试剂中的反义链序列至少基本上互补。在一些实施方案中，dsRNA任一项所述的正义链序列与所述dsRNA试剂中的反义链序列完全互补。
在一些实施方案中，与SEQ ID NO:1互补的区域包含与表1-3中所列出的反义序列之一相差不超过3个核苷酸的至少15、16、17、18或19个连续核苷酸。
在一些实施方案中，dsRNA的反义链与SEQ ID NO:1的任意靶区域至少基本互补，并且在表1-3之一中提供。
在一些实施方案中，dsRNA的反义链与SEQ ID NO:1的任意靶区域完全互补并且在表1-3之一中提供。
在一些实施方案中，dsRNA试剂包含表1-3中所列出的任一个正义链序列，其中正义链序列与dsRNA试剂中的反义链序列至少基本上互补。
在一些实施方案中，dsRNA试剂包含表1-3中列出的任一个正义链序列，其中正义链序列与dsRNA试剂中的反义链序列完全互补。
在一些实施方案中，dsRNA试剂包含表1-3中列出的任一个反义链序列。
在一些实施方案中，dsRNA试剂包含表1-3中作为双链体序列列出的任一个序列。
在一些实施方案中，正义链在3'或/和5'末端包含一个或两个反向无碱基残基。
在一些实施方案中，正义链在3'或/和5'末端包含一个或两个imann残基。
在一些实施方案中，正义链的3’和5’末端各自独立地包含一个imann残基。
在一些实施方案中，dsRNA试剂具有两个平末端。在一些实施方案中，至少一条链包含至少1个核苷酸长的3'突出端。在一些实施方案中，至少一条链包含至少2个核苷酸长的3'突出端。在一个实施例中，正义链在3’-末端和/或5’-末端具有1-10核苷酸突出端，如1、2、3、4、5、6、7、8、9或10个核苷酸的突出端。
在一些实施方案中，dsRNA试剂包含至少一种修饰的核苷酸并且还包含一个或更多个靶向基团或连接基团。在一些实施方案中，一个或更多个靶向基团或连接基团与正义链缀合。在一些实施方案中，靶向基团或连接基团包括N-乙酰基-半乳糖胺(GalNAc)。在一些实施方案中，靶向基团如本文所用，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和GLO-16中的任一种。
在一些实施方案中，正义链在3’和5’末端各自独立地包含一个imann残基，其中任意一个3’和5’末端残基可选地进一步与靶向基团连接，所述靶向基团优选地为前述GLS-15。
根据本发明的另一个方面，提供了一种组合物，其包含本发明上述dsRNA试剂方面的任意实施方案。
在某些实施方案中，组合物还包含药学上可接受的载体。在一些实施方案中，组合物还包含一种或更多种另外的治疗剂。
在某些实施方案中，组合物被包装在药盒、容器、包装物、分配器、预填充注射器或小瓶中。
在一些实施方案中，组合物被配制用于皮下给药或被配制用于静脉内(IV)给药。
根据本发明的另一方面，提供了一种细胞，其包含本发明上述dsRNA试剂方面的任意实施方案。
在一些实施方案中，细胞是哺乳动物细胞，任选地是人细胞。
根据本发明的另一方面，提供了一种抑制细胞中黄嘌呤脱氢酶(XDH)基因表达的方法，该方法包括：(i)制备包含有效量的上述dsRNA试剂或上述组合物方面的任意实施方案的细胞。
在某些实施方案中，该方法还包括：(ii)将制备的细胞维持足够的时间以获得黄嘌呤脱氢酶(XDH)基因的mRNA转录物的降解，从而抑制细胞中黄嘌呤脱氢酶(XDH)基因的表达。
在一些实施方案中，细胞在对象体内并且dsRNA试剂经皮下施用于对象。
在一些实施方案中，细胞在对象体内并且dsRNA试剂通过IV给药施用于对象。
在某些实施方案中，该方法还包括在向对象施用dsRNA试剂后评估对黄嘌呤脱氢酶 (XDH)基因的抑制，其中评估的手段包括：(i)确定对象中黄嘌呤脱氢酶(XDH)相关疾病或病症的一个或更多个生理特征，以及(ii)将所确定的生理特征与黄嘌呤脱氢酶(XDH)相关疾病或病症的基线治疗前生理特征和/或黄嘌呤脱氢酶(XDH)相关疾病或病症的对照生理特征进行比较，其中的比较结果指示对象中黄嘌呤脱氢酶(XDH)基因表达的抑制存在或不存在。
在一些实施方案中，所确定的生理特征是在血液中的尿酸水平；
在一些实施方案中，血液中尿酸水平的降低表明对象中黄嘌呤脱氢酶(XDH)基因表达的降低。
根据本发明的另一方面，提供了一种抑制对象中黄嘌呤脱氢酶(XDH)基因表达的方法，其包括向对象施用有效量的前述dsRNA试剂方面的实施方案或前述组合物的实施方案。
在一些实施方案中，将dsRNA试剂皮下施用于对象。在某些实施方案中，dsRNA试剂通过IV给药施用于对象。在一些实施方案中，该方法还包括：在施用dsRNA试剂后评估黄嘌呤脱氢酶(XDH)基因的抑制，其中评估手段包括：(i)确定对象的黄嘌呤脱氢酶(XDH)相关疾病或病症的一种或更多种生理特征；(ii)将确定的生理特征与黄嘌呤脱氢酶(XDH)相关疾病或病症的基线治疗前生理特征和/或黄嘌呤脱氢酶(XDH)相关疾病或病症的对照生理特征进行比较；其中比较结果指示对象中黄嘌呤脱氢酶(XDH)基因表达的抑制存在或不存在。在一些实施方案中，所确定的生理特征是在血液中的尿酸水平。血液中尿酸水平的降低表明对象中黄嘌呤脱氢酶(XDH)基因表达的降低。
根据本发明的另一方面，提供了一种制备治疗与黄嘌呤脱氢酶(XDH)蛋白相关之疾病或病症的药物的应用，所述药物包含本发明的前述dsRNA试剂方面的任意实施方案或本发明的前述组合物的任意实施方案，所述药物可以抑制黄嘌呤脱氢酶(XDH)基因表达。
根据本发明的另一方面，提供了一种治疗与黄嘌呤脱氢酶(XDH)蛋白相关之疾病或病症的方法，其包括：向对象施用有效量的本发明的前述dsRNA试剂方面的任意实施方案或本发明的前述组合物的任意实施方案，以抑制黄嘌呤脱氢酶(XDH)基因表达。
在某些实施方式中，黄嘌呤脱氢酶(XDH)相关疾病或病症选自：高尿酸血症，痛风，非酒精性脂肪性肝病(NAFLD)，非酒精性脂肪性肝炎(NASH)，代谢紊乱，胰岛素抵抗，心血管疾病，2型糖尿病，Lesch Nyhan综合征，糖原贮积病(GSD)，例如I型GSD以及与氧化应激有关的疾病，例如慢性低度炎症；或其他XDH相关疾病。
在某些实施方案中，与XDH相关的疾病是痛风。在某些实施方案中，与XDH相关的疾病是高尿酸血症。在某些实施方案中，与XDH相关的疾病是NASH或NAFLD。
在一些实施方案中，该方法还包括：向对象施用另外的治疗方案。
在一些实施方案中，另外的治疗方案包括黄嘌呤脱氢酶(XDH)相关疾病或病症的治疗。在某些实施方案中，另外的治疗方案包括：向对象施用一种或更多种本发明的黄嘌呤脱氢酶(XDH)反义多核苷酸；向对象施用非黄嘌呤脱氢酶(XDH)dsRNA治疗剂；以及在对象中进行行为改变。在一些实施方案中，非黄嘌呤脱氢酶(XDH)dsRNA治疗剂是以下另外的治疗剂中的一种：例如，别嘌醇，羟嘌呤，非布索坦或白介素-1β(IL-1β)拮抗剂，优选地，所述白介素-1β拮抗剂选自卡那单抗或利纳西普(rilonacept)，从而治疗将从黄嘌呤 脱氢酶(XDH)减少中受益的疾病的受试者。
在一些实施方案中，将dsRNA试剂皮下施用于对象。在某些实施方案中，dsRNA试剂通过IV给药施用于对象。在一些实施方案中，该方法还包括确定所施用的双链核糖核酸(dsRNA)试剂在对象中的功效。在一些实施方案中，确定治疗在对象中的功效的手段包括：(i)确定对象中黄嘌呤脱氢酶(XDH)相关疾病或病症的一种或更多种生理特征；(ii)将确定的生理特征与黄嘌呤脱氢酶(XDH)相关疾病或病症的基线治疗前生理特征进行比较，其中该比较结果指示对对象施用双链核糖核酸(dsRNA)试剂的功效存在、不存在和水平中的一种或更多种。在一些实施方案中，所确定的生理特征是在血液中的尿酸水平。血液中尿酸水平的降低表明对对象施用双链核糖核酸(dsRNA)试剂的有效性的存在。
根据本发明的另一方面，提供了与对象中黄嘌呤脱氢酶(XDH)蛋白的基线治疗前水平相比降低对象中黄嘌呤脱氢酶(XDH)蛋白水平的方法，其包括向对象施用有效量的本发明的前述dsRNA试剂方面的任意实施方案或本发明的前述组合物的任意实施方案，以降低黄嘌呤脱氢酶(XDH)基因表达的水平。在一些实施方案中，将dsRNA试剂皮下施用于对象或通过IV施用于对象。
根据本发明的另一方面，提供了与对象中黄嘌呤脱氢酶(XDH)相关疾病或病症的基线治疗前生理特征相比改变对象中黄嘌呤脱氢酶(XDH)相关疾病或病症的生理特征的方法，该方法包括向对象施用有效量的本发明前述dsRNA试剂方面的任意实施方案或本发明的前述组合物的任意实施方案，以改变对象中黄嘌呤脱氢酶(XDH)相关疾病或病症的生理特征。在一些实施方案中，将dsRNA试剂皮下施用于对象或通过IV施用于对象。在一些实施方案中，所确定的生理特征是尿酸的降低。
序列说明
双链体AV00206至AV00292显示在表1中并且显示了其正义链序列。
双链体AV00206至AV00292显示在表1中并且显示了其反义链序列。
SEQ ID NO:1是人黄嘌呤脱氢酶(XDH)mRNA[NCBI参考序列：NM_000379.4]。
在显示于表2的序列中，化学修饰表示为：大写：2'-氟；小写：2'-OMe；硫代磷酸酯：*。
在显示于表3的序列中，体内研究中使用的递送分子在每条正义链的3'或5'末端表示为“GLX-n”。化学修饰表示为：大写：2'-氟；小写：2'-OMe；硫代磷酸酯：*；Invab＝反向无碱基；imann：在每条链末端时:或当进一步偶联递送分子或靶向基团时：
具体实施方式
本发明的部分实施方案包括能够抑制黄嘌呤脱氢酶(XDH)基因表达的RNAi试剂，例如但不限于双链(ds)RNAi试剂。本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂可以靶向黄嘌呤脱氢酶(XDH)转录物，导致黄嘌呤脱氢酶(XDH)蛋白表达的抑制。本发明的部分实施方案还包括包含黄嘌呤脱氢酶(XDH)基因RNAi试剂的组合物和使用该组合物的方法。本文公开的黄嘌呤脱氢酶(XDH)基因RNAi试剂可接附于递送化合物以递送至细胞，包括递送至肝细胞。本发明的药物组合物可包含至少一种黄嘌呤脱氢酶(XDH)dsRNA试剂和递送化合物。在本发明的一些实施方案中，递送化合物是含GalNAc的递送化合物。递送至细胞的黄嘌呤脱氢酶(XDH)基因RNAi试剂能够抑制黄嘌呤脱氢酶(XDH)基因表达，从而降低基因的黄嘌呤脱氢酶(XDH)蛋白产物在细胞中的活性。本发明的dsRNAi试剂可用于治疗黄嘌呤脱氢酶(XDH)相关疾病和病症。这样的dsRNAi试剂包括例如表1中所显示的双链体AV00206至AV00292。在一些实施方案中，优选的dsRNAi试剂包括例如双链体AV00227、AV00260、AV00285、AV00266、AV00280、AV00238、AV00249、AV00289、AV00233、AV00225、AV00273或AV00283。在另一些实施方案中，优选的表2中dsRNAi试剂包括例如双链体AV00227、AV00260、AV00285、AV00266或AV00280。在另一些实施方案中，优选的表3中dsRNAi试剂包括例如双链体AD00353、AD00356、AD00358、AD00364、AD00365、AD00366、AD00368、AD00370、AD00430、AD00431、AD00432、AD00521、AD00521-1、AD00364-1、AD00365-2、AD00366-1和AD00432-1。
在本发明的一些实施方案中，降低细胞或对象中黄嘌呤脱氢酶(XDH)表达分别治疗与细胞或对象中黄嘌呤脱氢酶(XDH)表达相关的疾病或病症。可通过降低黄嘌呤脱氢酶(XDH)活性治疗的疾病和病症的非限制性实例是：高尿酸血症，痛风，NAFLD，NASH，代谢紊乱，胰岛素抵抗，心血管疾病，2型糖尿病，Lesch Nyhan综合征，糖原贮积病(GSD)，例如I型GSD，或与氧化应激有关的疾病，例如慢性低度炎症；或其他XDH相关疾病。
下面描述了如何制备和使用包含黄嘌呤脱氢酶(XDH)单链(ssRNA)和双链(dsRNA)试剂的组合物来抑制黄嘌呤脱氢酶(XDH)基因表达，以及用于治疗由黄嘌呤脱氢酶(XDH)基因表达引起或调节的疾病和病症的组合物和方法。如本文所用，由黄嘌呤脱氢酶(XDH)基因表达的存在和/或水平引起或调节的疾病和/或病症称为“黄嘌呤脱氢酶(XDH)相关疾病和/或病症”。术语“RNAi”也是本领域已知的，并且可以被称为“siRNA”。
如本文所用，“G”、“C”、“A”和“U”通常各自代表分别含有鸟嘌呤、胞嘧啶、腺嘌呤和尿嘧啶作为碱基的核苷酸。然而，可以理解的是，术语“核糖核苷酸”或“核苷酸”也可以指修饰的核苷酸，如下文进一步详述的，或可替换的替代部分。本领域技术人员理解，鸟嘌呤、胞嘧啶、腺嘌呤和尿嘧啶可以被其他部分替换，而不会实质性改变包含这种替换部分的核苷酸的寡核苷酸的碱基配对财产。例如，但不限于，包含肌苷作为其碱基的核苷酸可以与包含腺嘌呤、胞嘧啶或尿嘧啶的核苷酸碱基配对。因此，在本发明的核苷酸序列中，含有尿嘧啶、鸟嘌呤或腺嘌呤的核苷酸可以被含有例如肌苷的核苷酸取代。包含这样的替换部分 的序列是本发明的实施方案。
如本文所用，术语“RNAi”是指包含RNA并通过RNA诱导的沉默复合物(RISC)途径介导RNA转录物的靶向切割的试剂。如本领域已知的，RNAi靶区域是指在基因转录过程中形成的RNA分子的核苷酸序列的连续部分，其包括信使RNA(mRNA)，它是初级转录产物RNA的加工产物。该序列的靶标部分将至少足够长以用作在该部分处或附近进行RNAi定向切割的底物。靶序列可以是8-30个核苷酸长(包括端值)、10-30个核苷酸长(包括端值)、12-25个核苷酸长(包括端值)、15-23个核苷酸长(包括端值)、16-23个核苷酸长(包括端值)，或18-23个核苷酸长(包括端值)，并包括每个规定范围内的所有较短长度。在本发明的一些实施方案中，靶序列为9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25或26个核苷酸长。在某些实施方案中，靶序列的长度在9到26个核苷酸之间(包括端值)，包括其间的所有子范围和整数。例如，虽然不意在限制，但在本发明的某些实施方案中，靶序列为8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30个核苷酸长，该序列与黄嘌呤脱氢酶(XDH)基因的RNA转录物的至少一部分完全或至少基本上互补。
本发明的一些方面包括包含一种或更多种黄嘌呤脱氢酶(XDH)dsRNA试剂和药学上可接受的载体的药物组合物。在本发明的某些实施方案中，如本文所述的黄嘌呤脱氢酶(XDH)基因RNAi抑制黄嘌呤脱氢酶(XDH)蛋白的表达。
本发明的一些实施方案中，药物组合物包括一种、两种、三种或更多种独立的抗黄嘌呤脱氢酶(XDH)dsRNA试剂，并且还可以包括一种或多种独立选择的递送化合物。在一些实施方案中，将能够分别靶向黄嘌呤脱氢酶(XDH)mRNA的一个、两个、三个、四个或更多个不同位置/区域的两个、三个、四个或更多个黄嘌呤脱氢酶(XDH)dsRNA施用于细胞或对象中。
如本文所用，“dsRNA试剂”是指包含RNA或RNA样(例如，化学修饰的RNA)寡核苷酸分子的组合物，其能够降解或抑制靶mRNA转录物的翻译。尽管不希望限于特定理论，但本发明的dsRNA试剂可通过RNA干扰机制起作用(即，通过与哺乳动物细胞的RNA干扰途径机制(RNA诱导的沉默复合物或RISC)相互作用来诱导产生RNA干扰)，或通过任何替代机制或途径起作用。在植物、无脊椎动物和脊椎动物细胞中实现基因沉默的方法是本领域所公知的(参见例如，Sharp et al.,Genes Dev.2001,15:485；Bernstein,et al.,(2001)Nature 409:363；Nykanen,et al.,(2001)Cell 107:309；以及Elbashir,et al.,(2001)Genes Dev.15:188))，其各自的公开内容通过引用整体并入本文。本领域已知的基因沉默手段可与本文提供的公开内容结合使用以实现抑制黄嘌呤脱氢酶(XDH)的表达。
本文公开的dsRNA试剂由正义链和反义链组成，其包括但不限于：短干扰RNA(siRNA)、RNAi试剂、微RNA(miRNA)、短发夹RNA(shRNA)和切丁酶(Dicer)底物。本文描述的dsRNA试剂的反义链至少部分地与所靶向的mRNA互补，本领域能够理解，多种长度的dsRNA双链体结构可用于抑制靶基因表达。例如，已知具有19、20、21、22和23个碱基对的双链体结构的dsRNA可有效诱导RNA干扰(Elbashir et al.,EMBO 2001,20:6877-6888)。本领域还已知较短或较长的RNA双链体结构也可有效诱导RNA干扰。本 发明的某些实施方案中的黄嘌呤脱氢酶(XDH)dsRNA可以包含至少一条长度至少为21nt的链，或者双链体可以具有基于表1-3中任何列出的序列之一的长度减1、2、3nt或更短的长度。与分别在表1-3中列出的dsRNA相比，在其一端或两端减少4个核苷酸也可以是有效的。在本发明的一些实施方案中，黄嘌呤脱氢酶(XDH)dsRNA试剂可具有来自表1-3的一个或更多个序列的至少15、16、17、18、19、20或更多个连续核苷酸的部分序列，并且它们抑制黄嘌呤脱氢酶(XDH)基因表达的能力与由包含全序列(此处也称为“亲本”序列)的dsRNA产生的抑制水平相差不超过5％、10％、15％、20％、25％或30％。表1-3中公开的正义序列、反义序列和双链体在本文中可以称为“亲本”序列，意味着表1-3中公开的序列可以被修饰、缩短、延长、包括替换等，如本文所述，所得序列保留其亲本序列在本发明的方法和组合物中的全部或至少部分功效。
本发明的组合物和方法的某些实施方案在组合物中包含单链RNA和/或将单链RNA施用于对象。例如，表1-3任一项中所列的反义链可以作为一种组合物或在一种组合物内，该组合物施用给对象会降低对象中黄嘌呤脱氢酶(XDH)多肽活性和/或黄嘌呤脱氢酶(XDH)基因的表达。表1-3显示了某些黄嘌呤脱氢酶(XDH)dsRNA试剂的反义链和正义链核心延伸碱基序列。可以包含在本发明的某些组合物中和/或在本发明的某些方法中施用的单链反义分子在本文中称为“单链反义试剂”或“反义多核苷酸试剂”。可以包含在某些组合物中和/或在本发明的某些方法中施用的单链正义分子在本文中称为“单链正义试剂”或“正义多核苷酸试剂”。术语“碱基序列”在本文中是指没有化学修饰或递送化合物的多核苷酸序列。例如，表1所示的正义链对应于是表3中的相应碱基序列；但表3中的相应序列中显示了各自的化学修饰和递送化合物。在此公开的序列可以被分配标识符。例如，单链正义序列可以用“正义链SS#”来标识；单链反义序列可以用“反义链AS#”来标识；并且包含正义链和反义链的双链体可以用“双链体AD#”来标识。
表1包括正义链和反义链，并提供了由表1中同一行上的正义链和反义链形成的双链体的标识号。在本发明的某些实施方案中，反义序列在其第1位中包含核碱基u或核碱基a。在本发明的某些实施方案中，反义序列在反义序列的第1位包含核碱基u。如本文所用，术语“匹配位置”在某种意义上是指当两条链作为双链体时每条链中互相“配对”的位置。例如，在21核碱基正义链和21核碱基反义链中，正义链第1位与反义链第21位的核碱基处于“匹配位置”。在另一个非限制性实例中，对于23核碱基正义链和23核碱基反义链，正义链的第2位核碱基与反义链的22位处于匹配位置。在另一个非限制性实例中，在18核碱基正义链和18核碱基反义链中，正义链第1位核碱基与反义链第18位核碱基处于匹配位置；并且正义链中的第4位核碱基与反义链中的第15位核碱基处于匹配位置。技术人员会理解如何识别双链和成对链的正义和反义链之间的匹配位置。
表1中的第一列表示双链体的双链体AV#，该双链体在表格同一行中包含正义和反义序列。例如，表1公开了指定为“双链体AV00206”的双链体，其包含相应的正义链序列和反义链序列。因此，表1中的每一行都标识了本发明的双链体，每一个都包含显示在同一行中的正义和反义序列，每个双链体的指定标识符均显示在该行的第一列中。
在本发明方法的一些实施方案中，向对象施用包含表1中所示多核苷酸序列的RNAi 试剂。在本发明的一些实施方案中，向对象施用的RNAi试剂包括双链体，所述双链体包含表1中列出的碱基序列中的至少一个，并包含0、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23或24个序列修改。在本发明方法的一些实施方案中，还包括将表1中所示多核苷酸序列的RNAi试剂连接至递送分子上，其非限制性实例是包含GalNAc的递送化合物。
表1：无修饰的黄嘌呤脱氢酶(XDH)基因RNAi试剂反义链和正义链序列


表1所有序列均显示为5'到3'方向。双链体AV#是分配给表中同一行中两条链的双链体的编号。表1第三列中的“SEQ ID NO”是第二列中显示的正义序列的序列编号，表1第五列中的“SEQ ID NO”是第四列中显示的反义序列的序列编号。
表2显示了本发明的某些化学修饰的黄嘌呤脱氢酶(XDH)基因RNAi剂反义链和正义链序列。在本发明方法的一些实施方案中，将具有表2中所示多核苷酸序列的RNAi试剂施用于细胞和/或对象。在本发明方法的一些实施方案中，将具有表2中所示多核苷酸序列的RNAi试剂施用于对象。在本发明的一些实施方案中，向对象施用的RNAi试剂包含在表2第一列中标注的双链体，并且分别包含显示在表2中同一行第三列和第六列的正义和反义链序列中的序列修饰。在本发明方法的一些实施方案中，表2中所示的序列可以连接到(在本文中也称为“缀合到”)能够将RNAi试剂递送至对象的细胞和/或组织的化合物上。可用于本发明的某些实施方案中的递送化合物的非限制性实例是含GalNAc的化合物。在表2中，第一列表示碱基序列的双链体AV#，与表1对应。对于双链体AV#标识的碱基序列，不仅显示正义和反义链所包含的碱基序列，而且具有表2同一行中所示的指定化学修饰。例如，表1第一行显示了正义和反义碱基单链序列，它们一起构成双链体，标识为：双链体AV#AV00206；而表2列出的双链体AV#AV00206中，作为双链体，其包含AV00206-SS和AV00206-AS的碱基序列，而且分别包含在第三列和第六列中显示的正义和反义序列中的化学修饰。表2第二列中的“正义链SS#”是同一行中第三列所示正义序列(包括修饰)的指定标识符。表2第五列中的“反义链AS#”是第六列中显示的反义序列(包括修饰)的指定标识符。表2第四列中的“SEQ ID NO”是同一行中第三列所示正义序列(包括修饰)的序列编号。表2第七列中的“SEQ ID NO”是第六列中显示的反义序列(包括修饰)的序列编号。



表3显示了本发明的某些化学修饰的黄嘌呤脱氢酶(XDH)基因RNAi试剂反义链和正义链序列。在本发明方法的一些实施方案中，将表3中所示的RNAi试剂施用于细胞和/或对象。在本发明方法的一些实施方案中，将具有表3中所示多核苷酸序列的RNAi试剂施用于对象。在本发明的一些实施方案中，向对象施用的RNAi试剂包含在表3的第一列中标识的双链体，并且分别包含在表3中同一行第三列和第六列的正义和反义链序列中显示的序列修饰和/或递送化合物。该序列用于本文别处描述的某些体内测试研究。在本发明方法的一些实施方案中，表3中所示的序列可以连接到(在本文中也称为“缀合到”)用于递送的化合物上，其非限制性实例是含GalNAc的化合物，即在表3中第三列的正义链上具有标识为“GLX-n”的递送化合物。递送化合物的某些实施方案被标识为表3中第三列有义链上的“GLS-5”、“GLS-15”或“GLX-n”。如本文所用和表3中所示，“GLS-5”、“GLS-15”和“GLX-n”表示含有GalNAc的化合物。如本文所用，“GLX”用于表示“GLS”或“GLO”递送化合物(“X”可以是“S”或“O”)，并且GLX-n可以是任何GLS和GLO可以在合成过程中连接到寡核苷酸3'-或者5'-末端的递送化合物。举例但不局限于此：GLX-13和GLX-14可以在合成过程中连接到本发明的寡核苷酸的3'-末端；GLX-5和GLX-15可以在合成过程中连接到本发明的寡核苷酸的5'-末端。在一些实施例中，如本文所用和表3所示，“GLX-n”用于表示所连接的含GalNAc的化合物，其被代替为化合物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和GLO-16中的任一种。在一些实施中，GLX-n表示为举例但是不限于此现有技术中(Jayaprakash,et al.,(2014)J.Am.Chem.Soc.,136,16958-16961)公开的可用于所连接的含GalNAc的化合物，该化合物用于本文别处描述的某些体内测试研究。本领域技术人员将能够制备和使用本发明的dsRNA化合物，其中附着的递送化合物包括但是不局限于：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和GLO-16。其中每一个的结构在本文别处提供。表3的第一列提供了分配给表中该行中的正义和反义序列的双链体的双链体AD#。例如，双链体AD#AD00352是正义链AD00352-SS和反义链AD00352-AS构成的双链体。表3中的每一行提供了一条正义链和一条反义链，并公开了所示正义链和反义链构成的双链体。表3第二列中的“正义链SS#”是同一行第三列所示正义序列(包括修改)的指定标识符。表3第五列中的“反义链AS#”是第六列中显示的反义序列(包括修饰)的指定标识符。某些所连接的含GalNAc的GLO化合物的标识符显示为GLX-n，并且应当理解，GLO-n或GLS-n化合物中的另一种可以替代显示为GLX-n的化合物，所得化合物也包括所得化合物包括在本发明的方法和/或组合物的实施方案中。表3中SEQ ID NO:350至SEQ ID NO:397，SEQ ID NO:446至SEQ ID NO:451是正义链的序列并且SEQ ID NO:398至SEQ ID NO:445，SEQ ID NO:452至SEQ ID NO:457是反义链序列。在表3中，递送分子表示为在正义链NO:350至SEQ ID NO:397，SEQ ID NO:446至SEQ ID  NO:451的3'或者5'端的“GLS-5”、“GLS-15”或“GLX-n”。化学修饰表示为：大写：2'-氟；小写：2'-甲氧基；硫代磷酸盐：*；Invab：反向无碱基；imann：在每条链末端时:或当进一步偶联递送分子或靶向基团时：


错配
本领域技术人员已知，对于dsRNA的功效而言，错配是可以容忍的，尤其是错配在dsRNA的末端区域内的情况。某些错配具有更好的耐受性，例如具有摆动碱基对G:U和A:C的错配对功效的耐受性更好(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)。本发明的方法和化合物的一些实施方案中，黄嘌呤脱氢酶(XDH)dsRNA试剂可以含有一个或更多个与黄嘌呤脱氢酶(XDH)靶序列的错配。在一些实施方案中，本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂不包含错配。在某些实施方案中，本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂包含不超过1个错配。在一些实施方案中，本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂包含不超过2个错配。在某些实施方案中，本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂包含不超过3个错配。在本发明的一些实施方案中，黄嘌呤脱氢酶(XDH)dsRNA试剂的反义链包含与不位于互补区域中心的黄嘌呤脱氢酶(XDH)靶序列的错配。在一些实施方案中，黄嘌呤脱氢酶(XDH)dsRNA试剂的反义链包含1、2、3、4或更多个错配，其位于互补区域的5'或3'末端之一或两者的最末5、4、3、2或1个核苷酸内。本文所述的方法和/或本领域已知的方法可用于确定包含与黄嘌呤脱氢酶(XDH)靶序列错配的黄嘌呤脱氢酶(XDH)dsRNA试剂是否有效抑制黄嘌呤脱氢酶(XDH)基因的表达。
互补性
如本文所用，除非另有说明，否则术语“互补性/互补”当用于描述第一核苷酸序列(例如，黄嘌呤脱氢酶(XDH)dsRNA试剂正义链或靶标黄嘌呤脱氢酶(XDH)mRNA)与第二核苷酸序列(例如，黄嘌呤脱氢酶(XDH)dsRNA试剂反义链或单链反义多核苷酸)的相关性时，是指包含第一核苷酸序列的寡核苷酸或多核苷酸与包含第二核苷酸序列的寡核苷酸或多核苷酸杂交[在哺乳动物生理条件(或体外类似条件)下形成碱基对间氢键]、并且在某些条件下形成双螺旋或双螺旋结构的能力。其中也可以应用其他条件，例如在生物体内可能遇到的生理相关条件。技术人员将能够根据杂交核苷酸的最终应用确定最适合测试两个序列互补性的条件集。互补序列包括沃森-克里克碱基对或非沃森-克里克碱基对，并且包括天然或修饰的核苷酸或核苷酸模拟物，只要至少达到上述杂交要求的程度即可。序列同一性或互补性与修饰无关。
例如，在如本文所述的黄嘌呤脱氢酶(XDH)dsRNA内的互补序列包含含有第一核苷酸序列的寡核苷酸或多核苷酸与含有第二核苷酸序列的寡核苷酸或多核苷酸在一个或两个核苷酸序列的全长上的碱基配对。此类序列在本文中可被称为彼此“完全互补”。应当理解，在设计两个寡核苷酸以在杂交时形成一个或更多个单链突出端的实施方案中，这种突出端在本文中不被视为基于互补性确定的错配。例如，黄嘌呤脱氢酶(XDH)dsRNA试剂包含一个长度为19个核苷酸的寡核苷酸和另一个长度为20个核苷酸的寡核苷酸，其中较长的寡核苷酸包含与较短的寡核苷酸完全互补的19个核苷酸 的序列，出于本文所述的目的，此种情况可以称为“完全互补”。因此，如本文所用，“完全互补”是指第一多核苷酸的连续序列中的所有(100％)碱基会与第二多核苷酸的连续序列中的相同数目的碱基杂交。连续序列可以包含第一或第二核苷酸序列的全部或部分。
如本文所用，术语“基本互补”是指在核碱基序列的杂交对中，第一多核苷酸的连续序列中的碱基的至少约85％(但不是全部)会与第二多核苷酸的连续序列中相同数目的碱基杂交。如果两个序列在杂交时包含一个或更多个错配碱基对，例如至少1、2、3、4或5个错配碱基对，则可以使用术语“基本上互补”来指第一序列相对于第二序列形成多达15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30个碱基对(bp)的双链体，同时保留在与其最终应用最相关的条件下杂交的能力，例如，通过RISC途径抑制黄嘌呤脱氢酶(XDH)基因表达。术语“部分互补”在本文中可用于指核碱基序列的杂交对中，第一多核苷酸的连续序列中碱基的至少75％(但不是全部)会与第二多核苷酸的连续序列中相同数目的碱基杂交。在一些实施方案中，“部分互补”是指第一多核苷酸的连续序列中至少76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的碱基会与第二多核苷酸的连续序列中相同数量的碱基杂交。
术语“互补”、“完全互补”、“基本互补”和“部分互补”在本文中使用时可用于指黄嘌呤脱氢酶(XDH)dsRNA试剂的正义链与反义链之间的碱基匹配、黄嘌呤脱氢酶(XDH)dsRNA试剂的反义链与靶黄嘌呤脱氢酶(XDH)mRNA的序列之间的碱基匹配，或单链反义寡核苷酸与靶黄嘌呤脱氢酶(XDH)mRNA序列之间的碱基匹配。应当理解，术语“黄嘌呤脱氢酶(XDH)dsRNA试剂的反义链”可以指与“黄嘌呤脱氢酶(XDH)反义多核苷酸试剂”相同的序列。
如本文所用，在提及核酸序列时使用的术语“基本相同”或“基本同一性”是指核酸序列与参考序列相比，包含具有至少约85％或更高序列同一性的序列，优选至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％同一性。序列同一性的百分比通过在比对窗口上比较两个序列的最佳比对来确定。百分比是通过以下方式来计算的：确定在两个序列中出现相同核酸碱基的位置数以产生匹配位置的数量；将匹配位置的数量除以比对窗口中的位置总数，然后将结果乘以100，从而得出序列同一性的百分比。本文公开的发明包括与本文公开(例如，在表1-3中)的那些基本相同的核苷酸序列。在一些实施方案中，所述核苷酸序列与本文公开(例如，在表1-3中)的序列完全相同，或具有至少约85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性。
如本文所用，术语“包含序列的链”是指包含核苷酸链的寡核苷酸，所述核苷酸链由使用标准核苷酸命名法指代的序列描述。如本文所用，术语“双链RNA”或“dsRNA”指包含RNA分子或RNAi分子复合物的序列，所述分子或复合物具有包含 两条反向平行且基本或完全互补的核酸链的杂交双链区，其分别被称为相对于靶黄嘌呤脱氢酶(XDH)基因RNA具有“正义”和“反义”方向。双链区可以具有允许通过RISC途径特异性降解靶标黄嘌呤脱氢酶(XDH)基因RNA的任何所需长度，但通常长度为9至30个碱基对，例如长度为15-30个碱基对。考虑到9到30个碱基对之间的双链体，双链体可以是此范围内的任何长度，例如，9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30个碱基对，以及其中的任何子范围，包括但不限于15-30个碱基对、15-26个碱基对；15-23碱基对、15-22碱基对、15-21碱基对、15-20碱基对、15-19碱基对、15-18碱基对、15-17碱基对、18-30个碱基对、18-26个碱基对、18-23个碱基对、18-22个碱基对、18-21个碱基对、18-20个碱基对、19-30个碱基对、19-26个碱基对、19-23个碱基对、19-22个碱基对、19-21个碱基对、19-20个碱基对、20-30个碱基对、20-26个碱基对、20-25个碱基对、20-24个碱基对、20-23个碱基对、20-22个碱基对、20-21个碱基对、21-30个碱基对、21-26个碱基对、21-25个碱基对、21-24个碱基对、21-23个碱基对或21-22个碱基对。通过用切丁酶和类似酶加工在细胞中产生的黄嘌呤脱氢酶(XDH)dsRNA试剂的长度通常在19-22个碱基对的范围内。黄嘌呤脱氢酶(XDH)dsDNA剂的双链区的一条链包含与靶黄嘌呤脱氢酶(XDH)基因RNA的区域基本互补的序列。形成双链体结构的两条链可以来自具有至少一个自身互补区的单个RNA分子，或者可以由两个或更多个单独的RNA分子形成。在双链区由单个分子形成的情况下，该分子可以具有由单链核苷酸链在3'-末端的一条链和相应的5'-末端的另一条链形成的双链体结构(本文称为“发夹环”)。在本发明的一些实施方案中，发夹构型包含至少1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20个或更多个未配对的核苷酸。当黄嘌呤脱氢酶(XDH)dsRNA试剂的基本互补的两条链由单独的RNA分子组成时，这些分子不需要共价连接，但也可以共价连接。当两条链通过发夹环以外的方式共价连接时，连接结构被称为“接头”。术语“siRNA”在本文中也用于指如本文所述的dsRNA试剂。
在本发明的一些实施方案中，黄嘌呤脱氢酶(XDH)dsRNA试剂可以包含在dsRNA试剂的一个或两个末端具有未配对核苷酸或核苷酸类似物的正义和反义序列。没有未配对核苷酸的末端被称为“平末端”并且没有核苷酸突出端。如果dsRNA试剂的两端都是平末端，则dsRNA被称为“平末端的”。在本发明的一些实施方案中，dsRNA试剂的第一末端是平末端的，在一些实施方案中，dsRNA试剂的第二末端是平末端的，并且在本发明的某些实施方案中，黄嘌呤脱氢酶(XDH)dsRNA试剂的两个末端都是平末端的。
在本发明的dsRNA试剂的一些实施方案中，dsRNA不具有一个或两个平末端。在这种情况下，在dsRNA试剂的一条链的末端有至少一个未配对的核苷酸。例如，当dsRNA一条链的3'-末端延伸超出另一条链的5'-末端时，则存在核苷酸突出端，反之亦然。dsRNA可包含至少1、2、3、4、5、6或更多个核苷酸的突出端。核苷酸突出端可 包含核苷酸/核苷类似物或由其组成，包括脱氧核苷酸/核苷。应当理解，在一些实施方案中，核苷酸突出端在dsRNA试剂的正义链上、在dsRNA试剂的反义链上，或在dsRNA试剂的两端，突出端的核苷酸可存在于dsRNA的反义链或正义链的5'端、3'端或两端。在本发明的某些实施方案中，突出端中的一个或更多个核苷酸被核苷硫代磷酸酯替换。
如本文所用，术语“反义链”或“引导链”是指包含与黄嘌呤脱氢酶(XDH)靶序列基本互补的区域的黄嘌呤脱氢酶(XDH)dsRNA试剂的链。如本文所用，术语“正义链”或“过客链”是指包含与黄嘌呤脱氢酶(XDH)dsRNA试剂的反义链的区域基本互补的区域的黄嘌呤脱氢酶(XDH)dsRNA试剂的链。
修饰
在本发明的一些实施方案中，黄嘌呤脱氢酶(XDH)基因RNAi剂的RNA被化学修饰以获得增强的稳定性和/或一种或更多种其他有益特性。本发明的某些实施方案中的核酸可以通过本领域公知的方法合成和/或修饰，例如，见“Current protocols in Nucleic Acid Chemistry,"Beaucage,S.L.et al.(Eds.),John Wiley&Sons,Inc.,New York,N.Y.,USA，其作为参考在此并入本文。可以存在于本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂的某些实施方案中的修饰包括例如：(a)末端修饰，例如5'端修饰(磷酸化、缀合、反向连接等)、3'端修饰(缀合、DNA核苷酸、反向连接等)；(b)碱基修饰，例如用稳定碱基、去稳定碱基或与扩展的配偶体库进行碱基配对的碱基替换、缺失碱基(无碱基核苷酸)或缀合碱基；(c)糖修饰(例如，在2'位置或4'位置)或糖的替换；以及(d)骨架修饰，包括磷酸二酯键的修饰或替换。在本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂、黄嘌呤脱氢酶(XDH)反义多核苷酸和黄嘌呤脱氢酶(XDH)正义多核苷酸的某些实施方案中可用的RNA化合物的具体实例包括但不限于包含修饰骨架或没有天然核苷间键联的RNA。作为非限制性实例，具有骨架修饰的RNA在骨架中可以不具有磷原子。在其核苷间骨架中没有磷原子的RNA可称为寡核苷。在本发明的某些实施方案中，修饰的RNA在其核苷间骨架中具有磷原子。
应当理解，术语“RNA分子”或“RNA”或“核糖核酸分子”不仅包括在自然界中表达或发现的RNA分子，还包括RNA的类似物和衍生物，其包含一种或更多种如本文所述或本领域已知的核糖核苷酸/核糖核苷类似物或衍生物。术语“核糖核苷”和“核糖核苷酸”在本文中可互换使用。RNA分子可以在核碱基结构或核糖-磷酸骨架结构中进行修饰(例如，如下文所述)，并且包含核糖核苷类似物或衍生物的分子必须保留形成双链体的能力。作为非限制性实例，RNA分子还可包含至少一种修饰的核糖核苷，其包括但不限于2'-甲氧基修饰的核苷、包含5'硫代磷酸酯基团的核苷、与胆固醇衍生物或十二烷酸双癸酰胺基团相连的末端核苷、锁核苷、无碱基核苷、2'-脱氧-2'-氟修饰的核苷、2'-氨基修饰的核苷、2'-烷基修饰的核苷、吗啉代核苷、氨基磷酸酯或包含核苷的非天然碱基，或其任何组合。在本发明的一些实施方案中，RNA分子包含以下数量的修饰的核糖核苷：至少1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、 16、17、18、19、20或长达黄嘌呤脱氢酶(XDH)dsRNA试剂分子的核糖核苷的全长。对于这种RNA分子中的多个修饰的核糖核苷中的每一个，修饰不必相同。
在一些实施方案中，本发明的dsRNA试剂、黄嘌呤脱氢酶(XDH)反义多核苷酸和/或黄嘌呤脱氢酶(XDH)正义多核苷酸可以包含一个或更多个独立选择的修饰核苷酸和/或一个或更多个独立选择的非磷酸二酯键。本文所用的术语“独立选择”用于指选定的要素，例如修饰的核苷酸、非磷酸二酯键等，是指两个或更多个选定的要素可以彼此相同但不必彼此相同。如本文所用，“核苷酸碱基”、“核苷酸”或“核碱基”是杂环嘧啶或嘌呤化合物，其是所有核酸的标准成分，并且包括形成核苷酸的碱基：腺嘌呤(a)、鸟嘌呤(g)、胞嘧啶(c)、胸腺嘧啶(t)和尿嘧啶(u)。核碱基可进一步修饰以包括(但不旨在限制)：通用碱基、疏水性碱基、混杂碱基、尺寸扩大的碱基和氟化碱基。术语“核糖核苷酸”或“核苷酸”在本文中可用于指未修饰的核苷酸、修饰的核苷酸或替代部分。本领域技术人员将认识到，鸟嘌呤、胞嘧啶、腺嘌呤和尿嘧啶可以被其他部分替换，而不会显著改变包含带有这种替换部分的核苷酸的寡核苷酸的碱基配对特性。
在一个实施方案中，预期用于本文所述的方法和组合物中的修饰的RNA是肽核酸(PNA)，其具有形成所需双链体结构并且允许或介导靶RNA经由RISC途径的特异性降解的能力。在本发明的某些实施方案中，黄嘌呤脱氢酶(XDH)基因RNA干扰剂包括与靶黄嘌呤脱氢酶(XDH)基因RNA序列相互作用以指导靶黄嘌呤脱氢酶(XDH)基因RNA切割的单链RNA。
修饰的RNA骨架可以包含例如硫代磷酸酯、手性硫代磷酸酯、二硫代磷酸酯、磷酸三酯、氨基烷基磷酸三酯、甲基和其他烷基膦酸酯(包括3'-亚烷基膦酸酯和手性膦酸酯)、次膦酸酯、氨基磷酸酯(包括3'-氨基氨基磷酸酯和氨基烷基氨基磷酸酯)、硫代氨基磷酸酯、硫代烷基膦酸酯、硫代烷基磷酸三酯，和硼酸磷酸酯(其具有正常3'-5'连接的，以及这些的2'-5'连接类似物，以及具有倒置极性的那些，其中相邻的核苷单元对以3'-5'至5'-3'或2'-5'至5'-2'形式连接)。还包括各种盐、混合盐和游离酸形式。制备含磷键的方法是本领域的常规实施手段，并且此类方法可用于制备本发明的某些修饰的黄嘌呤脱氢酶(XDH)dsRNA试剂、某些修饰的黄嘌呤脱氢酶(XDH)反义多核苷酸和/或某些修饰的黄嘌呤脱氢酶(XDH)正义多核苷酸。
其中不包含磷原子的修饰的RNA骨架具有由短链烷基或环烷基核苷间键联、混合杂原子和烷基或环烷基核苷间键联、或一个或更多个短链杂原子或杂环核苷间键联形成的骨架。其包括具有吗啉键的那些(部分由核苷的糖部分形成)；硅氧烷骨架；硫化物、亚砜和砜骨架；甲乙酰和硫甲乙酰骨架；亚甲基甲乙酰和硫甲乙酰骨架；含有烯烃的骨架；氨基磺酸盐骨架；亚甲基亚氨基和亚甲基肼基骨架；磺酸盐和磺酰胺骨架；酰胺骨架；以及其他混合有N、O、S和CH2成分的部分。制备不含磷原子的修饰的RNA骨架的方法在本领域中是常规实践，并且此类方法可用于制备本发明的某些修饰的黄嘌呤脱氢酶(XDH)dsRNA试剂、某些修饰的黄嘌呤脱氢酶(XDH)反义多核苷酸和/或 某些修饰的黄嘌呤脱氢酶(XDH)正义多核苷酸。
在本发明的某些实施方案中，RNA模拟物被包括在黄嘌呤脱氢酶(XDH)dsRNA、黄嘌呤脱氢酶(XDH)反义多核苷酸和/或黄嘌呤脱氢酶(XDH)正义多核苷酸中，例如但不限于用新基团替换核苷酸单元的糖和核苷间键联(即骨架)。在此类实施方案中，保持碱基单位以与合适的黄嘌呤脱氢酶(XDH)核酸靶化合物杂交。一种这样的寡聚化合物(已被证明具有优异杂交特性的RNA模拟物)，被称为肽核酸(PNA)。在PNA化合物中，RNA的糖骨架被含有酰胺的骨架，特别是氨乙基甘氨酸骨架取代。核碱基被保留并直接或间接地与骨架酰胺部分的氮杂氮原子结合。制备RNA模拟物的方法是本领域常规实践的，并且此类方法可用于制备本发明的某些修饰的黄嘌呤脱氢酶(XDH)dsRNA试剂。
本发明的一些实施方案包括具有硫代磷酸酯骨架的RNA和具有杂原子骨架的寡核苷，特别是-CH2-NH-CH2-、-CH2-N(CH3)-O-CH2-[称为亚甲基(甲基亚氨基)或MMI骨架]、-CH2-O-N(CH3)-CH2-、-CH2-N(CH3)-N(CH3)-CH2-以及-N(CH3)-CH2-[其中天然磷酸二酯骨架表示为-O-P-O-CH2-]。制备具有硫代磷酸酯骨架的RNA和具有杂原子骨架的寡核苷的方法是本领域常规实践的，并且此类方法可用于制备本发明的某些修饰的黄嘌呤脱氢酶(XDH)dsRNA试剂、某些黄嘌呤脱氢酶(XDH)反义多核苷酸和/或某些黄嘌呤脱氢酶(XDH)正义多核苷酸。
修饰的RNA还可以包含一个或更多个取代的糖部分。本发明的黄嘌呤脱氢酶(XDH)dsRNA、黄嘌呤脱氢酶(XDH)反义多核苷酸和/或黄嘌呤脱氢酶(XDH)正义多核苷酸可在2'位置包含以下之一：OH；F；O-、S-、或N-烷基；O-、S-或N-烯基；O-、S-或N-炔基；或O-烷基-O-烷基，其中烷基、烯基和炔基可以是取代或未取代的C1至C10烷基或C2至C10烯基和炔基。示例性的合适的修饰包括：O[(CH2)nO]mCH3、O(CH2)nOCH3、O(CH2)nNH2、O(CH2)nCH3、O(CH2)nONH2、以及O(CH2)nON[(CH2)nCH3)]2，其中n和m为1至约10。在其他实施方案中，dsRNA在2'位置包括以下之一：C1至C10低级烷基、取代的低级烷基、烷芳基、芳烷基、O-烷芳基或O-芳烷基、SH、SCH3、OCN、Cl、Br、CN、CF3、OCF3、SOCH3、SO2CH3、ONO2、NO2、N3、NH2、杂环烷基、杂环烷芳基、氨基烷基氨基、聚烷基氨基；取代的甲硅烷基、RNA裂解基团、报告基团、嵌入剂；用于改善黄嘌呤脱氢酶(XDH)dsRNA试剂的药代动力学特性的基团；或用于改善黄嘌呤脱氢酶(XDH)dsRNA试剂、黄嘌呤脱氢酶(XDH)反义多核苷酸和/或黄嘌呤脱氢酶(XDH)正义多核苷酸的药效学特性的基团，和其他具有类似性质的取代基。在一些实施方案中，修饰包括2'-甲氧基乙氧基(2'-O-CH2CH2OCH3，也称为2'-O-(2-甲氧基乙基)或2'-MOE)(Martin et al.,Helv.Chim.Acta,1995,78:486-504)，即烷氧基-烷氧基。另一种示例性修饰是2'-二甲氨基乙氧基乙氧基，即O(CH2)2ON(CH3)2基团，也称为2'-DMAOE，如下文实施例中所述；以及2'-二甲氨基乙氧基乙氧基(在本领域中也称为2'-O-二甲氨基乙氧基乙基或2'-DMAEOE)，即2'-O-CH2-O-CH2-N(CH2)2。制备所描述的那些的修饰RNA的方法是本领 域常规实践的，并且此类方法可用于制备本发明的某些修饰的黄嘌呤脱氢酶(XDH)dsRNA试剂。
其他修饰包括2'-甲氧基(2'-OCH3)、2'-氨基丙氧基(2'-OCH2CH2CH2NH2)和2'-氟(2'-F)。类似的修饰也可以在本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂、黄嘌呤脱氢酶(XDH)反义多核苷酸的RNA上的其他位置、黄嘌呤脱氢酶(XDH)正义多核苷酸和/或黄嘌呤脱氢酶(XDH)正义多核苷酸的其他位置，特别是3'末端核苷酸上或2'-5'连接的黄嘌呤脱氢酶(XDH)dsRNA、黄嘌呤脱氢酶(XDH)反义多核苷酸或黄嘌呤脱氢酶(XDH)正义多核苷酸中的糖的3'位置、和5'末端核苷酸的5'位置进行。黄嘌呤脱氢酶(XDH)dsRNA试剂、黄嘌呤脱氢酶(XDH)反义多核苷酸和/或黄嘌呤脱氢酶(XDH)正义多核苷酸也可以具有糖模拟物，例如代替呋喃戊糖的环丁基部分。制备例如所描述的那些的修饰RNA的方法是本领域常规实践的，并且此类方法可用于制备本发明的某些修饰的黄嘌呤脱氢酶(XDH)dsRNA试剂、黄嘌呤脱氢酶(XDH)反义多核苷酸和/或黄嘌呤脱氢酶(XDH)正义多核苷酸。
在一些实施方案中，黄嘌呤脱氢酶(XDH)dsRNA试剂、黄嘌呤脱氢酶(XDH)反义多核苷酸和/或黄嘌呤脱氢酶(XDH)正义多核苷酸可以包括核碱基(在本领域中通常简称为“碱基”)修饰或取代。如本文所用，“未修饰的”或“天然”核碱基包括嘌呤碱基腺嘌呤(A)和鸟嘌呤(G)，以及嘧啶碱基胸腺嘧啶(T)、胞嘧啶(C)和尿嘧啶(U)。修饰的核碱基包括其他合成和天然核碱基，例如5-甲基胞嘧啶(5-me-C)、5-羟甲基胞嘧啶、黄嘌呤、次黄嘌呤、2-氨基腺嘌呤、6-甲基和其他腺嘌呤和鸟嘌呤的烷基衍生物、2-丙基和其他腺嘌呤和鸟嘌呤的烷基衍生物、2-硫尿嘧啶、2-硫胸腺嘧啶和2-硫胞嘧啶、5-卤尿嘧啶和胞嘧啶、5-丙炔基尿嘧啶和胞嘧啶、6-偶氮尿嘧啶、胞嘧啶和胸腺嘧啶、5-尿嘧啶(假尿嘧啶)、4-硫尿嘧啶；8-卤代、8-氨基、8-硫醇、8-硫代烷基、8-羟基以及其他8-取代的腺嘌呤和鸟嘌呤；5-卤代，特别是5-溴、5-三氟甲基和其他5-取代的尿嘧啶和胞嘧啶；7-甲基鸟嘌呤和7-甲基腺嘌呤、8-氮杂鸟嘌呤和8-氮杂腺嘌呤、7-氮杂鸟嘌呤和7-氮杂腺嘌呤以及3-氮杂鸟嘌呤和3-氮杂腺嘌呤。可以包含在本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂的某些实施方案中的另外的核碱基是本领域已知的，参见例如：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,J.L,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,S.T.and Lebleu,B.,Ed.,CRC Press,1993。制备包含核碱基修饰和/或取代的dsRNA、黄嘌呤脱氢酶(XDH)反义链多核苷酸和/或黄嘌呤脱氢酶(XDH)正义链多核苷酸(例如本文所述的那些)的方法是本领域常规实践的，并且此类方法可用于制备本发明的某些修饰的黄嘌呤脱氢酶(XDH)dsRNA试剂、黄嘌呤脱氢酶(XDH)正义多核苷酸和/或黄嘌呤脱氢酶(XDH)反义多核苷酸。
本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂、黄嘌呤脱氢酶(XDH)反义多核 苷酸和/或黄嘌呤脱氢酶(XDH)正义多核苷酸的某些实施方案包括经修饰以包括一种或更多种锁核酸(LNA)的RNA。锁核酸是具有这样的修饰核糖部分的核苷酸，其包含额外的连接2'和4'碳的桥。这种结构有效地将核糖“锁定”在3'-内结构构象中。在本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂、黄嘌呤脱氢酶(XDH)反义多核苷酸和/或黄嘌呤脱氢酶(XDH)正义多核苷酸中添加锁核酸可以增加血清中的稳定性，并减少脱靶效应(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)。制备包含锁核酸的dsRNA试剂、黄嘌呤脱氢酶(XDH)反义多核苷酸和/或黄嘌呤脱氢酶(XDH)正义多核苷酸的方法是本领域常规实施的，并且此类方法可用于制备本发明的某些修饰的黄嘌呤脱氢酶(XDH)dsRNA试剂。本发明的黄嘌呤脱氢酶(XDH)dsRNA化合物、正义多核苷酸和/或反义多核苷酸的某些实施方案包括至少一种修饰的核苷酸，其中所述至少一种修饰的核苷酸包含：2'-甲氧核苷酸、2'-氟核苷酸、2'-脱氧核苷酸、2',3'-seco核苷酸模拟物、锁核苷酸、2'-F-阿拉伯糖核苷酸、2'-甲氧基乙基核苷酸、2'-氨基修饰的核苷酸、2'-烷基修饰的核苷酸、吗啉代核苷酸和3'-Ome核苷酸、包含5'-硫代磷酸酯基团的核苷酸，或与胆固醇衍生物或十二烷酸双癸酰胺基团连接的末端核苷酸、2'-氨基修饰的核苷酸、氨基磷酸酯或包含核苷酸的非天然碱基。在一些实施方案中，黄嘌呤脱氢酶(XDH)dsRNA化合物在反义链(在本文中也称为引导链)的5’末端处包含E-乙烯基膦酸酯核苷酸。
本发明的某些实施方案中，在黄嘌呤脱氢酶(XDH)dsRNA化合物、正义多核苷酸的3'和5'末端和/或反义多核苷酸的3'末端包含至少一种修饰的核苷酸，其中至少一种修饰的核苷酸包括：无碱基核苷酸、核糖醇、反向核苷酸、反向无碱基核苷酸、反向2'-OMe核苷酸、反向2'-脱氧核苷酸。本领域技术人员已知，在寡核苷酸末端包含无碱基或反向无碱基核苷酸可增强稳定性(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)。
本发明的某些实施方案中，黄嘌呤脱氢酶(XDH)dsRNA化合物、反义多核苷酸包含至少一种修饰的核苷酸，其中所述至少一种修饰的核苷酸包含开环核酸核苷酸(UNA)或/和二醇核酸核苷酸(GNA)。本领域技术人员已知，UNA和GNA是热不稳定化学修饰，可以显著改善siRNA化合物的脱靶谱(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)。
在某些实施方案中，本发明涉及用于治疗的开环核酸(UNA)寡聚体。开环核酸(unlocked nucleic acid，UNA)是RNA的无环类似物，其中核糖环的C2'和C3'原子之间的键已被切断。已经证明，掺入UNA对siRNA基因沉默活性具有良好的耐受性，在 某些情况下甚至可以增强其活性(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)。
UNA是一种热不稳定修饰，用UNA替换核糖核苷酸会降低碱基配对强度和双链体稳定性。将UNA策略性地放置在siRNA反义链的种子区域可以降低通过microRNA(miRNA)介导的基因沉默机制中的脱靶活性。miRNA主要通过反义种子区(从5'端开始的第2-8位)与靶mRNA之间的碱基配对来识别靶基因，以进行基因抑制。每个miRNA都可能调节大量基因。RNA诱导沉默复合物(RISC)所加载的siRNA反义链也可以通过miRNA介导的机制潜在地调节大量非预期基因。因此，在siRNA的种子区域中加入热不稳定的核苷酸，如UNA，可以降低脱靶活性(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.)。具体而言，这样的RNA寡核苷酸或RNA寡核苷酸的复合物在种子区域含有至少一个UNA核苷酸单体(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)。
根据本技术方案，在RNA寡核苷酸或RNA寡核苷酸的复合物中并入UNA的潜在优势包括但不限于：
1.减少脱靶活性。在siRNA种子区添加UNA会降低种子区的碱基配对强度，从而降低由micro-RNA机制引起的潜在脱靶活性。
2.UNA在siRNA活性方面具有良好的耐受性。在某些情况下，UNA可以导致活性增强。
可用于本技术方案的示例性UNA单体包括但不限于：
Invab是反向无碱基(脱氧核糖)残基。例如，在一些实施方案中，有义链或反义链可包括“末端帽”，其如本文所用是可以在本文公开的RNAi试剂的链的一个或多个末端处并入的非核苷酸化合物或其他部分，并且在一些情况下可以为RNAi试剂提供某些有利特性，诸如例如针对核酸外切酶降解的保护。在一些实施方案中，反向无碱基残基(Invab)作为末端帽添加(参见，例如，
F.Czauderna,Nucleic Acids Res.,2003,31(11),2705-16)。末端帽通常是本领域中已知 的，在一些实施方案中，末端帽存在于有义链的5'末端处、3'末端处或5'和3'末端两者处。
本发明的某些实施方案中，黄嘌呤脱氢酶(XDH)dsRNA化合物、反义多核苷酸包含至少一种修饰的核苷酸，其中所述至少一种修饰的核苷酸包含异甘露糖醇核苷酸。在某些实施方案中，正义链在3'或和5'末端分别独立地包含一个异甘露糖醇残基及其立体异构体。特定的包含异甘露糖醇残基及其立体异构体的核苷酸的实例包括但不限于：
其中短语“Olig”各自独立地代表多核苷酸部分。示例性异甘露糖醇残基(imann)包括但不限于以下： 在某些实施方案中，异甘露醇残基(imann)还可以使用其立体异构体替代。 在某些实施方案中，正义链在3'或和5'末端分别独立地包含一个异甘露醇残基(imann)，并可选地进一步包含5'-末端缀合的靶向基团，例如，靶向基团N-乙酰-半乳糖胺，优选为上述GLS-15，示例性结构如下：.
其中短语“Olig”各自独立地代表多核苷酸部分。
可以在本发明的某些实施方案的黄嘌呤脱氢酶(XDH)dsRNA试剂、黄嘌呤脱氢酶(XDH)反义多核苷酸和/或黄嘌呤脱氢酶(XDH)正义多核苷酸的RNA中包含另一种修饰，其包括分别增强黄嘌呤脱氢酶(XDH)dsRNA试剂、黄嘌呤脱氢酶(XDH)反义多核苷酸和/或黄嘌呤脱氢酶(XDH)正义多核苷酸的一种或更多种特征的一种或更多种配体、部分或与RNA化学连接的缀合物。可以增强的特征的非限制性实例是：黄嘌呤脱氢酶(XDH)dsRNA试剂、黄嘌呤脱氢酶(XDH)反义多核苷酸和/或黄嘌呤脱氢酶(XDH)正义多核苷酸活性、细胞分布、黄嘌呤脱氢酶(XDH)dsRNA试剂的递送、黄嘌呤脱氢酶(XDH)dsRNA试剂的药代动力学特性以及黄嘌呤脱氢酶(XDH)dsRNA试剂的细胞摄取。在本发明的一些实施方案中，黄嘌呤脱氢酶(XDH)dsRNA试剂包含一个或更多个靶向基团或连接基团，在本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂的某些实施方案中，其与正义链缀合。靶向基团的非限制性实例是包含N-乙酰基-半乳糖胺(GalNAc)的化合物。术语“递送分子”、“靶向基团”、“靶向剂”、“连接剂”、“靶向化合物”和“靶向配体”在本文中可互换使用。在本发明的某些实施方案中，黄嘌呤脱氢酶(XDH)dsRNA试剂包含与正义链的5'-末端缀合的靶向化合物。在本发明的某些实施方案中，黄嘌呤脱氢酶(XDH)dsRNA试剂包含与正义链的3'-末端缀合的靶向化合物。在本发明的一些实施方案中，黄嘌呤脱氢酶(XDH)dsRNA试剂包含含有GalNAc的靶向基团。在本发明的某些实施方案中，黄嘌呤脱氢酶(XDH)dsRNA试剂不包含与正义链的3'-末端和5'-末端之一或两者缀合的靶向化合物。在本发明的某些实施方案中，黄嘌呤脱氢酶(XDH)dsRNA试剂不包含与正义链的5'-末端和3'-末端之一或两者缀合的含有GalNAc的靶向化合物。
另外的靶向和连接剂是本领域众所周知的，例如，可用于本发明的某些实施方案中的靶向和连接剂包括但不限于脂质部分，例如胆固醇部分(Letsinger et al.,Proc.Natl.Acid.Sci.USA,1989,86:6553-6556)、胆酸(Manoharan et al.,Biorg.Med.Chem.Let.,1994,4:1053-1060)、硫醚，例如beryl-S-三苯甲基硫醇(Manoharan et al.,Ann.N.Y.Acad. Sci.,1992,660:306-309；Manoharan et al.,Biorg.Med.Chem.Let.,1993,3:2765-2770)、硫胆固醇(Oberhauser et al.,Nucl.Acids Res.,1992,20:533-538)、脂肪链，例如十二烷二醇或十一烷基残基(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)、磷脂，例如二-十六烷基-rac-甘油或三乙基-铵1,2-二-O-十六烷基-rac-甘油-3-膦酸酯(Manoharan et al.,Tetrahedron Lett.,1995,36:3651-3654；Shea et al.,Nucl.Acids Res.,1990,18:3777-3783)、聚胺或聚乙二醇链(Manoharan et al.,Nucleosides&Nucleotides,1995,14:969-973)或金刚烷乙酸(Manoharan et al.,Tetrahedron Lett.,1995,36:3651-3654)、棕榈酰部分(Mishra et al.,Biochim.Biophys.Acta,1995,1264:229-237)或十八胺或己氨基-羰氧基胆固醇部分(Crooke et al.,J.Pharmacol.Exp.Ther.,1996,277:923-937)。
包含黄嘌呤脱氢酶(XDH)dsRNA试剂、黄嘌呤脱氢酶(XDH)反义多核苷酸和/或黄嘌呤脱氢酶(XDH)正义多核苷酸的组合物的某些实施方案可包含改变黄嘌呤脱氢酶(XDH)dsRNA试剂的分布、靶向等性质的配体。在包含本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂的组合物的一些实施方案中，例如与不存在此类配体的物种相比，配体增加对选定靶标(例如分子、细胞或细胞类型、区室，例如细胞或器官区室、组织、器官或身体区域)的亲和力。在本发明的组合物和/或方法中有用的配体可以是天然存在的物质，例如蛋白质(例如人血清白蛋白(HSA)、低密度脂蛋白(LDL)或球蛋白)、碳水化合物(例如，葡聚糖、支链淀粉、几丁质、壳聚糖、菊粉、环糊精或透明质酸)或脂质。配体也可以是重组或合成分子，例如合成聚合物，例如合成聚氨基酸或聚胺。聚氨基酸的实例是聚赖氨酸(PLL)、聚L-天冬氨酸、聚L-谷氨酸、苯乙烯-马来酸酐共聚物、聚(L-丙交酯-共-乙醇酸)共聚物、二乙烯基醚-马来酸酐共聚物、N-(2-羟丙基)甲基丙烯酰胺共聚物(HMPA)、聚乙二醇(PEG)、聚乙烯醇(PVA)、聚氨酯、聚(2-乙基丙烯酸)、N-异丙基丙烯酰胺聚合物，或聚磷嗪。多胺的示例包括：聚乙烯亚胺、聚赖氨酸(PLL)、精胺、亚精胺、多胺、假肽-多胺、拟肽多胺、树枝状多胺、精氨酸、脒、鱼精蛋白、阳离子脂质、阳离子卟啉、多胺的季盐或α螺旋肽。
本发明的组合物和/或方法中包含的配体可包含靶向基团，其非限制性实例为细胞或组织靶向剂，例如，凝集素、糖蛋白、脂质或蛋白质，例如结合特定细胞类型如肾细胞或肝细胞的抗体。靶向基团可以是促甲状腺素、促黑素、凝集素、糖蛋白、表面活性蛋白A、黏蛋白碳水化合物、多价乳糖、多价半乳糖、N-乙酰-半乳糖胺、N-乙酰-葡糖胺多价甘露糖、多价岩藻糖、糖基化聚氨基酸、多价半乳糖、转铁蛋白、双膦酸盐、聚谷氨酸盐、聚天冬氨酸、脂质、胆固醇、类固醇、胆汁酸、叶酸、维生素B12、维生素A、生物素或RGD肽或RGD肽模拟物。
配体的其他实例包括染料、嵌入剂(例如吖啶)、交联剂(例如补骨脂素、丝裂霉素C)、卟啉(TPPC4、texaphyrin、Sapphyrin)、多环芳烃(例如吩嗪、二氢吩嗪)；人工核酸内切酶(例如EDTA)、亲脂性分子，例如胆固醇、胆酸、金刚烷乙酸、1-芘丁酸、二氢睾酮、1,3-双-O(十六烷基)甘油、香叶氧基己基、十六烷基甘油、冰片、薄荷 醇、1,3-丙二醇、十七烷基、棕榈酸、肉豆蔻酸、O3-(油酰基)石胆酸、O3-(油酰基)胆酸、二甲氧基三苯甲基或吩噁嗪和肽缀合物(例如，触角肽、Tat肽)、烷化剂、磷酸盐、氨基、巯基、PEG(例如，PEG-40K)、MPEG、[MPEG]2、聚氨基、烷基、取代烷基、放射性标记物、酶、半抗原(例如生物素)、转运/吸收促进剂(例如阿司匹林、维生素E、叶酸)、合成核糖核酸酶(例如咪唑、双咪唑、组胺、咪唑簇、吖啶-咪唑偶联物、四氮杂大环的Eu3+复合物)、二硝基苯基、HRP或AP。
本发明的组合物和/或方法中包括的配体可以是蛋白质，例如糖蛋白或肽，例如对共配体具有特定亲和力的分子，或抗体，例如与特定细胞类型如癌细胞、内皮细胞、心肌细胞或骨细胞结合的抗体。在本发明的组合物和/或方法的实施方案中有用的配体可以是激素或激素受体。在本发明的组合物和/或方法的实施方案中有用的配体可以是脂质、凝集素、碳水化合物、维生素、辅酶、多价乳糖、多价半乳糖、N-乙酰-半乳糖胺、N-乙酰-葡糖胺多价甘露糖或多价岩藻糖。在本发明的组合物和/或方法的实施方案中有用的配体可以是例如通过破坏细胞的细胞骨架(例如，通过破坏细胞的微管、微丝和/或中间丝)而增加黄嘌呤脱氢酶(XDH)dsRNA试剂向细胞中的摄取的物质。此类试剂的非限制性实例是：taxon、长春新碱、长春碱、细胞松弛素、诺考达唑、japlakinolide、latrunculin A、鬼笔环肽、swinholide A、indanocine和myoservin。
在一些实施方案中，与本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂连接的配体用作药代动力学(PK)调节剂。可用于本发明的组合物和方法的PK调节剂的实例包括但不限于：亲脂剂、胆汁酸、类固醇、磷脂类似物、肽、蛋白质结合剂、PEG、维生素、胆固醇、脂肪酸、胆酸、石胆酸、二烷基甘油酯、二酰基甘油酯、磷脂、鞘脂、萘普生、布洛芬、维生素E、生物素、结合血清蛋白的适体等。还已知包含许多硫代磷酸酯键的寡核苷酸与血清蛋白结合，因此，在骨架中包含多个硫代磷酸酯键的短寡核苷酸，例如约5个碱基、10个碱基、15个碱基或20个碱基的寡核苷酸也可用作本发明的组合物和/或方法中的配体。
黄嘌呤脱氢酶(XDH)dsRNA试剂组合物
在本发明的一些实施方案中，黄嘌呤脱氢酶(XDH)dsRNA试剂在组合物中。本发明的组合物可包含一种或更多种黄嘌呤脱氢酶(XDH)dsRNA试剂和任选的一种或更多种药学上可接受的载体、递送剂、靶向剂、可检测标签等，根据本发明方法的一些实施方案可用的靶向剂的非限制性实例是将本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂引导至和/或进入待治疗细胞的试剂。靶向剂的选择将取决于以下要素：黄嘌呤脱氢酶(XDH)相关疾病或病症的性质，以及靶细胞类型。在一个非限制性实例中，在本发明的一些实施方案中，可能需要将黄嘌呤脱氢酶(XDH)dsRNA试剂靶向至和/或进入肝细胞。应当理解，在本发明方法的一些实施方案中，治疗剂包含仅具有递送剂的黄嘌呤脱氢酶(XDH)dsRNA试剂，例如包含N-乙酰半乳糖胺(GalNAc)的递送剂，而没有任何附加的连接元件。例如，在本发明的一些方面，黄嘌呤脱氢酶(XDH) dsRNA试剂可以连接到包含GalNAc的递送化合物上，并且包含在含有药学上可接受载体的组合物中，并且在没有任何连接至黄嘌呤脱氢酶(XDH)dsRNA试剂的可检测标记或靶向剂等的情况下施用至细胞或对象。
在本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂与一种或更多种递送剂、靶向剂、标记剂等一起施用和/或连接到其上的情况下，本领域技术人员能够了解并能够选择和使用适合的试剂用于本发明的方法中。在本发明的某些方法中可以使用标记试剂来确定黄嘌呤脱氢酶(XDH)dsRNA试剂在细胞和组织中的位置，并且可用于确定已在本发明的方法中施用的包含黄嘌呤脱氢酶(XDH)dsRNA试剂的治疗组合物的细胞、组织或器官位置。接附和使用标记试剂如酶标记、染料、放射性标记等的手段是本领域公知的。应当理解，在本发明的组合物和方法的一些实施方案中，标记试剂连接至黄嘌呤脱氢酶(XDH)dsRNA试剂中所包含的正义多核苷酸和反义多核苷酸之一或两者。
黄嘌呤脱氢酶(XDH)dsRNA试剂和黄嘌呤脱氢酶(XDH)反义多核苷酸试剂的递送
本发明方法的某些实施方案包括将黄嘌呤脱氢酶(XDH)dsRNA试剂递送到细胞中。如本文所用，术语“递送”是指促进或影响细胞摄取或吸收。黄嘌呤脱氢酶(XDH)dsRNA试剂的吸收或摄取可通过独立的扩散或活性细胞过程来发生，或通过使用可与本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂相关的递送剂、靶向剂等来进行。适用于本发明方法的递送方式包括但不限于体内递送，其中将黄嘌呤脱氢酶(XDH)dsRNA试剂注射到组织部位或全身给药。在本发明的一些实施方案中，黄嘌呤脱氢酶(XDH)dsRNA试剂连接至递送剂。
可用于将黄嘌呤脱氢酶(XDH)dsRNA试剂递送至细胞、组织和/或对象的方法的非限制性实例包括：黄嘌呤脱氢酶(XDH)dsRNA-GalNAc缀合物、SAMiRNA技术、基于LNP的递送方法和裸RNA递送。这些和其他递送方法已在本领域成功地用于递送治疗性RNAi试剂以治疗各种疾病和病症，例如但不限于：肝病、急性间歇性卟啉症(AIP)、血友病、肺纤维化等。多种递送方式的详细信息可在出版物中找到，例如：Nikam,R.R.&K.R.Gore(2018)Nucleic Acid Ther,28(4),209-224 Aug 2018；Springer A.D.&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；和Nair,J.K.et al.,(2014)J.Am.Chem.Soc.136:16958-16961，其内容均以引用方式并入本文。
本发明的一些实施方案包括使用脂质纳米颗粒(LNP)将本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂递送至细胞、组织和/或对象。LNP通常用于体内递送黄嘌呤脱氢酶(XDH)dsRNA试剂，包括治疗性黄嘌呤脱氢酶(XDH)dsRNA试剂。使用LNP或其他递送剂的一个好处是，当使用LNP或其他递送剂递送至对象时，黄嘌呤脱氢酶(XDH)基因RNA剂的稳定性增加。在本发明的一些实施方案中，LNP包含负载有一种或更多种本发明的黄嘌呤脱氢酶(XDH)基因RNAi分子的阳离子LNP。将包 含黄嘌呤脱氢酶(XDH)基因RNAi分子的LNP施用于对象，LNP及其接附的黄嘌呤脱氢酶(XDH)基因RNAi分子通过胞吞作用被细胞摄取，它们的存在导致RNAi触发分子的释放，从而介导RNAi。
可用于本发明的实施方案以将本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂递送至细胞、组织和/或对象的递送剂的另一个非限制性实例是：包含GalNAc的试剂，其与本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂连接并将黄嘌呤脱氢酶(XDH)dsRNA试剂递送至细胞、组织和/或对象。PCT申请WO2020191183A1中公开了可用于本发明的方法和组合物的某些实施方案中的某些包含GalNAc的其他递送剂的实例。可用于本发明的组合物和方法中以将黄嘌呤脱氢酶(XDH)dsRNA试剂递送至细胞的GalNAc靶向配体的非限制性实例是靶向配体簇。在此提出的靶向配体簇的实例如：具有磷酸二酯键的GalNAc配体(GLO)和具有硫代磷酸酯键的GalNAc配体(GLS)。术语“GLX-n”在本文中可用于表示所连接的含GalNAc的化合物举例并不局限于是以下化合物：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和GLO-16中的任一种，每个的结构如下所示，下图中GalNAc靶向配体与本发明的RNAi剂的连接位置在每个靶向配体的最右侧。应当理解，本发明的任何RNAi和dsRNA分子都可以连接到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和GLO-16上，以下是GLO-1到GLO-16和GLS-1到GLS-16的结构。



“杂环烷基”表示具有指定数目的环原子的非芳族的部分饱和的或完全饱和的环(例如，3-10或3-7元杂环烷基)，所述环原子由一个或多个选自N、O和S的杂原子(例如，1、2、3或4个杂原子)组成，且其余的环原子为碳。5元杂环烷基是具有5个环原子的杂环烷基。6元杂环烷基是具有6个环原子的杂环烷基。杂环烷基可以是单环或多环(例如，二环、三环)的。杂环烷基的例子包括氧杂环丙基、氮杂环丙基、氮杂环丁基、吡咯烷基、咪唑烷基、吡唑烷基、哌啶基、哌嗪基、吗啉基和硫代吗啉基。此外，多环杂环烷基的一个环可以为芳族的(例如，芳基或杂芳基)，只要所述多环杂环烷基经由非芳族碳或氮原子结合至母体结构。例如，1,2,3,4-四氢喹啉-1-基(其中所 述部分经由非芳族氮原子结合至母体结构)被认为是杂环烷基，而1,2,3,4-四氢喹啉-8-基(其中所述部分经由芳族碳原子结合至母体结构)不被认为是杂环烷基。低级杂环烷烃一般是指C3-6个单环，在无特殊说明情况下，低级杂环烷基一般可优先为完全饱和的碳环。在本发明的一些实施方案中，体内递送也可以通过β-葡聚糖递送系统，例如美国专利No.5,032,401和5,607,677,以及美国公布No.2005/0281781中描述的那些，它们的全部内容通过引用并入本文。也可以使用本领域已知的方法例如电穿孔和脂质转染将黄嘌呤脱氢酶(XDH)基因RNAi试剂体外引入细胞。在本发明方法的某些实施方案中，黄嘌呤脱氢酶(XDH)dsRNA在没有靶向剂的情况下被递送。这些RNA可以作为“裸”RNA分子递送。作为非限制性实例，本发明的黄嘌呤脱氢酶(XDH)dsRNA可以在包含RNAi试剂但不包含靶向剂(例如GalNAc靶向化合物)的药物组合物中施用于对象，以治疗对象的黄嘌呤脱氢酶(XDH)相关疾病或病症，例如痛风。
应当理解，除了本文描述的某些递送方式之外，其他RNAi递送方式可以与本文描述的黄嘌呤脱氢酶(XDH)基因RNAi试剂和治疗方法的实施方案结合使用，例如但不限于本文描述的那些和本领域中使用的那些。
本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂可以以有效降低细胞和/或对象中黄嘌呤脱氢酶(XDH)多肽的水平和活性的量和方式施用于对象。在本发明方法的一些实施方案中，将一种或更多种黄嘌呤脱氢酶(XDH)dsRNA试剂施用于细胞和/或对象以治疗与黄嘌呤脱氢酶(XDH)表达和活性相关的疾病或病症。在一些实施方案中，本发明的方法包括向需要此类治疗的对象施用一种或更多种黄嘌呤脱氢酶(XDH)dsRNA试剂以减轻对象中与黄嘌呤脱氢酶(XDH)表达相关的疾病或病症。可以施用本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂以降低体外、离体和体内细胞中的一种或更多种中的黄嘌呤脱氢酶(XDH)表达和/或活性。
在本发明的一些实施方案中，通过将黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂递送(例如引入)细胞中来降低细胞中黄嘌呤脱氢酶(XDH)多肽的水平并因此降低其活性。靶向剂和方法可用于帮助将黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸剂递送至对象内的特定细胞类型、细胞亚型、器官、空间区域，和/或细胞内的亚细胞区域。黄嘌呤脱氢酶(XDH)dsRNA试剂可以在本发明的某些方法中单独地或与一种或更多种另外的黄嘌呤脱氢酶(XDH)dsRNA试剂组合施用。在一些实施方案中，向对象施用2、3、4或更多种独立选择的黄嘌呤脱氢酶(XDH)dsRNA试剂。在本发明的某些实施方案中，将黄嘌呤脱氢酶(XDH)dsRNA试剂与一种或更多种用于治疗黄嘌呤脱氢酶(XDH)相关疾病或病症的另外的治疗方案联合施用于对象以治疗黄嘌呤脱氢酶(XDH)相关疾病或病症。另外的治疗方案的非限制性实例是：施用一种或更多种本发明的黄嘌呤脱氢酶(XDH)反义多核苷酸、施用非黄嘌呤脱氢酶(XDH)dsRNA治疗剂和行为改变。可以在以下一个或更多个时间施用另外的治疗方案：在施用本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂之前、同时和之后。应当理解，本文所用的“同时”是指在零时 间的5分钟内、零时间的10分钟内、零时间的30分钟内、零时间的45分钟内和零时间的60分钟内，其中“零时间”是向对象施用本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂的时间。非黄嘌呤脱氢酶(XDH)dsRNA治疗剂的非限制性实例是：另外的治疗剂，例如别嘌醇，羟嘌呤，非布索坦或白介素-1β(IL-1β)拮抗剂，优选地，所述白介素-1β拮抗剂选自卡那单抗或利纳西普(rilonacept)，从而治疗患有将从黄嘌呤脱氢酶(XDH)减少中受益的疾病的受试者表达，或上述任何的组合，及配制成药剂组合的治疗高尿酸血症，痛风，非酒精性脂肪肝疾病(NAFLD)，非酒精性脂肪性肝炎(NASH)，代谢紊乱，胰岛素抵抗，心血管疾病，2型糖尿病，Lesch Nyhan综合征，糖原贮积病(GSD)，以及与氧化应激有关的疾病或其他XDH相关疾病的治疗剂。行为改变的非限制性实例是：饮食方案、咨询和锻炼方案。这些和其他治疗剂以及行为改变是本领域已知的，并且可用于治疗对象的黄嘌呤脱氢酶(XDH)疾病或病症，并且还可以与一种或更多种本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂组合，向对象给药以治疗黄嘌呤脱氢酶(XDH)疾病或病症。向细胞或对象施用以治疗黄嘌呤脱氢酶(XDH)相关疾病或病症的本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂可以与一种或更多种其他治疗剂或活性成分以协同方式起作用，从而增加一种或更多种治疗剂或活性成分的有效性和/或增加黄嘌呤脱氢酶(XDH)dsRNA试剂治疗黄嘌呤脱氢酶(XDH)相关疾病或病症的有效性。
本发明的治疗方法包括施用黄嘌呤脱氢酶(XDH)dsRNA试剂，可在黄嘌呤脱氢酶(XDH)相关疾病或病症发作之前和/或当存在黄嘌呤脱氢酶(XDH)相关疾病或病症时使用，包括在疾病或病症的早期、中期、晚期阶段以及任何这些阶段之前和之后的所有时间使用。本发明的方法还可以治疗先前已经接受过一种或更多种其他治疗剂和/或治疗活性成分的黄嘌呤脱氢酶(XDH)相关疾病或病症治疗的对象，其中一种或更多种其他治疗剂和/或治疗活性成分在治疗对象的黄嘌呤脱氢酶(XDH)相关疾病或病症方面不成功、成功性最小和/或不再成功。
载体编码的dsRNA
在本发明的某些实施方案中，可以使用载体将黄嘌呤脱氢酶(XDH)dsRNA试剂递送到细胞中。黄嘌呤脱氢酶(XDH)dsRNA试剂转录单位可包含在DNA或RNA载体中。用于将序列递送到细胞和/或对象中的此类编码转基因的载体的制备和使用是本领域公知的。可在本发明的方法中使用导致黄嘌呤脱氢酶(XDH)dsRNA瞬时表达的载体，瞬时表达例如至少1、2、3、4、5、6、7、8、9、10小时或更多小时、1、2、3、4、5、6、7、8、9、10周或更多周。瞬时表达的长度可以使用基于以下要素的常规方法确定：例如但不限于所选的特定载体构建体和靶细胞和/或组织。此类转基因可作为线性构建体、环状质粒或病毒载体引入，其可为整合或非整合载体。也可以构建转基因以使其作为染色体外质粒遗传(Gassmann,et al.,Proc.Natl.Acad.Sci.USA(1995)92:1292)。
黄嘌呤脱氢酶(XDH)dsRNA试剂的一条或更多条单链可以从表达载体上的启动子转录。在要表达两条单独的链以产生例如dsRNA的情况下，可以使用例如转染或感染的方式将两个单独的表达载体共同引入细胞。在某些实施方案中，本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂的每条单独链都可以被包含在同一表达载体上的启动子转录。在本发明的某些实施方案中，黄嘌呤脱氢酶(XDH)dsRNA试剂被表达为通过接头多核苷酸序列连接的反向重复多核苷酸，使得黄嘌呤脱氢酶(XDH)dsRNA试剂具有茎环结构。
RNA表达载体的非限制性实例是DNA质粒或病毒载体。在本发明的实施方案中有用的表达载体可以与真核细胞相容。真核细胞表达载体在本领域是常规使用的，并且可从许多商业来源获得。黄嘌呤脱氢酶(XDH)dsRNA表达载体的递送可以是全身性的，例如通过静脉内或肌肉内给药、通过给药至从对象移出的靶细胞然后将靶细胞重新引入对象，或通过允许引入所需靶细胞的任何其他方式来进行。
可包括在该方法的实施方案中的病毒载体系统包括但不限于：(a)腺病毒载体；(b)逆转录病毒载体，包括但不限于慢病毒载体、莫洛尼鼠白血病病毒等；(c)腺相关病毒载体；(d)单纯疱疹病毒载体；(e)SV 40载体；(f)多瘤病毒载体；(g)乳头状瘤病毒载体；(h)小核糖核酸病毒载体；(i)痘病毒载体，例如正痘病毒载体，例如痘苗病毒载体或禽痘病毒载体，例如金丝雀痘或家禽痘病毒载体；(j)辅助依赖型或无肠腺病毒载体。用于重组表达黄嘌呤脱氢酶(XDH)dsRNA试剂的构建体可以包含调节元件，例如启动子、增强子等，它们可以被选择以提供组成型或调节型/诱导型表达。病毒载体系统以及启动子和增强子的使用等在本领域是常规的并且可以与本文所述的方法和组合物结合使用。
本发明的某些实施方案包括使用病毒载体将黄嘌呤脱氢酶(XDH)dsRNA试剂递送到细胞中。许多基于腺病毒的递送系统在本领域中常规用于递送至例如肺、肝、中枢神经系统、内皮细胞和肌肉。可用于本发明方法的病毒载体的非限制性实例是：AAV载体、痘病毒如痘苗病毒、改良安卡拉病毒(MVA)、NYVAC、禽痘如禽痘或金丝雀痘病毒。
本发明的某些实施方案包括使用载体将黄嘌呤脱氢酶(XDH)dsRNA试剂递送到细胞中的方法，并且此类载体可以在药学上可接受的载体中，所述载体可以但不必包括其中嵌入基因递送载体的缓释基质。在一些实施方案中，用于递送黄嘌呤脱氢酶(XDH)dsRNA的载体可以由重组细胞产生，并且本发明的药物组合物可以包括一种或更多种产生黄嘌呤脱氢酶(XDH)dsRNA递送系统的细胞。
含有黄嘌呤脱氢酶(XDH)dsRNA或ssRNA试剂的药物组合物
本发明的某些实施方案包括含有黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂和药学上可接受的载体的药物组合物的用途。包含黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸剂的药物组合物 可用于本发明的方法中以降低细胞中的黄嘌呤脱氢酶(XDH)基因表达和黄嘌呤脱氢酶(XDH)活性，并可用于治疗黄嘌呤脱氢酶(XDH)相关疾病或病症。此类药物组合物可以基于递送方式来配制。用于递送方式的制剂的非限制性实例是：配制用于皮下递送的组合物、配制用于通过肠胃外递送全身给药的组合物、配制用于静脉内(IV)递送的组合物、配制用于鞘内递送的组合物、配制用于直接递送至脑中的组合物等。可以使用一种或更多种方式施用本发明的药物组合物以将黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂递送到细胞中，例如：表面(例如，通过透皮贴剂)；肺部，例如通过吸入或吹入粉末或气雾剂，包括通过雾化器；气道内、鼻内、表皮和透皮、口服或肠胃外。肠胃外给药包括静脉内、动脉内、皮下、腹膜内或肌肉内注射或输注；表皮下，例如通过植入装置；或颅内，例如通过实质内；鞘内或心室内施用。黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸剂也可以直接递送至靶组织，例如直接递送至肝脏等。可以理解的是，“递送黄嘌呤脱氢酶(XDH)dsRNA试剂”或“递送黄嘌呤脱氢酶(XDH)反义多核苷酸试剂”到细胞中分别包括递送黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸剂、直接在细胞中表达黄嘌呤脱氢酶(XDH)dsRNA试剂以及从递送到细胞中的编码载体表达黄嘌呤脱氢酶(XDH)dsRNA试剂，或使得黄嘌呤脱氢酶(XDH)dsRNA或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂出现在细胞中的任何合适的方式。制剂的制备和使用以及用于递送抑制性RNA的手段是本领域公知的和常规使用的。
如本文所用，“药物组合物”包含药理学有效量的本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸剂和药学上可接受的载体。术语“药学上可接受的载体”是指用于施用治疗剂的载体。此类载体包括但不限于盐水、缓冲盐水、葡萄糖、水、甘油、乙醇及其组合。该术语明确排除细胞培养基。对于口服给药的药物，药学上可接受的载体包括但不限于药学上可接受的赋形剂，例如惰性稀释剂、崩解剂、黏合剂、润滑剂、甜味剂、调味剂、着色剂和防腐剂。合适的惰性稀释剂包括碳酸钠和碳酸钙、磷酸钠和磷酸钙以及乳糖，而玉米淀粉和藻酸是合适的崩解剂。黏合剂可包括淀粉和明胶，而润滑剂(如果存在)通常是硬脂酸镁、硬脂酸或滑石粉。如果需要，片剂可以用例如单硬脂酸甘油酯或二硬脂酸甘油酯之类的材料包衣，以延迟在胃肠道中的吸收。包含在药物制剂中的试剂在下文进一步描述。如本文所用的术语，例如“药理学有效量”、“治疗有效量”和“有效量”，是指本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂产生预期的药理学、治疗或预防结果的量。例如，如果与疾病或障碍相关的可测量参数至少降低10％时，则认为给定的临床治疗有效，那么用于治疗该疾病或病症的药物的治疗有效量是使该参数降低至少10％所需的量。例如，治疗有效量的黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸剂可以将黄嘌呤脱氢酶(XDH)多肽水平降低至少10％。药物组合物可以包含这样的dsRNAi试剂，其包括例如表1中所显示的双链体AV00206至AV00174。优选的dsRNAi试剂包括例如双链体AV00227、AV00260、AV00285、 AV00266、AV00280、AV00238、AV00249、AV00289、AV00233、AV00225、AV00273或AV00283。在某些实施方案中，优选表2中dsRNAi试剂包括例如双链体AV00227、AV00260、AV00285、AV00266或AV00280。在某些实施方案中，优选的表3中dsRNAi试剂包括例如双链体AD00353、AD00356、AD00358、AD00364、AD00365、AD00366、AD00368、AD00370、AD00430、AD00431、AD00432、AD00521、AD00521-1、AD00364-1、AD00365-2、AD00366-1和AD00432-1。
有效量
在一些方面，本发明的方法包括将细胞与有效量的黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂接触以减少所接触细胞中的黄嘌呤脱氢酶(XDH)基因表达。本发明方法的某些实施方案包括以有效降低黄嘌呤脱氢酶(XDH)基因表达和治疗对象的黄嘌呤脱氢酶(XDH)相关疾病或病症的量向对象施用黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸剂。就减少黄嘌呤脱氢酶(XDH)的表达和/或用于治疗黄嘌呤脱氢酶(XDH)相关疾病或病症而言，所使用的“有效量”是实现所需生物学效果所必需或足够的量。例如，治疗黄嘌呤脱氢酶(XDH)相关疾病或病症的黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂的有效量可以是：(i)减缓或停止疾病或病症的进展所需的量；(ii)逆转、减少或消除疾病或病症的一种或更多种症状。在本发明的一些方面，有效量是当施用于需要治疗黄嘌呤脱氢酶(XDH)相关疾病或病症的对象时，导致疾病或病症的预防和/或治疗的治疗响应的黄嘌呤脱氢酶(XDH dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸剂的量。根据本发明的一些方面，有效量是本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂当与针对黄嘌呤脱氢酶(XDH)相关疾病或病症的另一种治疗性治疗组合或共同施用时，导致预防和/或治疗该疾病或病症的治疗响应的量。在本发明的一些实施方案中，用本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂治疗对象的生物学效应可以是由黄嘌呤脱氢酶(XDH)相关疾病或病症引起的症状的改善和/或完全消除。在本发明的一些实施方案中，生物学效应是黄嘌呤脱氢酶(XDH)相关疾病或病症的完全消除，例如通过指示对象没有黄嘌呤脱氢酶(XDH)相关疾病或病症的诊断测试来证明。可检测的生理症状的非限制性实例包括在施用本发明的试剂后对象临床上血液或尿液中尿酸水平的降低。其他评估黄嘌呤脱氢酶(XDH)相关疾病或病症状态的本领域已知方式可用于确定本发明的试剂和/或方法对黄嘌呤脱氢酶(XDH)相关疾病或病症的影响。
通常在临床试验中确定将黄嘌呤脱氢酶(XDH)多肽活性降低至治疗黄嘌呤脱氢酶(XDH)相关疾病或病症的水平的黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂的有效量，这样的临床试验在盲法研究中为测试人群与对照人群建立有效剂量。在一些实施方案中，有效量是导致所需响应的量，例如减少细胞、 组织和/或患有疾病或病症的对象中的黄嘌呤脱氢酶(XDH)相关疾病或病症的量。因此，用于治疗可通过降低黄嘌呤脱氢酶(XDH)多肽活性治疗的黄嘌呤脱氢酶(XDH)相关疾病或病症的黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂的有效量可以是这样的量：当施用时，将对象中黄嘌呤脱氢酶(XDH)多肽活性的量降低至低于在未施用黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸剂的情况下将存在于细胞、组织和/或对象中的量。在本发明的某些方面，存在于未接触或施用过本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸剂的细胞、组织和/或对象中的黄嘌呤脱氢酶(XDH)多肽活性和/或黄嘌呤脱氢酶(XDH)基因表达的水平被称为“对照”量。在本发明方法的一些实施方案中，对象的对照量是对象的治疗前量；换言之，对象在施用黄嘌呤脱氢酶(XDH)试剂之前的水平可以是该对象的对照水平，并且用于与其在向对象施用siRNA后的黄嘌呤脱氢酶(XDH)多肽活性和/或黄嘌呤脱氢酶(XDH)基因表达水平相比较。在治疗黄嘌呤脱氢酶(XDH)相关疾病或病症的情况下，期望的响应可以是减少或消除细胞、组织和/或对象中疾病或病症的一种或更多种症状。减少或消除可以是暂时的，也可以是永久性的。应当理解，可以使用确定黄嘌呤脱氢酶(XDH)多肽活性、黄嘌呤脱氢酶(XDH)基因表达、症状评估、临床测试等的方法来监测黄嘌呤脱氢酶(XDH)相关疾病或病症的状态。在本发明的一些方面，对治疗黄嘌呤脱氢酶(XDH)相关疾病或病症的期望响应是延迟疾病或病症的发作或甚至预防疾病或病症的发作。
降低黄嘌呤脱氢酶(XDH)多肽活性的化合物的有效量也可以通过以下方式确定：评估施用黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂对细胞或对象的生理作用，例如施用后黄嘌呤脱氢酶(XDH)相关疾病或病症的减少。对象的测定和/或症状监测可用于确定本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸剂的功效(其可以在本发明的药物化合物中给药)，并确定对治疗是否有响应。另一个非限制性示例是：在用本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂治疗对象之前和之后，可以使用一种或更多种本领域已知的血清尿酸水平测试来确定对象的黄嘌呤脱氢酶(XDH)相关疾病或病症的状态。在另一个非限制性实例中，使用一种或更多种本领域已知的尿酸水平测试来确定对象中黄嘌呤脱氢酶(XDH)相关疾病的状态。在该实施例中，疾病包括高尿酸血症，痛风，非酒精性脂肪肝疾病(NAFLD)，非酒精性脂肪性肝炎(NASH)，代谢紊乱，胰岛素抵抗，心血管疾病，2型糖尿病，Lesch Nyhan综合征，糖原贮积病(GSD)，以及与氧化应激有关的疾病或其他XDH相关疾病，并且该测试用于确定在用本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂治疗对象之前和之后对象中的尿酸水平。
本发明的一些实施方案包括确定向对象施用的本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂来治疗黄嘌呤脱氢酶(XDH)相关疾病或病症之功效的方法，其通过评估和/或监测对象中黄嘌呤脱氢酶(XDH)相关疾病或病症的一种或更多种“生理特征”来进行。黄嘌呤脱氢酶(XDH)相关疾病或 病症的生理特征的非限制性实例是许多患者还会出现血液和尿液中尿酸水平升高，疼痛的增加(例如由痛风引起的疼痛)，炎症风险增加事件。确定这种生理特征的标准方法是本领域已知的，包括但不限于血液测试、成像研究、身体检查等，该综合征的定义是存在血液和尿液中尿酸水平的降低，疼痛的减轻(例如由痛风引起的疼痛)，炎症的减少。
可以理解的是，可以至少部分地基于这种对对象确定的疾病和/或病症状态和/或生理特征的测定结果来修改向对象施用的黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸剂的量。治疗量可以通过例如以下方式改变：通过改变施用黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸剂的组合物、通过改变给药途径、通过改变给药时间等，来增加或减少黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂的量。黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸剂的有效量将随着所治疗的特定病症、所治疗对象的年龄和身体状况、病情的严重程度、治疗的持续时间、共同治疗的性质(如果有的话)、具体的给药途径以及健康从业者知识和专业知识范围内的其他因素而变化。例如，有效量可取决于对治疗黄嘌呤脱氢酶(XDH)相关疾病或病症有效的黄嘌呤脱氢酶(XDH)多肽活性和/或黄嘌呤脱氢酶(XDH)基因表达的所需水平。技术人员可以凭经验确定用于本发明方法的特定黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂的有效量，而无需过度实验。结合本文提供的教导，通过从本发明的多种黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂中进行选择，并权衡例如效力、相对生物利用度、患者体重、不良副作用的严重程度和优选的给药方式等因素，可以规划有效的预防性或治疗性治疗方案以有效治疗特定对象。如在本发明的实施方案中使用的，本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂的有效量可以是当与细胞接触时在细胞中产生所需生物学效应的量。
应当认识到，黄嘌呤脱氢酶(XDH)基因沉默可以在表达黄嘌呤脱氢酶(XDH)的任何细胞中通过组成型或通过基因组工程进行，并通过任何合适的测定来确定。在本发明的一些实施方案中，通过施用本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂，黄嘌呤脱氢酶(XDH)基因表达降低至少5％、6％、7％、8％、9％、10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或100％。在本发明的一些实施方案中，通过施用本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂，黄嘌呤脱氢酶(XDH)基因表达减少5％至10％、5％至25％、10％至50％、10％至75％、25％至75％、25％至100％或50％至100％。
给药
黄嘌呤脱氢酶(XDH)dsRNA试剂和黄嘌呤脱氢酶(XDH)反义多核苷酸试剂以足以抑制黄嘌呤脱氢酶(XDH)基因表达的剂量在药物组合物中递送。在本发明的某些实施方案中，黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷 酸剂的剂量为每千克接受者体重每天0.01至200.0毫克，一般为每天1至50mg/kg体重、5至40mg/kg体重、10至30mg/kg体重、1至20mg/kg体重、1至10mg/kg体重、4至15mg/kg体重，包括端值。例如，黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂的每单次给药可以以从约0.01mg/kg、0.05mg/kg、0.1mg/kg、0.2mg/kg、0.3mg/kg、0.4mg/kg、0.5mg/kg、1mg/kg、1.1mg/kg、1.2mg/kg、1.3mg/kg、1.4mg/kg、1.5mg/kg、1.6mg/kg、1.7mg/kg、1.8mg/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/kg、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/kg、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/kg、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至50mg/kg体重的量来施用。
在确定本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂的递送剂量和时间时可以考虑多种因素。递送的黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸剂的绝对量将取决于多种因素，包括共同治疗、剂量数和个体对象参数，包括年龄、身体状况、体格大小和体重。这些是本领域普通技术人员众所公知的因素，并且可以通过常规实验解决。在一些实施方案中，可以使用最大剂量，即根据合理的医学判断的最高安全剂量。
在一些实施方案中，本发明的方法可包括向对象施用1、2、3、4、5、6、7、8、9、10或更多个剂量的黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂。在一些情况下，可以至少每天、每隔一天、每周、每隔一周、每月等向对象施用药物化合物(例如，包含黄嘌呤脱氢酶(XDH)dsRNA试剂或包含黄嘌呤脱氢酶(XDH)反义多核苷酸剂)的剂量，可以每天给药一次或每天给药多于一次，例如在一个24小时周期内给药2、3、4、5或更多次。本发明的药物组合物可以每天给药一次；或者黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂可以在一天中以适当的间隔以两个、三个或更多个亚剂量给药，或者甚至使用连续 输注或通过控释制剂递送。在本发明方法的一些实施方案中，将本发明的药物组合物每天一次或更多次、每周一次或更多次、每月一次或更多次或每年一次或更多次施用给对象。
在某些方面，本发明的方法包括单独施用药物化合物；与一种或更多种其他黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂组合；和/或与对患有黄嘌呤脱氢酶(XDH)相关疾病或病症的对象施用的其他药物疗法或治疗活动或方案组合。药物化合物可以以药物组合物的形式给药。本发明方法中使用的药物组合物可以是无菌的，并且含有一定量的黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂，其会将黄嘌呤脱氢酶(XDH)多肽的活性降低到足以在适合施用于对象的重量或体积单位中产生所需响应的水平。可以根据不同参数选择向对象施用的包含黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸剂药物组合物的剂量以降低黄嘌呤脱氢酶(XDH)蛋白活性，特别是根据所使用的给药方式和对象的状态来进行选择。其他因素包括所需的治疗时间。如果对象在初始剂量下的响应不足，则可以在患者耐受性允许的范围内采用更高的剂量(或通过不同的、更局部的递送途径有效地提高剂量)。
治疗
如本文所用的，术语“预防”或“进行预防”，当用于指将受益于黄嘌呤脱氢酶(XDH)基因表达降低的疾病、病症或其病况时，是指对象发生与此类疾病、病症或病况相关的症状的可能性降低，所述与此类疾病、病症或病况相关的症状是例如与由黄嘌呤脱氢酶(XDH)活化引起或与之相关的疾病或病症相关的症状，例如痛风。在这样的情况下发生痛风的可能性被降低：例如，当个体具有一种或更多种痛风风险因素，其相对于具有相同风险因素且未接受本文所述治疗的人群而言，未能发展出相关疾病、病症或病症，或与此类疾病、病症或病症相关的症状的发展程度降低(例如，在临床上患有该疾病或病症的量表上降低至少约10％)，或延迟症状的表现(例如，延迟数天、数周、数月或数年)，则被认为是有效的预防。
对于黄嘌呤脱氢酶(XDH)相关疾病和病症，其中黄嘌呤脱氢酶(XDH)多肽的水平和/或活性的降低可有效治疗该疾病或病症，可以使用本发明的方法和黄嘌呤脱氢酶(XDH)dsRNA试剂来治疗以抑制黄嘌呤脱氢酶(XDH)表达。可以用本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂和本发明的治疗方法治疗的疾病和病症的实例包括但不限于：高尿酸血症，痛风，NAFLD，NASH，代谢紊乱，胰岛素抵抗，心血管疾病，2型糖尿病，Lesch Nyhan综合征，糖原贮积病(GSD)，例如I型GSD以及与氧化应激有关的疾病，例如慢性低度炎症；或其他XDH相关疾病。此类疾病和病症在本文中可称为“黄嘌呤脱氢酶(XDH)相关疾病和病症”和“由黄嘌呤脱氢酶(XDH)引起和/或调节的疾病和病症”。
在本发明的某些方面，可以在诊断黄嘌呤脱氢酶(XDH)相关疾病或病症之前或 之后的一个或更多个时间向对象施用本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂。在本发明的一些方面，对象处于患有或发展黄嘌呤脱氢酶(XDH)相关疾病或病症的风险中。与发展黄嘌呤脱氢酶(XDH)相关疾病或病症的对照风险相比，有发展黄嘌呤脱氢酶(XDH)相关疾病或病症风险的对象是发展黄嘌呤脱氢酶(XDH)相关疾病或病症的可能性提高的对象。在本发明的一些实施方案中，与风险的对照水平相比，风险水平在统计上是显著的。有风险的对象可包括，例如：是或将是具有预先存在的疾病和/或遗传异常的对象，其使得该对象比没有预先存在的疾病或遗传异常的对照对象更易患黄嘌呤脱氢酶(XDH)相关疾病或病症；具有黄嘌呤脱氢酶(XDH)相关疾病或病症的家族和/或个人病史的对象；以及先前已接受黄嘌呤脱氢酶(XDH)相关疾病或病症治疗的对象。应当理解，使对象对黄嘌呤脱氢酶(XDH)相关疾病或病症更易感的预先存在的疾病和/或遗传异常可以是这样的疾病或遗传异常：当存在时，其先前已被确定为与发展黄嘌呤脱氢酶(XDH)相关疾病或病症的更高可能性具有相关关系。
应当理解，可以基于个体对象的医学状况向对象施用黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂。例如，为对象提供的医疗保健可以评估从对象获得的样品中测量的黄嘌呤脱氢酶(XDH)水平，并确定通过施用本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂来降低对象的黄嘌呤脱氢酶(XDH)水平是可期望的。在一个非限制性实例中，可以从对象获得生物样品，例如血液或血清样品，并且在样品中确定对象的尿酸水平。向对象施用黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂，并且在给药后从对象获得血液或血清样品，并且使用该样品测定尿酸水平，并将该结果与对象给药前(先前)样品中确定的结果进行比较。与给药前水平相比，随后样品中对象的尿酸水平降低则表明所施用的黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂在降低对象的黄嘌呤脱氢酶(XDH)水平方面的功效。在一个非限制性实例中，尿酸水平升高可以被认为是黄嘌呤脱氢酶(XDH)相关病症的生理特征，即使对象没有被诊断为患有黄嘌呤脱氢酶(XDH)相关疾病，例如本文公开的疾病。医疗保健提供者可以监测对象的尿酸水平的变化，作为施用的本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸剂的功效的量度。在一个非限制性实例中，黄嘌呤脱氢酶(XDH)相关疾病是高尿酸血症，痛风，NAFLD，NASH，代谢紊乱，胰岛素抵抗，心血管疾病，2型糖尿病，Lesch Nyhan综合征，糖原贮积病(GSD)，例如I型GSD以及与氧化应激有关的疾病，例如慢性低度炎症；或其他XDH相关疾病。
本发明方法的某些实施方案包括调整治疗，所述治疗包括至少部分地基于对对象中由治疗引起的黄嘌呤脱氢酶(XDH)相关疾病或病症一种或更多种生理特征的变化的评估，来向对象施用本发明的dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂。例如，在本发明的一些实施方案中，可以确定对对象施用的本发明的dsRNA试剂或黄 嘌呤脱氢酶(XDH)反义多核苷酸剂的作用，并用于帮助调节随后向对象施用的本发明的dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸剂的量。在一个非限制性实例中，对对象施用本发明的dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂，并在施用后测定对象尿酸水平；并且至少部分基于所确定的水平，确定是否需要更高量的dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂以提高所施用试剂的生理作用，例如降低或进一步降低对象的尿酸水平。在另一个非限制性实例中，向对象施用本发明的dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸剂，并在给药后确定对象的尿酸水平，并且至少部分地基于所确定的水平，预期向对象施用更低量的dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂。
因此，本发明的一些实施方案包括评估由对象先前治疗引起的一种或更多种生理特征的变化，包括血液和尿液中尿酸水平的降低，疼痛的减轻(例如由痛风引起的疼痛)，炎症的减少等，以调整随后施用于对象的本发明的dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸剂的量。本发明方法的一些实施方案包括对黄嘌呤脱氢酶(XDH)相关疾病或病症的生理特征的1、2、3、4、5、6或更多次测定；评估和/或监测施用的本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂的功效；并任选地使用所测定的结果来调整以下一项或更多项：本发明的dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂治疗对象中黄嘌呤脱氢酶(XDH)相关疾病或病症的剂量、给药方案和/或给药频率。在本发明方法的一些实施方案中，向对象施用有效量的本发明的dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂的期望结果是：与为对象确定的先前相比，对象黄嘌呤脱氢酶(XDH)转录物水平、血浆尿酸水平、黄嘌呤脱氢酶(XDH)基因表达的降低。
如本文所用，术语“治疗”、“治疗性”或“治疗的”当用于黄嘌呤脱氢酶(XDH)相关疾病或病症时可指预防性治疗、降低对象发展黄嘌呤脱氢酶(XDH)相关疾病或病症的可能性，并且也可以指在对象已经发展出黄嘌呤脱氢酶(XDH)相关疾病或病症之后为了消除或降低黄嘌呤脱氢酶(XDH)相关疾病或病症的水平而进行的治疗、防止黄嘌呤脱氢酶(XDH)相关疾病或病症变得更严重，和/或与在不存在降低对象中黄嘌呤脱氢酶(XDH)多肽活性的疗法的情况下的对象相比，减缓对象中黄嘌呤脱氢酶(XDH)相关疾病或病症的进展。
本发明的试剂、组合物和方法的某些实施方案可用于抑制黄嘌呤脱氢酶(XDH)基因表达。如本文所用，关于黄嘌呤脱氢酶(XDH)基因的表达，术语“抑制”、“沉默”、“减少”、“下调”和“敲低”是指例如通过以下一种或更多种情况改变黄嘌呤脱氢酶(XDH)基因的表达：分别与由黄嘌呤脱氢酶(XDH)基因转录的RNA的对照水平、所表达的黄嘌呤脱氢酶(XDH)的活性对照水平或由mRNA翻译的黄嘌呤脱氢酶(XDH)的对照水平相比，当细胞、细胞群、组织、器官或对象与本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂接触(例如，用其处理)时，其中由基因转录的RNA的水平、表达的黄嘌呤脱氢酶(XDH)的活性水平， 以及由细胞、细胞群、组织、器官或对象中的mRNA翻译的黄嘌呤脱氢酶(XDH)多肽、蛋白质或蛋白质亚基的水平降低。在一些实施方案中，对照水平是未接触黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂(例如用其处理)的细胞、组织、器官或对象中的水平。
施用方法
黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸剂的多种给药途径可用于本发明的方法。特定递送模式的选择将至少部分取决于所治疗的特定病症和治疗功效所需的剂量。一般而言，本发明的方法可以使用医学上可接受的任何给药模式来实施，这意味着产生黄嘌呤脱氢酶(XDH)相关疾病或病症的有效治疗水平而不引起临床上不可接受的副作用的任何模式。在本发明的一些实施方案中，黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸剂可以通过口服、肠内、黏膜、皮下和/或肠胃外途径施用。术语“肠胃外”包括皮下、静脉内、鞘内、肌内、腹膜内和胸骨内注射或输注技术。其他途径包括但不限于鼻(例如，通过胃鼻管)、经皮、阴道、直肠、舌下和吸入。本发明的递送途径可包括鞘内、心室内或颅内。在本发明的一些实施方案中，黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸剂可以放置在缓释基质中并通过将基质放置在对象中来施用。在本发明的一些方面，黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸剂可以使用涂覆有靶向特定细胞或细胞器的递送剂的纳米颗粒递送至对象细胞。多种递送方式、方法、试剂是本领域已知的。递送方法和递送剂的非限制性实例在本文别处另外提供。在本发明的一些方面，关于黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂的术语“递送”可以是指：向细胞或对象施用一种或更多种“裸”黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸剂序列。在本发明的某些方面，“递送”是指通过转染方式给予细胞或对象、将包含黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂的细胞递送给对象、将编码黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂的载体递送到细胞和/或对象等中。使用转染方式递送黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸剂可包括向细胞和/或对象施用载体。
在本发明的一些方法中，一种或更多种黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂可以以制剂形式给药，也可以在药学上可接受的溶液中给药，其通常可以含有药学上可接受浓度的盐、缓冲剂、防腐剂、相容的载体、佐剂和任选的其他治疗成分。在本发明的一些实施方案中，黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸剂可以与另一种治疗剂一起配制成用于同时给药。根据本发明的方法，黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂可以以药物组合物的形式给药。通常，药物组合物包含黄 嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸剂和任选的药学上可接受的载体。药学上可接受的载体是本领域普通技术人员公知的。如本文所用，药学上可接受的载体是指不干扰活性成分生物活性(例如，黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸剂抑制细胞或对象中黄嘌呤脱氢酶(XDH)基因表达的能力)有效性的无毒材料。施用和递送用于治疗用途的黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂的多种方法是本领域已知的并且可用于本发明的方法中。
药学上可接受的载体包括稀释剂、填充剂、盐、缓冲剂、稳定剂、增溶剂和本领域公知的其他材料。示例性的药学上可接受的载体描述于美国专利No.5,211,657中，而其他载体是本领域技术人员已知的。这种制剂通常可以含有盐、缓冲剂、防腐剂、相容的载体和任选的其他治疗剂。用于医药时，该盐应当是药学上可接受的，但非药学上可接受的盐可以方便地用于制备其药学上可接受的盐，不排除在本发明的范围之外。此类药理学和药学上可接受的盐包括但不限于由以下酸制备的盐：盐酸、氢溴酸、硫酸、硝酸、磷酸、马来酸、乙酸、水杨酸、柠檬酸、甲酸、丙二酸、琥珀酸等。此外，药学上可接受的盐可以制备为碱金属盐或碱土金属盐，例如钠盐、钾盐或钙盐。
本发明方法的一些实施方案包括将一种或更多种黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂直接施用于组织。在一些实施方案中，施用化合物的组织是其中存在或可能出现黄嘌呤脱氢酶(XDH)相关疾病或病症的组织，其非限制性实例是肝脏或肾脏。直接组织给药可以通过直接注射或其他方式实现。许多口服递送的化合物自然进入并通过肝脏和肾脏，本发明的治疗方法的一些实施方案包括向对象口服施用一种或更多种黄嘌呤脱氢酶(XDH)dsRNA试剂。黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸剂，单独或与其他治疗剂联合，可以施用一次，或者它们可以多次施用。如果多次给药，黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸剂可以通过不同途径给药。例如，虽然不打算限制，第一次(或前几次)给药可以通过皮下方式进行，并且一次或更多次额外给药可以是口服和/或全身给药。
对于其中希望全身性施用黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸剂的本发明实施方案，可以配制黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸剂用于通过注射例如通过推注或连续输注肠胃外施用。注射制剂可以以单位剂型存在，例如安瓿或多剂量容器，其添加或不添加防腐剂。黄嘌呤脱氢酶(XDH)dsRNA试剂制剂(也称为药物组合物)可采用油性或水性载体中的混悬液、溶液或乳液等形式，并且可含有配制剂，例如混悬剂、稳定剂和/或分散剂。
肠胃外给药的制剂包括无菌水溶液或非水溶液、混悬液和乳液。非水溶剂的例子是丙二醇、聚乙二醇、植物油如橄榄油和可注射的有机酯如油酸乙酯。水性载体包括水、酒精/水溶液、乳液或混悬液，包括盐水和缓冲介质。肠胃外载体包括氯化钠溶液、林 格氏葡萄糖溶液、葡萄糖和氯化钠溶液、乳酸林格氏液或固定油。静脉内赋形剂包括流体和营养补充剂、电解质补充剂(例如基于林格氏葡萄糖溶液的那些)等。也可以存在防腐剂和其他添加剂，例如抗微生物剂、抗氧化剂、螯合剂和惰性气体等。其他形式的给药，例如静脉给药，将导致较低的剂量。如果对象在初始剂量下的反应不足，则可以在患者耐受性允许的范围内采用更高的剂量(或通过不同的、更局部的递送途径有效地提高剂量)。可以根据需要每天使用多次剂量以实现一种或更多种黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂的适当全身或局部水平，并实现黄嘌呤脱氢酶(XDH)活性的适当降低。
在其他实施方案中，本发明的方法包括使用递送载体，例如生物相容性微粒、纳米颗粒或适合植入受体例如对象的植入物。PCT公开WO 95/24929(通过引用并入本文)中描述了可根据该方法使用的示例性可生物降解植入物，其描述了用于包含生物大分子的生物相容的、可生物降解的聚合物基质。
不可生物降解的和可生物降解的聚合物基质都可用于本发明的方法中，以将一种或更多种黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂递送给对象。在一些实施方案中，基质可以是可生物降解的。基质聚合物可以是天然或合成聚合物。可以基于期望释放的时间段来选择聚合物，通常在几小时到一年或更长时间的数量级。通常，可以使用在几小时到三到十二个月之间的一段时间内的释放。聚合物任选地呈水凝胶形式，其可以吸收高达其重量约90％的水，并且还任选地与多价离子或其他聚合物交联。
通常，黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂在本发明的一些实施方案中可以使用可生物降解的植入物通过扩散或通过聚合物基质的降解来递送。用于这种用途的示例性合成聚合物是本领域公知的。使用本领域已知的方法，可生物降解的聚合物和不可生物降解的聚合物可用于递送黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂。生物黏附聚合物如可生物侵蚀的水凝胶(H.S.Sawhney,C.P.Pathak and J.A.Hubell in Macromolecules,1993,26,581-587)也可用于递送黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂，以治疗黄嘌呤脱氢酶(XDH)相关疾病或病症。其他合适的递送系统可以包括定时释放、延迟释放或持续释放递送系统。此类系统可避免重复施用黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸剂，从而提高对象和医疗保健专业人员的便利性。许多类型的释放递送系统是可用的并且是本领域普通技术人员已知的。见例如美国专利No.5,075,109、4,452,775、4,675,189、5,736,152、3,854,480、5,133,974和5,407,686。此外，可以使用基于泵的硬件输送系统，其中一些也适用于植入。
长期持续释放植入物的使用可以适用于对象的预防性治疗和具有发生复发性黄嘌呤脱氢酶(XDH)相关疾病或病症的风险的对象。如本文所用，长期释放是指将植入物构建和布置成以至少长达10天、20天、30天、60天、90天、六个月、一年或更长时 间递送治疗水平的黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂。长期持续释放植入物是本领域普通技术人员众所公知的并且包括上述的一些释放系统。
黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂的治疗制剂可以通过将具有所需纯度的分子或化合物与任选的药学上可接受的载体、赋形剂或稳定剂[Remington's Pharmaceutical Sciences 21st edition,(2006)]以冻干制剂或水溶液的形式混合来制备用于储存。可接受的载体、赋形剂或稳定剂在所采用的剂量和浓度下对接受者是无毒的，并且包括缓冲剂，例如磷酸盐、柠檬酸盐和其他有机酸；抗氧化剂，包括抗坏血酸和蛋氨酸；防腐剂(例如十八烷基二甲基苄基氯化铵；六甲铵氯化物；苯扎氯铵、苄索氯铵；苯酚、丁醇或苯甲醇；对羟基苯甲酸酯类，例如对羟基苯甲酸甲酯或丙酯；邻苯二酚；间苯二酚；环己醇；3-戊醇；和间甲酚)；低分子量(少于约10个残基)多肽；蛋白质，例如血清白蛋白、明胶或免疫球蛋白；亲水性聚合物，如聚乙烯吡咯烷酮；氨基酸，例如甘氨酸、谷氨酰胺、天冬酰胺、组氨酸、精氨酸或赖氨酸；单糖、二糖和其他碳水化合物，包括葡萄糖、甘露糖或糊精；螯合剂如EDTA；蔗糖、甘露糖醇、海藻糖或山梨糖醇等糖类；形成盐的反离子，如钠；金属配合物(例如，锌-蛋白质配合物)；和/或非离子表面活性剂，例如或聚乙二醇(PEG)。
细胞、对象和对照
本发明的方法可以与细胞、组织、器官和/或对象结合使用。在本发明的一些方面，对象是人或脊椎动物哺乳动物，包括但不限于狗、猫、马、牛、山羊、小鼠、大鼠和灵长类动物，例如猴。因此，本发明可用于治疗人和非人对象的黄嘌呤脱氢酶(XDH)相关疾病或病症。在本发明的一些方面，对象可以是农场动物、动物园动物、驯养动物或非驯养动物，并且本发明的方法可用于兽医预防和治疗方案。在本发明的一些实施方案中，对象是人并且本发明的方法可用于人预防和治疗方案。
可应用本发明的对象的非限制性实例是被诊断患有、怀疑患有或有风险患有与以下疾病或病症相关的疾病或病症的对象：高于期望的黄嘌呤脱氢酶(XDH)表达和/或活性，也称为“升高的黄嘌呤脱氢酶(XDH)表达水平”。与高于期望水平的黄嘌呤脱氢酶(XDH)表达和/或活性相关的疾病和病症的非限制性实例在本文别处描述。本发明的方法可应用于在治疗时已被诊断为患有该疾病或病症的对象、与高于期望的黄嘌呤脱氢酶(XDH)表达和/或活性相关的对象，或被认为处于患有或发展与高于期望的黄嘌呤脱氢酶(XDH)表达和/或活性相关的疾病或病症的风险中的对象。在本发明的一些方面，与高于期望的黄嘌呤脱氢酶(XDH)表达和/或活性水平相关的疾病或病症是急性疾病或病症；在本发明的某些方面，与高于期望的黄嘌呤脱氢酶(XDH)表达和/或活性水平相关的疾病或病症是慢性疾病或病症。
在一个非限制性实例中，将本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂施用于 被诊断患有XDH相关疾病，包括但是不局限于：高尿酸血症，痛风，NAFLD，NASH，代谢紊乱，胰岛素抵抗，心血管疾病，2型糖尿病，Lesch Nyhan综合征，糖原贮积病(GSD)，例如I型GSD以及与氧化应激有关的疾病，例如慢性低度炎症；或其他XDH相关疾病。本发明的方法可应用于在治疗时已被诊断为患有该疾病或病症的对象，或被认为有患或发展该疾病或病症的风险的对象。
在另一个非限制性实例中，将本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂施用以治疗指因黄嘌呤脱氢酶(XDH)被激活导致或与其相关的疾病或障碍，或其症状或进展响应于黄嘌呤脱氢酶(XDH)失活的疾病或障碍。术语“黄嘌呤脱氢酶(XDH)相关疾病”包括因黄嘌呤脱氢酶(XDH)表达降低而受益的疾病、障碍或病症。这类疾病包括但是不局限于：高尿酸血症，痛风，NAFLD，NASH，代谢紊乱，胰岛素抵抗，心血管疾病，2型糖尿病，Lesch Nyhan综合征，糖原贮积病(GSD)，例如I型GSD以及与氧化应激有关的疾病，例如慢性低度炎症；或其他XDH相关疾病。
可应用本发明方法的细胞包括体外、体内、离体细胞。细胞可以在对象中、在培养物中和/或混悬液中，或处于任何其他合适的状态或条件中。可以应用本发明的方法的细胞可以是：肝脏细胞(liver cell)、肝细胞(hepatocyte)、心脏细胞、胰腺细胞、心血管细胞、肾细胞或其他类型的脊椎动物细胞，包括人和非人哺乳动物细胞。在本发明的某些方面，可应用本发明方法的细胞是健康的正常细胞，其未知为疾病细胞。在本发明的某些实施方案中，将本发明的方法和组合物应用于肝脏细胞、肝细胞、心脏细胞、胰腺细胞、心血管细胞和/或肾细胞的细胞。在本发明的某些方面，对照细胞是正常细胞，但应当理解，具有疾病或病症的细胞也可以在特定情况下用作对照细胞，例如在比较具有疾病或病症的经处理细胞与具有疾病或病症的未处理细胞的结果等的情况下。
根据本发明的方法，可以确定黄嘌呤脱氢酶(XDH)多肽活性的水平并将其与黄嘌呤脱氢酶(XDH)多肽活性的对照水平进行比较。对照可以是预定值，其可以采取多种形式。它可以是单个截止值，例如中位数或平均值。它可以基于比较组来建立，例如在具有正常水平的黄嘌呤脱氢酶(XDH)多肽和/或黄嘌呤脱氢酶(XDH)多肽活性的组和具有增加的黄嘌呤脱氢酶(XDH)多肽和/或黄嘌呤脱氢酶(XDH)多肽活性水平的组中。比较组的另一个非限制性实例可以是具有黄嘌呤脱氢酶(XDH)相关疾病或病症的一种或更多种症状或诊断的群体与没有疾病或病症的一种或更多种症状或诊断的群体；已对其施用本发明的siRNA治疗的对象组与未对其施用本发明的siRNA治疗的对象组。通常，对照可以基于适当年龄组中的明显健康的正常个体或明显健康的细胞。应当理解，除了预定值之外，根据本发明的对照可以是与实验材料平行测试的材料样品。示例包括来自对照群体的样品或通过制造产生的对照样品，以用于与实验样品进行平行测试。在本发明的一些实施方案中，对照可包括未用本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂接触或处理的细胞或对象，在这种情况下，可以比较黄嘌呤脱氢酶(XDH)多肽和/或黄嘌呤脱氢酶(XDH)多肽活性的对照水平以及与本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂接触的细胞或对象中 黄嘌呤脱氢酶(XDH)多肽和/或黄嘌呤脱氢酶(XDH)多肽活性的水平。
在本发明的一些实施方案中，对照水平可以是为对象确定的黄嘌呤脱氢酶(XDH)多肽水平，其中将在不同时间为同一对象确定的黄嘌呤脱氢酶(XDH)多肽水平与该对照水平进行比较。在一个非限制性实例中，在从未接受过本发明的黄嘌呤脱氢酶(XDH)治疗的对象获得的生物样品中确定黄嘌呤脱氢酶(XDH)的水平。在一些实施方案中，生物样品是血清样品。从对象获得的样品中测定的尿酸水平可作为对象的基线或对照值。在本发明的治疗方法中向对象施用一次或更多次黄嘌呤脱氢酶(XDH)dsRNA试剂之后，可以从对象获得一个或更多个另外的血清样品，并且可以将随后的一个或更多个样品中的尿酸水平与对象的对照/基线水平进行比较。此类比较可用于评估对象中黄嘌呤脱氢酶(XDH)相关疾病或病症的发作、进展或消退。例如，从对象获得的基线样品中黄嘌呤脱氢酶(XDH)多肽的水平高于在给予对象本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂后从同一对象获得的水平，则表示黄嘌呤脱氢酶(XDH)相关疾病或病症的消退并且表示施用的本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂治疗黄嘌呤脱氢酶(XDH)相关疾病或病症产生的功效。
在本发明的某些方面，生物样品是肝细胞样品。为对象确定的黄嘌呤脱氢酶(XDH)多肽和/或黄嘌呤脱氢酶(XDH)多肽活性水平中的一个或更多个值可以作为对照值，并用于稍后在同一对象中比较黄嘌呤脱氢酶(XDH)多肽和/或黄嘌呤脱氢酶(XDH)活性水平，从而允许评估对象中“基线”黄嘌呤脱氢酶(XDH)多肽活性的变化。因此，将初始水平用作该对象的对照水平的情况下，初始黄嘌呤脱氢酶(XDH)多肽水平和/或初始黄嘌呤脱氢酶(XDH)多肽活性水平可以用于显示和/或确定本发明的方法和化合物在对象中所能够降低对象中黄嘌呤脱氢酶(XDH)多肽和/或黄嘌呤脱氢酶(XDH)多肽活性的水平。
使用本发明的方法，可以将本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂和/或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂施用于对象。这样的dsRNAi试剂包括例如表1中所显示的双链体AV00206至AV00292。在一些实施方案中，优选的dsRNAi试剂包括例如双链体AV00227、AV00260、AV00285、AV00266、AV00280、AV00238、AV00249、AV00289、AV00233、AV00225、AV00273或AV00283。在另一些实施方案中，优选的表2中dsRNAi试剂包括例如双链体AV00227、AV00260、AV00285、AV00266或AV00280。在另一些实施方案中，优选的表3中dsRNAi试剂包括例如双链体AD00353、AD00356、AD00358、AD00364、AD00365、AD00366、AD00368、AD00370、AD00430、AD00431、AD00521、AD00521-1、AD00364-1、AD00365-2、AD00366-1和AD00432-1。可以如下评估本发明的施用和治疗的功效：与先前时间点从对象获得的血清样品中尿酸的给药前水平相比，或与非接触对照水平(例如对照血清样品中的尿酸水平)相比，当施用和治疗后，从对象获得的血清样品中尿酸的水平降低至少0.5％、1％、5％、10％、20％、30％、40％、50％、60％、70％、80％、90％、95％或更多。应当理解，尿酸的水平与黄嘌呤脱氢酶(XDH)基因表达的水平相关。本发明方法的某些实施方案 包括以有效抑制黄嘌呤脱氢酶(XDH)基因表达的量向对象施用本发明的黄嘌呤脱氢酶(XDH)dsRNA和/或黄嘌呤脱氢酶(XDH)反义试剂，从而降低对象中黄嘌呤脱氢酶(XDH)多肽的水平并降低黄嘌呤脱氢酶(XDH)多肽活性的水平。在本发明的方法的一些实施方案中，细胞与本发明的siRNA试剂的接触(在本文中也称为处理)导致细胞中黄嘌呤脱氢酶(XDH)基因表达抑制至少约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％或约100％，例如，至低于化验检测水平。
本发明的一些实施方案包括从一个或更多个对象获得的一个或更多个生物样品中确定黄嘌呤脱氢酶(XDH)多肽的存在、不存在和/或量(本文也称为水平)。该测定可用于评估本发明的治疗方法的功效。例如，本发明的方法和组合物可用于确定生物样品中黄嘌呤脱氢酶(XDH)多肽的水平，该生物样品获自先前用施用本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂和/或黄嘌呤脱氢酶(XDH)反义剂治疗的对象。与先前时间点从对象获得的肝细胞样品中黄嘌呤脱氢酶(XDH)多肽的给药前水平相比，或与非接触对照水平(例如对照肝细胞样品中的黄嘌呤脱氢酶(XDH)多肽水平)相比，当施用和治疗后，从对象获得的肝细胞样品中黄嘌呤脱氢酶(XDH)多肽的水平降低至少0.5％、1％、5％、10％、20％、30％、40％、50％、60％、70％、80％、90％、95％或更多，则表明给予对象的治疗的功效水平。
在本发明的一些实施方案中，针对对象确定的黄嘌呤脱氢酶(XDH)相关疾病或病症的生理特征可以作为对照结果，并将同一对象在不同时间的生理特征的确定结果与对照结果进行比较。在一个非限制性实例中，病理特征尿酸水平测量自从未给予本发明的黄嘌呤脱氢酶(XDH)治疗的对象，其可用作对象的基线或对照值。在本发明的治疗方法中向对象施用一次或更多次黄嘌呤脱氢酶(XDH)dsRNA试剂之后，尿酸水平分别与对象的对照/基线水平进行比较。此类比较可用于评估对象中黄嘌呤脱氢酶(XDH)相关疾病或病症的发作、进展或消退。例如，从对象获得的基线尿酸水平高于在对对象施用本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂后从同一对象测定的尿酸水平，则表示黄嘌呤脱氢酶(XDH)相关疾病或病症的消退并且表示施用的本发明的黄嘌呤脱氢酶(XDH)dsRNA试剂治疗黄嘌呤脱氢酶(XDH)相关疾病或病症的功效。
本发明的一些实施方案包括使用例如但不限于以下方法确定黄嘌呤脱氢酶(XDH)相关疾病或病症的生理特征的存在、不存在和/或变化：(1)测量对象的尿酸水平；(2)评估从一名或更多名对象获得的一份或更多份生物样品的生理特征；(3)或对对象进行身 体检查。该测定可用于评估本发明的治疗方法的功效。
药盒
包含一种或更多种黄嘌呤脱氢酶(XDH)dsRNA试剂和/或黄嘌呤脱氢酶(XDH)反义多核苷酸试剂及其在本发明方法中的使用说明的药盒也在本发明的范围内。本发明的药盒可包含可用于治疗黄嘌呤脱氢酶(XDH)相关疾病或病症的黄嘌呤脱氢酶(XDH)dsRNA试剂、黄嘌呤脱氢酶(XDH)正义多核苷酸和黄嘌呤脱氢酶(XDH)反义多核苷酸试剂中的一种或更多种。可以制备包含一种或更多种黄嘌呤脱氢酶(XDH)dsRNA试剂、黄嘌呤脱氢酶(XDH)正义多核苷酸和黄嘌呤脱氢酶(XDH)反义多核苷酸试剂的药盒以用于本发明的治疗方法。本发明药盒的组分可以以水性介质或冻干形式包装。本发明的药盒可以包含被分隔开以在其中封闭地收纳一个或更多个容器装置或一系列容器装置(例如试管、小瓶、烧瓶、瓶子、注射器等)的载体。第一容器装置或一系列容器装置可包含一种或更多种化合物，例如黄嘌呤脱氢酶(XDH)dsRNA试剂和/或黄嘌呤脱氢酶(XDH)正义或反义多核苷酸试剂。第二容器装置或一系列容器装置可包含靶向剂、标记剂、递送剂等，其可作为在本发明的治疗方法的实施方案中施用的黄嘌呤脱氢酶(XDH)dsRNA试剂和/或黄嘌呤脱氢酶(XDH)反义多核苷酸的一部分包括在内。
本发明的药盒还可包含说明书。说明书通常采用书面形式，并且将为执行由药盒体现的治疗和基于该治疗做出决定提供指导。
提供以下实施例以说明本发明实践的具体实例，其并不旨在限制本发明的范围。对本领域普通技术人员来说明显的是，本发明可应用于多种组合物和方法。
实施例
实施例1.RNAi试剂的合成
上表1-3中所示的黄嘌呤脱氢酶(XDH)基因RNAi试剂双链体是根据以下通用程序合成的：
使用基于亚磷酰胺化学的成熟固相合成方法，在寡核苷酸合成仪上合成siRNA的正义和反义链序列。寡核苷酸链的增长是通过4步循环实现的：去保护、缩合、加帽和用于添加每个核苷酸的氧化或硫化步骤。合成是在由可控多孔玻璃(CPG,)制成的固体支持物上进行的。单体亚磷酰胺购自商业来源。根据本文实施例2-3的程序合成具有GalNAc配体簇的亚磷酰胺(GLPA1、GLPA2和GLPA15作为非限制性实例)。对于用于体外筛选的siRNA(表2)，合成是在2μmol规模下进行的；对于用于体内测试的siRNA(表3)，合成规模为5μmol或更大。在GalNAc配体(作为非限制性实例的GLO-n)连接在正义链的3'-末端的情况下，使用连接有GalNAc配体的CPG固体支持物。在GalNAc配体(GLS-1或GLS-2作为非限制性实例)连接在正义链的5'-末端的情况下，将GalNAc亚磷酰胺(GLPA1或GLPA2作为非限制性实例)用于最后的偶 联反应。将3％二氯甲烷中的三氯乙酸(TCA)用于4,4'-二甲氧基三苯甲基保护基(DMT)的脱保护。5-乙硫基-1H-四唑用作活化剂。THF/Py/H2O中的I2和吡啶/MeCN中的苯乙酰二硫化物(PADS)分别用于氧化和硫化反应。在最后的固相合成步骤之后，通过用1:1体积的20wt％甲胺水溶液和28％氢氧化铵溶液处理来切割固体载体结合的低聚物并去除保护基团。为了合成用于体外筛选的siRNA，将粗混合物浓缩。将剩余的固体溶解在1.0M NaOAc中，加入冰冷的EtOH以沉淀出作为钠盐的单链产物，其无需进一步纯化即可用于退火。为了合成用于体内测试的siRNA，粗单链产物通过离子对反相HPLC(IP-RP-HPLC)进一步纯化。通过将来自IP-RP-HPLC的纯化单链寡核苷酸产物溶解在1.0M NaOAc中并通过添加冰冷的EtOH进行沉淀，将其转化为钠盐。在水中通过等摩尔互补进行正义链和反义链寡核苷酸的退火，以形成双链siRNA产物，将其冻干以提供蓬松的白色固体。
实施例2.中间体-A和中间体-B的制备
如以下方案1所示，通过在二氯甲烷(DCM)中用三甲基甲硅烷基三氟甲磺酸酯(TMSOTf)处理市售的半乳糖胺五乙酸酯来合成中间体-A。然后用Cbz保护的2-(2-氨基乙氧基)乙-1-醇进行糖基化，得到化合物II。通过氢化除去Cbz保护基团以提供作为三氟乙酸盐(TFA)盐的中间体-A。除了使用Cbz保护的2-(2-(2-氨基乙氧基)乙氧基)乙-1-醇作为原料之外，中间体B基于相同的方案合成。
方案1
向化合物I(20.0g,51.4mmol)在100mL 1,2-二氯乙烷(DCE)中的溶液中加入TMSOTf(17.1g,77.2mmol)。将所得反应溶液在60℃下搅拌2小时，然后在25℃下搅拌1小时；Cbz保护的2-(2-氨基乙氧基)乙-1-醇(13.5g,56.5mmol)在DCE(100mL)中用粉末分子筛(10g)干燥，在N2气氛下在0℃滴加到上述反应溶液中。在N2气氛下，将所得反应混合物在25℃下搅拌16小时。过滤反应混合物并用饱和NaHCO3(200mL)、水(200mL)和饱和盐水(200mL)洗涤。有机层经无水Na2SO4干燥，过滤并减压浓缩，得到粗产物，将其与2-甲基四氢呋喃/庚烷(5/3，v/v，1.80L)一起研磨2小时。将所得混合物过滤并干燥以得到呈白色固体状的化合物II(15.0g，产率50.3％)。
将10％Pd/C(1.50g)小心地加入到干燥和氩气吹扫的氢化瓶中，然后加入10毫升四氢呋喃(THF)，然后是化合物II(15.0克，26.4毫摩尔)在THF(300毫升)和TFA(三氟乙酸，3.00克，26.4毫摩尔)中的溶液。将所得混合物脱气并用H2吹扫3次并在H2(45psi)气氛下在25℃下搅拌3小时。薄层色谱法(TLC，溶剂：DCM:MeOH＝10:1)表明化合物II已完全消耗。过滤反应混合物并减压浓缩。将残余物溶解在无水DCM(500mL)中并浓缩。该过程重复3次以得到呈泡沫状白色固体的中间体-A(14.0g，产率96.5％)。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)。
使用与合成中间体-A类似的程序合成中间体-B。1H NMR(400MHz DMSO-d6):δppm 7.90(br d,J＝9.03Hz,4H),5.21(d,J＝3.51Hz,1H),4.97(dd,J＝11.1Hz,1H),4.54(d,J＝8.53Hz,1H),3.98-4.06(m,3H),3.88(dt,J＝10.9Hz,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).计算质谱C20H34N2O11:478.22；实测:479.3(M+H+)。
实施例3.GalNAc配体簇亚磷酰胺GLPA1、GLPA2和GLPA15的合成
按照以下方案2制备GLPA1和GLPA2。从苄基保护的丙烷-1,3-二胺开始，用2-溴乙酸叔丁酯对其进行烷基化，得到三酯化合物I。通过氢化除去苄基保护基，得到仲胺化合物II。将酰胺与6-羟基己酸偶联得到化合物III。然后在用二氧六环中的HCl处理时除去叔丁基保护基以生成三酸化合物IV。进行三酸化合物IV和中间体-A或中间体-B之间的酰胺偶联以提供化合物Va或Vb。亚磷酰胺GLPA1或GLPA2是通过化合物Va或Vb与2-氰乙基N,N-二异丙基氯亚磷酰胺和催化量的1H-四唑的亚磷酸化合成的。
方案2
向N-苄基-1,3-丙二胺(5.00g,30.4mmol)在二甲基甲酰胺(DMF,100mL)中的溶液中加入2-溴乙酸叔丁酯(23.7g,121mmol)；然后滴加二异丙基乙胺(DIEA，23.61g，182mmol)。将所得反应混合物在25-30℃搅拌16小时。LCMS显示N-苄基-1,3-丙二胺被完全消耗。反应混合物用H2O(500mL)稀释并用EtOAc(500mL×2)萃取。合并的有机物用饱和盐水(1L)洗涤，用无水Na2SO4干燥，过滤，减压浓缩，得到粗产物；经硅胶柱层析纯化(梯度：石油醚:乙酸乙酯20:1至5:1)。获得无色油状化合物I(12.1g，产率78.4％)。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)。
干燥的氢化瓶用氩气吹扫3次。加入Pd/C(200mg，10％)，然后加入MeOH (5mL)，然后加入化合物I(1.00g，1.97mmol)在MeOH(5mL)中的溶液。反应混合物在真空下脱气并重新填充H2。这个过程重复3次。将混合物在H2(15psi)气氛下在25℃搅拌12小时。LCMS显示化合物I被完全消耗。在N2气氛下减压过滤反应混合物。减压浓缩滤液，得到黄色油状化合物II(655mg，产率79.7％)，其无需进一步纯化即可用于下一步。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)。
化合物II(655mg，1.57mmol)、6-羟基己酸(249mg，1.89mmol)、DIEA(1.02g，7.86mmol)、1-乙基-3-(3-二甲氨基丙基)碳二亚胺盐酸盐(EDCI，904mg，4.72mmol)和1-羟基苯并三唑(HOBt，637mg，4.72mmol)的DMF(6mL)溶液的混合物脱气并用N2吹扫3次；然后在N2气氛下在25℃搅拌3小时。LCMS指示所需产物。反应混合物用H2O(10mL)稀释并用EtOAc 20mL(10mL×2)萃取。合并有机物并用饱和盐水(20mL)洗涤，经无水Na2SO4干燥，过滤并浓缩以得到粗产物；将其通过硅胶柱色谱法(梯度：石油醚:乙酸乙酯从5:1至1:1)纯化，得到呈黄色油状的化合物III(650mg，产率77.8％)。1H NMR(400MHz,CDCl3):δppm 3.90-3.95(s,2H),3.63(t,J＝6.40Hz,2H),3.38-3.45(m,6H),2.72(t,J＝6.65Hz,2H),2.40(t,J＝7.28Hz,2H),1.55-1.75(m,8H),1.44(s,27H).计算质谱C27H50N2O8:530.36；实测:531.3(M+H+)。
将化合物III(5.5g，10.3mmol)在HCl/二氧六环(2M，55mL)中的混合物在25℃下搅拌3小时。LCMS显示完全消耗化合物III。过滤反应混合物，用EtOAc(50mL)洗涤，减压干燥，得到粗产物。将其溶解在CH3CN(50mL)中，真空除去挥发物。重复该过程3次以得到呈白色固体状的化合物IV(2.05g，产率54.5％)。1H 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)。
将化合物IV(500mg,1.05mmol)、中间体-A(2.02g,3.67mmol)、DIEA(813mg,6.30mmol)、EDCI(704mg,3.67mmol)和在DMF(10毫升)中的HOBt(496mg,3.67mmol)的混合物脱气并用N2吹扫3次，然后将混合物在N2气氛下在25℃搅拌3小时。LCMS指示所需产物。通过加入H2O(10mL)淬灭反应混合物，用DCM(10mL×2)萃取。合并的有机物用10％柠檬酸(20mL)萃取。水相用饱和NaHCO3溶液中和并用DCM(10mL x 2)再萃取。有机物经硫酸钠干燥，过滤并在减压下浓缩以产生呈白色固体状的化合物Va(570mg，0.281mmol，产率26.8％)。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)。
向化合物Va(260mg,0.161mmol)的无水DCM(5mL)溶液中加入四唑二异丙基铵(30.3mg,0.177mmol)；然后在环境温度和N2下滴加3-双(二异丙基氨基)膦酰氧基丙腈(194mg,0.645mmol)。将反应混合物在20至25℃搅拌2小时。LCMS表明化合物Va被完全消耗。冷却至-20℃后，将反应混合物在0℃加入搅拌的盐水/饱和NaHCO3(1:1，5mL)溶液。搅拌1分钟后，加入DCM(5mL)。出现分层。有机层用盐水/饱和NaHCO3水溶液(1:1,5mL)洗涤，用Na2SO4干燥，过滤并浓缩至体积约1mL。在搅拌下将残余溶液滴加到20mL甲基叔丁基醚(MTBE)中。这导致白色固体沉淀。将混合物离心，收集固体。将固体重新溶解在1mL DCM中并通过加入MTBE(20mL)沉淀。再次通过离心分离固体。将收集的固体溶解在无水CH3CN中。除去挥发物。该过程再重复两次，得到呈白色固体状的GalNAc配体亚磷酰胺化合物GLPA1(153mg,84.4μmol)。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.
除了使用中间体-B之外，使用相同的程序合成GalNAc配体亚磷酰胺化合物GLPA2。1H NMR(400MHz,CDCl3):ppmδ7.94-8.18(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),2.22-2.45(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。
按照以下方案3制备GLPA15。

向中间体化合物II(275g,660mmol,1.00当量(eq))的二氯甲烷(2.75L)溶液中加入三乙胺(133g,1.32mol,2.00eq.),随后滴加入Cbz-Cl(169g,990mmol,1.50eq.)。反应液在25℃搅拌2小时，LCMS显示化合物II完全转化。反应液依次用NaHCO3(800mL)饱和溶液、饱和食盐水(500mL)洗涤，有机相用无水Na2SO4干燥。过滤除去干燥剂后，滤液浓缩至干。该粗品经柱层析后(SiO2,PE/EA＝100/1to 5/1)得到无色油状化合物III-1(290g,527mmol,产率75.7％)。1H 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。
向化合物III-1(145g,263mmol,1.00eq)中加入HCOOH(2.9L)，该溶液在60℃下搅拌12小时，LCMS显示化合物III-1转化完全。向反应液中加入1.5L甲苯和1.5L乙腈，减压浓缩至约500mL,随后加入甲苯/乙腈(1:1，～750mL)并浓缩至约500mL,然后加入乙腈(～1000mL)并浓缩至干，粗品用700mL乙腈在60℃粉碎2小时,过滤。收集固体，干燥得白色固体化合物IV-1(105g,定量)。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。
向化合物IV-1(100g,261mmol.)和中间体-A(502g,915.mmol,3.50eq.)的DMF(1.0L)溶液中加入TBTU(327g,1.02mol,3.90eq.),三乙胺(212g,2.09mol,8.00eq.)，反应在25℃进行1小时，LCMS显示化合物IV-1转化完成。将反应液加入到4000mL水中,并用甲基叔丁基醚(2000mL分两次)萃取除去杂质，剩余水相用二氯甲烷(3000mL分两次)萃取。二氯甲烷相依次用10％柠檬酸水溶液(2000mL分两次)、饱和NaHCO3(2.0L分两次),饱和盐水(2.0L)洗涤,无水Na2SO4干燥。过滤得滤液，减压浓缩得到白色固体化合物V-1(260g,159mmol,产率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。
用氩气惰性化2L氢化釜并小心加入干Pd/C(9g)，加入MeOH(50mL)润湿Pd/C,然后在氩气气氛下缓慢加入化合物V-1(90g,55.1mmol,1.00eq.)和三氟乙酸 (6.29g,55.1mmol,1.00eq.)的MeOH(850mL)溶液。混合物经脱气/加H2三次置换为氢气气氛，在25℃搅拌10小时。LCMS显示化合物V-1转化完全，过滤除去Pd/C，滤液经减压浓缩得到化合物VI-1(80g,产率90.2％).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。
向化合物VI-1(270g,168mmol,1.00eq.)和戊二酸酐(28.6g,252mmol,1.50eq)的二氯甲烷(2.7L)溶液中加入三乙胺(67.8g,672mmol,4.00eq)，该溶液在25℃搅拌1小时，LCMS显示化合物VI-1完全转化为化合物VII。向反应液中加入4-羟基哌啶(42.4g,420mmol,2.50eq.)和TBTU(107g,335mmol,2.00eq.)，并在25℃继续搅拌1小时。LCMS显示化合物VII转化完全。缓慢加入饱和NH4Cl(3.0L)淬灭反应，分层，水相用二氯甲烷(2x 1000mL)萃取并与先前的有机相合并。合并的有机相用饱和NaHCO3(aq)和饱和盐水的1:1混合液(3.0L)洗涤,用无水Na2SO4干燥,过滤减压浓缩。粗品溶于1.5L二氯甲烷,滴加到甲基叔丁基醚(7.5L)中,半透明的白色沉淀在滴加过程中逐渐形成。真空过滤沉淀，收集固体真空干燥得到白色固体化合物VIII(207g,产率72.8％)。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。
向化合物VIII(200g,118mmol,1.00eq.)、四唑二异丙基铵(8.08g,47.2mmol,0.40eq)的二氯甲烷(2.0L)溶液中加入3-双(二异丙基氨基)膦酰氧基丙腈，(53.3g,177mmol,1.50eq.)，反应液在40℃搅拌2小时，LCMS显示化合物VIII转化完成。反应液用饱和NaHCO3和饱和食盐水的1:1混合液(2.0L)洗涤,经无水Na2SO4干燥，滤液浓缩后所得粗品溶于二氯甲烷(1.2L),滴加到搅拌的甲基叔丁基醚(6.0L)中,过滤悬浊液,滤饼用基叔丁基醚淋洗,收集固体进行真空干燥,将产品溶于二氯甲烷(1.0L)并浓缩至干，重复操作4次以除去残留叔丁基醚得到GLPA15(164g,产率73.3％).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),1.99(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。
在某些研究中，提供了用于将包含GalNAc(本文也称为GalNAc递送化合物)的 靶向基团连接到正义链的5'-末端的方法，其包括在固相合成的最后一个偶联步骤中使用GalNAc亚磷酰胺(例如GLPA1)，使用这样的合成工艺，例如在寡核苷酸链延长时使用的(即在正义链的5'末端添加核苷酸)工艺，以将其连接到正义链的5'-末端。
在一些研究中，将包含GalNAc的靶向基团连接到正义链的3'-末端的方法包括使用包含GLO-n的固体支持物(CPG)。在一些研究中，将包含GalNAc的靶向基团连接到正义链的3'-末端的方法包括：将GalNAc靶向基团通过酯键连接到CPG固体支持物上，并在合成正义链时使用带有连接的GalNAc靶向基团的所得CPG，这导致GalNAc靶向基团连接在正义链的3'-末端。其他GalNAc亚磷酰胺化合物(GLPAn)也同样可以在使用合理对应的中间体后，采用本文类似或者本领域中熟知的方法获得，并作为靶向基团连接到siRNA双链体合适的位置。
实施例4.异甘露醇亚磷酰胺(化合物2)的合成
将在吡啶(400毫升)中的DMTrCl(232克，684毫摩尔，1.0当量)，加入到化合物A异甘露醇(100克，684毫摩尔，1.0当量)的吡啶(600毫升)溶液中，将混合物在25℃下搅拌16小时。LC-MS显示化合物A被完全消耗，并且检测到一个具有所需质量的主峰。将所得反应混合物用水(500毫升)稀释，用二氯甲烷(500毫升*2)萃取，合并的有机相，用盐水(500毫升)洗涤，用Na2SO4干燥并在真空中浓缩以获得残留物。残余物用柱层析(DCM/MeOH＝100/1至50/1，0.1％Et3N)纯化，得到化合物B(150克，收率48.9％)的黄色固体。1H 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).
在N2气氛下，在向化合物B(80.0克，178毫摩尔，1.0当量)的二氯甲烷(5.0毫升)溶液中，在25℃下滴加2H-四氮唑(0.45M，436毫升，1.1当量)，然后滴加化合物C(2-氰基乙基二异丙基氯代亚磷酰胺，80.6克，267毫摩尔，85.0毫升，1.5当量)中的二氯甲烷(200毫升)的溶液；将反应混合物在25℃下搅拌1.0小时；LC-MS显示化合物B被完全消耗，并且检测到一个具有所需质量的主峰。将所得反应混合物冷却至-20℃并倒入冰冷的坐下；NaHCO3(500毫升)，用二氯甲烷(500毫升*3)提取，合并的有机层用NaHCO3/盐水＝1：1(300毫升/300毫升)洗涤，用Na2SO4干燥，并在真空(35℃)中浓缩以获得残留物(100毫升)。残余物用柱层析法纯化(Al2O3， DCM/MeOH＝100/1至50/1，0.1％Et3N)，得到化合物2(77g，119mmol，收率66.5％)为白色固体。
1H NMR:EC4783-423-P1B1_C(400MHz,DMSO-d6)
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).
其他的亚磷酰胺化合物可以根据本领域技术人员熟知方法进行制备，例如但不限于现有技术US426,220和WO02/36743中的方法。
实施例5.包含异甘露醇单体的固体载体的制备
表示大孔胺甲基聚乙烯树脂载体部分
将50L玻璃釜在氮气保护下，向玻璃釜中加入二氯甲烷(19.50kg)，开动搅拌。控温20～30℃，向玻璃釜中加入DMTr-imann(1.47kg)，向反应釜中加入三乙胺(1.50kg)、4-二甲氨基吡啶(0.164kg)，以及加入丁二酸酐(1.34kg)。体系保温20～30℃反应18h后取样，结束反应。向反应完体系中加入饱和碳酸氢钠溶液(22.50kg)，搅拌10～20min后静置至分层，下层有机相转移至，上层水相用二氯甲烷萃取2次，合并有机相，采用无水硫酸钠干燥，过滤滤液，再转移旋蒸浓缩至无馏分，形成固体灰色至类白色固体1.83kg。
向100L玻璃釜中加入N,N-二甲基甲酰胺(23.50kg)，开动搅拌。控温20～30℃，氮气保护下，通过固体加料漏斗向上述100L玻璃釜中加入上步产物、O-苯并三氮唑-四甲基脲六氟磷酸盐(0.33kg)、加入N,N-二异丙基乙胺(0.13kg)，加料完毕搅拌10～30分钟后放出至50L镀锌桶待用。通过固体加料漏斗向上述100L固相合成釜中加入大孔胺甲基树脂(3.25kg)(购于天津南开和成科技有限公司，批号HA2X1209,负载量0.48mmol/g)，控温20～30℃，向固相合成釜中加入N,N-二甲基甲酰胺(21.00kg+21.00kg)，和上步镀锌桶待用反应液。体系于保温反应，，跟踪至固体载量≥250umol/g，载量检测方法为UV。体系用氮气压滤，滤饼用N,N-二甲基甲酰胺淋洗三次(26.00kg+26.10kg+26.00kg)，滤饼留在釜中。向80L玻璃釜中加入CAP.A(50％乙腈和50％乙酸酐，4.40kg+4.42kg+4.30kg)和CAP.B(20％吡啶和30％N-甲基咪唑和50％乙腈，4.40kg+4.40kg+4.47kg)，搅拌3～8min后待用。此操作重复进行3次进行盖帽，向固相合成釜中加入乙腈(18.00kg+18.00kg+18.00kg+17.50kg+17.50kg)，氮气鼓泡10～30min后压滤。重复此操作四次，滤饼在固相合成釜中使用氮气吹扫2～4h后转移至50L压滤罐，控温15～30℃，继续干燥，将烘干后黄色至白色固体产品，重量:3.516kg。
异甘露醇残基(imann)可以通过本领域技术人员熟知方法，如反向无碱基(invab)同样的工艺方法，将其添加到寡核苷酸链的5'末端或者3'末端，并进一步添加靶向基团。
实施例6.黄嘌呤脱氢酶(XDH)siRNA双链体的体外筛选
CAL27细胞用胰蛋白酶消化并调整到合适的密度，然后接种到24孔板中，1×105细胞/孔。在接种的同时，使用Lipofectamine RNAiMax(Invitrogen-13778-150)以2μL每孔用待测siRNA或对照siRNA转染细胞。无义对照-2(CON)和待测siRNA以两个浓度(0.2nM和5nM)进行测试。转染无义对照-2(CON)siRNA的孔也被用作阴性对照。
转染后24小时，去除培养基并收获细胞用于RNA提取。根据手册使用TRIzolTM Reagent(Invitrogen-15596018)提取总RNA。
根据手册，使用PrimeScriptTM RT试剂盒和gDNA Eraser(Perfect Real Time)(TaKaRa-RR047A)合成cDNA。通过qPCR检测黄嘌呤脱氢酶(XDH)cDNA。平行检测GAPDH cDNA作为内部对照。PCR如下进行：在95℃下进行30秒，然后以在95℃下10秒、在60℃下30秒的循环进行40个循环。
数据分析
使用比较性Ct(ΔΔCt)方法，通过相对定量(RQ)确定每个样品中目标基因的表达；该方法测量目标基因和看家基因(ACTIN)之间的Ct差异(ΔCt)。
方程式列出如下：
ΔCT＝目标基因平均Ct–ACTIN平均Ct
ΔΔCT＝ΔCT(样品)–ΔCT(无义siRNA对照)；
目标基因mRNA的相对定量＝2-ΔΔCT
抑制％＝(对照的相对定量–样品的相对定量)/对照的相对定量×100％。
表4提供了使用多种XDH RNAi试剂抑制XDH表达的体外研究的实验结果；使用的双链序列对应于表2中所示的化合物。
表4 XDH RNAi试剂抑制XDH表达的体外研究的实验结果


实施例7.黄嘌呤脱氢酶(XDH)siRNA双链体的体内测试
为了评估黄嘌呤脱氢酶(XDH)siRNA的体内活性，使用了感染了编码人黄嘌呤脱氢酶(XDH)和萤光素酶基因的AAV的小鼠(每组4只小鼠)。在siRNA给药前14天，对雌性C57BL/6J小鼠通过静脉注射1x10^11或者2x10^11viral particle of编码人黄嘌呤脱氢酶(XDH)和萤光素酶基因的腺相关病毒8(AAV8)载体的原液来进行感染。在第0天，小鼠皮下注射单剂量3mg/kg，6mg/kg或者9mg/kg的黄嘌呤脱氢酶(XDH)siRNA试剂或PBS。在第0天siRNA给药前和第7和14天终止时收集血样。测量荧光素酶活性。通过比较siRNA处理组给药前血样的荧光素酶活性和第7，14 天收集血样的荧光素酶活性并且基于来自PBS处理组的血清样品中的荧光素酶活性变化进行归一化，以计算敲低百分比。
实施例7使用对应于表3中所示的序列、化学修饰和递送的化合物,GLO-n为Jayaprakash,et al.,(2014)J.Am.Chem.Soc.,136,16958-16961中的GalNAc3所示的递送化合物。
在AAV-XDH-荧光素酶转导小鼠中进行XDH siRNA单次3mpk皮下剂量筛选。比较siRNA处理组给药前血样(第0天)的荧光素酶活性和第7天收集血样的荧光素酶活性并且基于来自PBS处理组的血清样品中的荧光素酶活性变化进行归一化，以计算敲低百分比。数据列在表5.
表5 XDH siRNA单次3mpk在AAV-XDH-荧光素酶转导小鼠中皮下剂量筛选
在AAV-XDH-荧光素酶转导小鼠中进行XDH siRNA单次6mpk皮下剂量筛选。比较siRNA处理组给药前血样(第0天)的荧光素酶活性和第7天收集血样的荧光素酶活性并且基于来自PBS处理组的血清样品中的荧光素酶活性变化进行归一化，以计算敲低百分比。两次实验数据分别列在表6和表7。
表6 XDH siRNA单次6mpk在AAV-XDH-荧光素酶转导小鼠中皮下剂量筛选
表7 XDH siRNA单次6mpk在AAV-XDH-荧光素酶转导小鼠中皮下剂量筛选
在AAV-XDH-荧光素酶转导小鼠中进行XDH siRNA单次9mpk皮下剂量筛选。比较siRNA处理组给药前血样(第0天)的荧光素酶活性和第14天收集血样的荧光素酶活性并且基于来自PBS处理组的血清样品中的荧光素酶活性变化进行归一化，以计算敲低百分比。数据列在表8.
表8 XDH siRNA单次9mpk在AAV-XDH-荧光素酶转导小鼠中皮下剂量筛选
实施例8.黄嘌呤脱氢酶(XDH)siRNA双链体的体内测试
为了评估siRNA的体内活性，本研究共选择入组18只雄性食蟹猴(13-22岁，体重7～9公斤)，每组3只，每只动物皮下注射给予7.5mg/kg供试品，使用的供试品对应于下表所示的化合物。
禁食过夜后，在D-14、D21、D42和D78，分别将动物麻醉(氯胺酮，SC，10mg/kg)，采用超声引导进行活体穿刺从而获得肝脏样品约10mg，放入RNAlater中保存(样品4℃固定过夜后，转移到-80℃保存)，用于后续的RT-qPCR检测。结果如表9所示。
表9通过qPCR测量的猴子肝脏中XDH mRNA的剩余百分比
等同物
尽管本文已经描述和说明了本发明的几个实施例，但本领域普通技术人员很容易理解，用于执行此处描述的功能和/或获得结果和/或一个或更多个优点的多种其他手段和/或结构，以及这些变化和/或修改中的每一个都被认为在本发明的范围内。更一般地，本领域技术人员将容易理解，此处描述的所有参数、尺寸、材料和配置都是示例性的，并且实际参数、尺寸、材料和/或配置将取决于使用本发明教导的具体应用。本领域技术人员将认识到或能够仅使用常规实验来确定本文描述的本发明的特定实施例的许多等价物。因此，应当理解，前述实施例仅通过示例的方式呈现并且属于所附权利要求及其等效物的范围内，本发明可以以不同于具体描述和要求保护的方式实施。本发明针对在此描述的每个单独的特征、系统、物品、材料和/或方法。此外，两个或更多个此类特征、系统、物品、材料和/或方法的任何组合，如果此类特征、系统、物品、材料和/或方法相互不矛盾，则也包括在本发明的范围内。
如本文所定义和使用的所有定义应理解为对照字典定义、通过引用并入的文件中的定义和/或所定义术语的普通含义。
在说明书和权利要求中并未使用数量限定的情况，除非明确指出相反，否则应理解为“至少一个”。
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Claims (69)

  1. 一种用于抑制黄嘌呤脱氢酶(XDH)表达的双链核糖核酸(dsRNA)试剂，其中所述dsRNA试剂包含正义链和反义链，所述反义链中的核苷酸第2至18位包含与黄嘌呤脱氢酶(XDH)基因RNA转录物互补的区域，其中互补区域包含与表1-3之一中所列出的反义序列之一相差0、1、2或3个核苷酸的至少15个连续核苷酸，并且任选地包含靶向配体。
  2. 权利要求1所述的dsRNA试剂，其中所述与黄嘌呤脱氢酶(XDH)基因RNA转录物互补的区域包含至少15、16、17、18或19个连续核苷酸，其与表1-3之一中所列出的任一反义序列相差不超过3个核苷酸。
  3. 权利要求1或2所述的dsRNA试剂，其中所述dsRNA的反义链与SEQ ID NO：1中的任一个靶区域至少基本上互补，并且在表1-3的任一个中提供。
  4. 权利要求3所述的dsRNA试剂，其中所述dsRNA的反义链与SEQ ID NO：1中的任一靶区完全互补，并在表1-3的任一个中提供。
  5. 权利要求1所述的dsRNA试剂，其中所述dsRNA试剂包含表1-3中任一项所述的正义链序列，其中所述正义链序列与所述dsRNA试剂中的反义链序列至少基本上互补。
  6. 权利要求1所述的dsRNA试剂，其中所述dsRNA试剂包含表1-3中任一项所述的正义链序列，其中所述正义链序列与所述dsRNA试剂中的反义链序列完全互补。
  7. 权利要求1所述的dsRNA试剂，其中所述dsRNA试剂包含表1-3中任一项所列出的反义链序列。
  8. 权利要求1所述的dsRNA试剂，其中所述dsRNA试剂包含表1-3中任一项中作为双链体序列所列出的序列。
  9. 权利要求1所述的dsRNA试剂，其中所述dsRNA试剂包含至少一个修饰的核苷酸。
  10. 权利要求1所述的dsRNA试剂，其中所述反义链中的所有或基本上所有核苷酸是修饰的核苷酸。
  11. 权利要求9或10所述的dsRNA试剂，其中所述至少一种修饰的核苷酸包括：2'-O-甲基核苷酸、2'-氟核苷酸、2'-脱氧核苷酸、2'3'-seco核苷酸模拟物、锁定核苷酸、开环核酸核苷酸(UNA)、乙二醇核酸核苷酸(GNA)、2'-F-阿拉伯糖核苷酸、2'-甲氧基乙基核苷酸、无碱基核苷酸、核糖醇、反向核苷酸、反向无碱基核苷酸、反向2'-OMe核苷酸、反向2'-脱氧核苷酸、2'-氨基修饰核苷酸、2'-烷基修饰核苷酸、吗啉代核苷酸和3'-OMe核苷酸、包含5'-硫代磷酸酯基团的核苷酸，或与胆固醇衍生物或十二烷酸双癸酰胺基团连接的末端核苷酸、2'-氨基修饰的核苷酸、氨基磷酸酯，或包含核苷酸的非天然碱基。
  12. 权利要求9或10所述的dsRNA试剂，其中在引导链的5'末端包含E-乙烯基膦 酸酯核苷酸。
  13. 权利要求1所述的dsRNA试剂，其中所述dsRNA试剂包含至少一个硫代磷酸酯核苷间键联。
  14. 权利要求1所述的dsRNA试剂，其中所述正义链包含至少一个硫代磷酸酯核苷间键联。
  15. 权利要求1所述的dsRNA试剂，其中所述反义链包含至少一个硫代磷酸酯核苷间键联。
  16. 权利要求1所述的dsRNA试剂，其中所述正义链包含1、2、3、4、5或6个硫代磷酸酯核苷间键联。
  17. 权利要求1所述的dsRNA试剂，其中所述反义链包含1、2、3、4、5或6个硫代磷酸酯核苷间键联。
  18. 权利要求1所述的dsRNA试剂，其中所述正义链和反义链的全部或基本上全部核苷酸是修饰的核苷酸。
  19. 权利要求1所述的dsRNA试剂，其中修饰的正义链是表2-3之一中所列出的修饰的正义链序列。
  20. 权利要求1所述的dsRNA试剂，其中修饰的反义链是表2-3之一中所列出的修饰的反义链序列。
  21. 权利要求1所述的dsRNA试剂，其中所述正义链与反义链互补或基本上互补，并且互补区域的长度为16至23个核苷酸。
  22. 权利要求21所述的dsRNA试剂，其中所述互补区域的长度为19至21个核苷酸。
  23. 权利要求1所述的dsRNA试剂，其中每条链的长度为不超过30个核苷酸。
  24. 权利要求1所述的dsRNA试剂，其中每条链的长度为不超过25个核苷酸。
  25. 权利要求1所述的dsRNA试剂，其中每条链的长度为不超过23个核苷酸。
  26. 权利要求1所述的dsRNA试剂，其中所述dsRNA试剂包含至少一种修饰的核苷酸，并且还包含一种或更多种靶向基团或连接基团。
  27. 权利要求26所述的dsRNA试剂，其中所述一种或更多种靶向基团或连接基团与所述正义链缀合。
  28. 权利要求26或27所述的dsRNA试剂，其中所述靶向基团或连接基团包括N-乙酰基-半乳糖胺(GalNAc)。
  29. 权利要求28所述的dsRNA试剂，其中所述靶向基团或连接基团如式(X)所示的结构，包括靶向部分、链接键以及接头W，其中靶向部分选自N-乙酰基-半乳糖胺衍生物(GalNAc)，其通过链接键与接头W链接，接头W具有如式(XI)所示的结构，X选自O、NH2或者S，Y﹣选自：O﹣、S﹣、甲基或者NRaRb，Ra和Rb分别独立的选自氢、取代或者未取代的C1-C6的烷基、取代或者未取代的C3-C6的环烷基，或者Ra和Rb与附着的原子一起链接形成含有1-3个N、O、S杂原子组成的3-12元杂环烷基,优选的 取代或者未取代的C1-C6的烷基、取代或者未取代的C3-C6的环烷基中，所述的取代基选自羟基、氨基；
  30. 权利要求29所述的dsRNA试剂，靶向基团中的链接键选自聚乙二醇、取代或未取代的C2-C12烷基,取代或者未取代的C3-C12环烷基、取代或者未取代的C3-C12杂环烷基、取代或者未取代的C3-C12酰胺。
  31. 权利要求30所述的dsRNA试剂，所述取代或者未取代的C2-C12烷基,取代或者未取代的C3-C12环烷基、取代或者未取代的C3-C12杂环烷基、取代或者未取代的C3-C12酰胺中，其中所述的取代基选自羟基、羰基。
  32. 权利要求30所述的dsRNA试剂，靶向基团中的链接键优选自以下片段：
    其中每个m独立的为1-6的整数，每个n、o、p独立的为0或者1，每个q1与q2分别独立的为0、1或者2。
  33. 权利要求32所述的dsRNA试剂，靶向基团中的链接键更优选自以下片段：
  34. 权利要求29所述的dsRNA试剂，靶向基团中的靶向部分具有为以下结构片段， n’为1或者2。
  35. 权利要求29所述的dsRNA试剂，X选自O或者S，Y﹣选自：O﹣或者S﹣。
  36. 权利要求26或27所述的dsRNA试剂，其中所述靶向基团具有以下结构：



  37. 权利要求1-36任一项所述的dsRNA试剂，其中所述dsRNA试剂包含与所述正义链的5'-末端缀合的靶向基团。
  38. 权利要求1-36任一项所述的dsRNA试剂，其中所述dsRNA试剂包含与所述正义链的3'-末端缀合的靶向基团。
  39. 权利要求1-38任一项所述的dsRNA试剂，其中所述反义链在3'-末端包含一个反向无碱基残基。
  40. 权利要求1-38任一项所述的dsRNA试剂，其中所述正义链在3'或/和5'末端包含一个或两个反向无碱基残基，和/或，正义链在3'和/或5'末端各自独立地包含一个或两个异甘露醇残基，优选为异甘露醇残基imann。
  41. 权利要求1-40任一项所述的dsRNA试剂，其中所述dsRNA试剂具有两个平末端。
  42. 权利要求1-40任一项所述的dsRNA试剂，其中至少一条链包含至少1个核苷酸的3'突出端。
  43. 权利要求1-40任一项所述的dsRNA试剂，其中至少一条链包含至少2个核苷酸的3'突出端。
  44. 权利要求1-43任一项所述的dsRNA试剂，正义链在3’-末端和/或5’-末端具有 1、2、3、4、5、6、7、8、9、或10个核苷酸的突出端。
  45. 一种抑制黄嘌呤脱氢酶(XDH)表达的双链核糖核酸(dsRNA)试剂，其中所述dsRNA试剂包含正义链和反义链，其中每条链的长度是14个至30个核苷酸，在反义链中的核苷酸位置2至18处包含与黄嘌呤脱氢酶(XDH)基因RNA转录物互补的区域，其中互补区域包含与式I中所列出的双链核糖核酸(dsRNA)试剂反义序列之一相差0、1、2或3个核苷酸的至少15个连续核苷酸，并且任选地包含靶向配体，所述的双链核糖核酸(dsRNA)试剂反义链如式I所述:
    5’-Na-(A1 A2 A3 A4)i-Nb–(B1 B2 B3 B4)-Nb-(C1 C2 C3 C4 C5)j-Na-3’
    (式I)
    其中:i和j各自独立地选自0或1；
    每个Na和Nb分别独立的代表0-17寡核苷酸，所述寡核苷酸可选地被化学修饰；
    优选的，所述化学修饰选自2'-OMe或2'-氟修饰；
    A1 A2 A3 A4表示四个连续的核苷酸依次被代表小写2'-OMe、大写2'-氟、小写2'-OMe、小写2'-OMe修饰的一个基序，优选的A1与A2之间、A2与A3之间进一步均包含硫代磷酸酯核苷间键联；
    B1 B2 B3 B4表示四个连续的核苷酸依次被1个大写2'-氟，和连续3个小写2'-OMe修饰的一个基序；
    C1 C2 C3 C4 C5表示五个连续的核苷酸均被小写2'-OMe修饰的一个基序，优选的C3与C4之间、C4与C5之间进一步包含硫代磷酸酯核苷间键联。
  46. 权利要求45所述的dsRNA试剂，Na各自独立的代表0个寡核苷酸，Nb各自独立的代表2-5个经化学修饰的寡核苷酸，i和j各自独立地代表1；所述化学修饰选自2'-OMe或2'-氟。
  47. 权利要求45-46任一项所述的式(I)，其特征在于，进一步如式(II)所示：
    5’-xYxxxxYxxxxYxYxYxxxxx-3’
    (式II)
    其中，x代表小写2'-OMe修饰的寡核苷酸，Y代表大写2'-氟修饰的寡核苷酸，优选的x与Y和/或x与x之间进一步包含硫代磷酸酯核苷间键联。
  48. 权利要求45-47任一项所述的dsRNA试剂，dsRNA的反义链与SEQ ID NO:1的任意靶区域至少基本互补。
  49. 权利要求45-47任一项所述的dsRNA试剂，dsRNA的反义链与SEQ ID NO:1的任意靶区域完全互补。
  50. 权利要求45-47任一项所述的dsRNA试剂，其中所述dsRNA任一项所述的正义链序列与所述dsRNA试剂中的反义链序列至少基本上互补。
  51. 权利要求45-47任一项所述的dsRNA试剂，其中所述dsRNA任一项所述的正义链序列，与所述dsRNA试剂中的反义链序列完全互补。
  52. 权利要求45-47任一项所述的dsRNA试剂，其中所述dsRNA正义链在3'或/和 5'末端包含一个或两个反向无碱基残基,和/或，正义链在3'和/或5'末端各自独立地包含一个或两个异甘露醇残基，优选为异甘露醇残基imann。
  53. 权利要求45-47任一项所述的dsRNA试剂，其中所述dsRNA具有两个平末端。
  54. 权利要求45-47任一项所述的dsRNA试剂，其中所述dsRNA正义链在3’-末端和/或5’-末端具有一个1、2、3、4、5、6、7、8、9、或10个核苷酸的突出端。
  55. 包含权利要求1至54中任一项所述的dsRNA试剂的组合物。
  56. 权利要求55所述的组合物，其还包含药学上可接受的载体。
  57. 权利要求55-56任一项所述的组合物，还包含一种或更多种另外的治疗剂。
  58. 权利要求55-57任一项所述的组合物，其中所述组合物被包装在药盒、容器、包装物、分配器、预填充注射器或小瓶中。
  59. 权利要求55-57任一项所述的组合物，其中所述组合物被配制成用于皮下给药或被配制成用于静脉内(IV)给药。
  60. 包含权利要求1至54中任一项所述的dsRNA试剂的细胞。
  61. 权利要求60所述的细胞，其特征在于，所述细胞是哺乳动物细胞，任选地是人细胞。
  62. 抑制细胞中黄嘌呤脱氢酶(XDH)基因表达的方法，其包括：
    (i)制备包含有效量的权利要求1至54中任一项所述的双链核糖核酸(dsRNA)试剂或权利要求55至59中任一项所述的组合物的细胞；
    (ii)将第(i)中制备的细胞维持足够的时间，以获得黄嘌呤脱氢酶(XDH)基因的mRNA转录物的降解，从而抑制细胞中黄嘌呤脱氢酶(XDH)基因的表达。
  63. 权利要求62所述的方法，其特征在于，所述细胞在对象体内并且将所述dsRNA试剂皮下施用至所述对象。
  64. 治疗与黄嘌呤脱氢酶(XDH)蛋白相关的疾病或病症的方法，所述方法包括向对象施用有效量的权利要求1至54中任一项所述的双链核糖核酸(dsRNA)试剂或权利要求55至59中任一项所述的组合物，以抑制黄嘌呤脱氢酶(XDH)基因表达。
  65. 权利要求64所述的方法，其中所述疾病或病症与黄嘌呤脱氢酶(XDH)表达相关，所述疾病选自以下一种或多种：高尿酸血症，痛风，非酒精性脂肪肝疾病(NAFLD)，非酒精性脂肪性肝炎(NASH)，代谢紊乱，胰岛素抵抗，心血管疾病，2型糖尿病，Lesch Nyhan综合征，糖原贮积病(GSD)，以及与氧化应激有关的疾病或其他XDH相关疾病；优选地，所述糖原贮积病(GSD)选自I型GSD，所述与氧化应激有关的疾病选自慢性低度炎症。
  66. 权利要求64-65任一项所述的方法，所述方法降低血清尿酸水平。
  67. 权利要求64-65任一项所述的方法，其还包括对所述对象施用另外的治疗方案。
  68. 权利要求67所述的方法，其中所述另外的治疗方案包括：向所述对象施用一种或更多种的黄嘌呤脱氢酶(XDH)反义多核苷酸或向所述对象施用非黄嘌呤脱氢酶(XDH)dsRNA治疗剂。
  69. 权利要求68所述的方法，其中所述非黄嘌呤脱氢酶(XDH)dsRNA治疗剂选自以下中的一种或多种：别嘌醇，羟嘌呤，非布索坦或白介素-1β(IL-1β)拮抗剂，优选地，所述白介素-1β拮抗剂选自卡那单抗或利纳西普(rilonacept)。
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