An iRNA composition and methods of use thereof

Abstract

An iRNA composition and methods of use thereof.

Description

An iRNA composition and its use method

[0001] This application claims priority to Chinese Patent Application No. 2023112899120 filed on October 7, 2023, and Chinese Patent Application No. 2024113751290 filed on September 29, 2024. This application incorporates the entirety of the aforementioned Chinese patent applications. Technical Field

[0002] The present disclosure relates to RNA interference (RNAi) agents, such as double-stranded RNAi agents, for inhibiting xanthine dehydrogenase (XDH), pharmaceutical compositions comprising XDH RNAi agents, and methods of using the same. Background Art

[0003] Xanthine dehydrogenase (XDH) is a molybdenum-containing hydroxylase that catalyzes the conversion of xanthine to uric acid. XDH is highly expressed in the liver and gastrointestinal tract.

[0004] Xanthine oxidoreductase (xanthine oxidase (XO) and xanthine dehydrogenase (XDH)) is closely related to purine catabolism and uric acid (UA) formation, and is the key enzyme in the last step of uric acid synthesis. Its catalytic center includes the molybdenum pterin center (Mo-pt), the iron-sulfur center (Fe-S), and the flavin adenine dinucleotide (FAD) domain. The active center of the enzyme is located in the molybdenum pterin center. The natural substrate xanthine is hydroxylated in the Mo-pt center to produce uric acid. At the same time, the metal molybdenum in the Mo-pt center is reduced, and electrons are transferred to the FAD domain. Xanthine oxidase uses O2 as a substrate to produce reactive oxygen species in the FAD domain. XDH and XO are different forms of the same gene product. The enzyme is reversibly converted into its oxidase (XO) form by modification of cysteine ​​residues in its dehydrogenase (XDH) form. In this process, XDH helps transfer the two electrons produced by purine metabolism to NAD + XO helps transfer electrons to oxygen to form hydrogen peroxide or superoxide anion. Purines from endogenous cellular nucleic acid metabolism and exogenous dietary intake are converted to uric acid in the liver by xanthine oxidoreductase, which catalyzes the final two steps of uric acid conversion: the conversion of hypoxanthine to xanthine and the conversion of xanthine to uric acid.

[0005] Small molecule inhibitors of XDH have been widely used in uric acid-lowering treatments. Commonly used uric acid-lowering drugs in China are allopurinol, febuxostat, and benzbromarone. Benzbromarone is associated with severe hepatotoxicity and gastrointestinal side effects. Some patients taking allopurinol experience severe hypersensitivity reactions. Recent studies have shown that long-term use of febuxostat may induce heart disease, leading to a black box warning from the FDA. Furthermore, because small molecule drugs require daily administration, over half of patients fail to adhere to the required medication schedule or regular checkups, resulting in inconsistent responses to uric acid treatment. However, many gout patients cannot tolerate or have difficulty accepting these therapies, and these drugs can cause serious side effects, including an increased risk of death. There remains an unmet need for new XDH inhibitors, such as XDH RNAi reagents, to reduce hepatic XDH levels and treat hyperuricemia and gout. siRNA drugs offer a new approach to uric acid-lowering therapy. Following subcutaneous injection, they can be administered for a long period of time, for example, three to six months, significantly improving patient compliance. They also offer manageable safety and side effects, potentially offering the potential for effective treatment of hyperuricemia or gout.

[0006] SUMMARY OF THE INVENTION

[0007] The present disclosure provides an iRNA composition that causes cleavage of RNA transcripts of the gene for XDH mediated by the RNA-induced silencing complex (RISC). XDH can be located within a cell, such as a cell of a subject (eg, a human subject).

[0008] The present disclosure relates to a double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of xanthine dehydrogenase (XDH), wherein the dsRNA agent comprises a sense strand and an antisense strand, and the sense strand is as follows: 5'-Q1-X-Q2-3'. Wherein, Q1 and Q2 are 0, 1, or 2 nucleotide sequences selected from A, U, C, and G nucleotide motifs; X is a sequence comprising positions 1 to n1 of the sense strand sequence shown in Table 1-1 or Table 1-3, where n1 is 18 or 19; and the antisense strand is as follows: 5'-Q3-Y-Q4-3'. Wherein, Q3 and Q4 are 0, 1, or 2 nucleotide sequences selected from A, U, C, and G nucleotide motifs; and Y is a sequence comprising positions n2 to n3 of the antisense strand sequence shown in Table 1-1 or Table 1-3, where n2 is 1 or 2, and n3 is 18, 19, 20, or 21.

[0009] The present disclosure also relates to a double-stranded ribonucleic acid (dsRNA) agent that inhibits the expression of xanthine dehydrogenase (XDH), wherein the dsRNA agent comprises a sense strand and an antisense strand, wherein the nucleotides in the antisense strand comprise a region complementary to the XDH transcript, wherein the complementary region comprises at least 15, at least 16, at least 17, at least 18, or at least 19 consecutive nucleotides that differ from one of the antisense sequences listed in Table 1-1 or one of Tables 1-3 by 0, 1, 2, or 3 nucleotides. The dsRNA agent comprises at least one modified nucleotide, or all or substantially all nucleotides in the antisense strand are modified nucleotides.

[0010] The present disclosure also relates to modified dsRNA agents, such as nucleic acids characterized as described herein, which can be synthesized and / or modified using methods known in the art, for example, at least one modified nucleotide thereof includes: 2'-O-methyl nucleotides, 2'-fluoro nucleotides, 2'-deoxy nucleotides, locked nucleotides (LNA), open-circular nucleic acid nucleotides (UNA), glycol nucleic acid nucleotides (GNA), 2'-F-arabino nucleotides, 2'-methoxyethyl nucleotides, abasic nucleotides, ribitol, inverted nucleotides, inverted abasic nucleotides, inverted 2'-OMe nucleotides, inverted 2'-deoxy nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides and 3'-OMe nucleotides, nucleotides containing a 5'-phosphorothioate group, or a terminal nucleotide linked to a cholesterol derivative or a dodecanoic acid bisdecylamide group, a 2'-amino modified nucleotide, a phosphoramidate, or a non-natural base containing nucleotide. Preferably, the method further comprises an E-vinylphosphonate nucleotide at the 5' end of the sense strand and / or the antisense strand; or at least one phosphorothioate internucleoside linkage in the sense strand and / or the antisense strand.

[0011] In a specific embodiment, the length of the double-stranded region of the dsRNA agent can be 19 to 30 nucleotide pairs; or 19 to 25 nucleotide pairs; or 19 to 21 nucleotide pairs; or 19 to 23 nucleotide pairs; or 21 to 23 nucleotide pairs; or each strand is independently no more than 30 nucleotides in length; or each strand is independently no more than 25 nucleotides in length; or each strand is independently no more than 23 nucleotides in length.

[0012] In a specific embodiment, the dsRNA agent has two blunt ends; or at least one strand comprises a 3' overhang of at least 1 nucleotide; or at least one strand comprises a 3' overhang of at least 2 nucleotides.

[0013] In a specific embodiment, the dsRNA agent further comprises a ligand. Because RNA cannot directly penetrate the cell membrane, a carrier is required to carry the nucleic acid drug to the cytoplasm of the target cell of the target organ. Any carrier that can bring the nucleic acid drug of the present invention into the cytoplasm is applicable to the present invention. Methods known in the art of vectors include, but are not limited to: GalNac vectors, viral delivery (retrovirus, adenovirus, lentivirus, baculovirus, AAV), liposome nanoparticles LNP, polymer vectors, cell-penetrating peptide vectors, bacterial delivery (tkRNAi), and the use of vectors is not limited. Those skilled in the art should understand that displacement and mutation can be made on the basis of the core sequence of the present invention, which can achieve the active effect of the present invention, or even better; so as long as the core sequence X or Y of the present invention is used, or the nucleic acid drug that is displaced or mutated on the basis of the core sequence X or Y of the present invention is within the scope of protection of the present invention; or nucleic acid drugs that only differ from the sequence of the present invention by 0, 1, 2, or 3 nucleotides are also within the scope of protection of the present invention.

[0014] Representative patent applications for oligonucleotide preparation herein include (but are not limited to): WO1986005518A1, WO1986005519A1, WO1991009033A1, WO1991009073A1, WO1993000352A1, WO1994012517A1, WO1995031459A1, US5489677B, US5541307A, US5610289A, US5677437A and US5633360A, etc.

[0015] In one embodiment, the strands in the dsRNA are unmodified and do not comprise chemical modifications and / or conjugations, eg, as known in the art and described herein.

[0016] In another embodiment, the dsRNA is chemically modified to enhance stability or other beneficial properties.

[0017] In another embodiment, all nucleotides in the double-stranded dsRNA are modified.

[0018] In another embodiment, the dsRNA herein is not completely modified on both strands and may include no more than 5, 4, 3, 2, or 1 unmodified nucleotides.

[0019] Another modification of the RNA in the iRNAs herein involves chemically linking the RNA to one or more ligands, moieties, or conjugates that enhance the activity, cellular distribution, or cellular uptake of the iRNA.

[0020] In one embodiment, the incorporation of a ligand into a dsRNA alters its distribution, targeting, or half-life. In preferred embodiments, the ligand enhances affinity for a selected target (e.g., a molecule, cell or cellular form, compartment (e.g., a cellular or organ compartment), tissue, organ, or body region) compared to, for example, the absence of the ligand. Preferably, the ligand does not participate in base pairing within a duplex nucleic acid.

[0021] In some embodiments, the ligand attached to the dsRNA described herein acts as a pharmacokinetic modulator (PK modulator). PK modulators include lipophiles, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, and the like. Examples of PK modulators include, but are not limited to, cholesterol, fatty acids, bile acid, lithocholic acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, and the like.

[0022] Representative patent applications for the preparation of RNA conjugates herein include, but are not limited to, WO1984001394A1, WO1984002360A1, WO1984003285A1, WO1984002362A1, WO1986002929A1, WO1988009810A1, WO1989012110A1, WO1996010030A1, US5218105A, US5512439A, US5214136A, US5317098A, US5512667A, US5688941A, US6825331B2, US7037646B1, US20030113769A1, etc.

[0023] In one embodiment, the ligand is conjugated to the sense strand. Preferably, the ligand is an N-acetylgalactosamine (GalNAc) derivative. More preferably, the ligand is one or more GalNAc derivatives attached via a monovalent, divalent, trivalent or tetravalent branched linker.

[0024] The present disclosure relates to a pharmaceutical composition for inhibiting the expression of a gene encoding xanthine dehydrogenase (XDH). Optionally, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, a buffer solution, a non-buffered solution, or another therapeutic agent. Alternatively, the composition is packaged in a medicine box, a container, a wrapper, a dispenser, a prefilled syringe, or a vial.

[0025] In one embodiment, the pharmaceutical composition can be formulated for subcutaneous administration or formulated for intravenous (IV) administration.

[0026] The present disclosure relates to a cell comprising a dsRNA agent, preferably, the cell is a mammalian cell, optionally a human cell.

[0027] The present disclosure relates to a method for inhibiting the expression of a xanthine dehydrogenase (XDH) gene, the method comprising contacting a target cell with a dsRNA agent or pharmaceutical composition of the present disclosure, thereby inhibiting the expression of the XDH gene in the target cell, wherein the expression of XDH in the target cell is reduced by at least 40%, at least 50%, at least 60%, or at least 70%.

[0028] The present disclosure relates to a method of treating or preventing a subject who would benefit from a condition that would benefit from reduced expression of xanthine dehydrogenase (XDH), the method comprising contacting a target cell with a dsRNA agent or pharmaceutical composition of the present disclosure, thereby inhibiting expression of the XDH gene in the target cell.

[0029] In a specific embodiment, the cell is in a subject, preferably a human, and / or the subject suffers from a xanthine dehydrogenase (XDH)-related disorder; and / or the cell is in a subject and the dsRNA agent is administered subcutaneously to the subject; and / or the cell is in a subject and the dsRNA is administered subcutaneously or IV to the subject; and / or contact of the cell with the dsRNA agent inhibits the expression of xanthine dehydrogenase by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%.

[0030] In a specific embodiment, the disorder is an XDH-related disease, including but not limited to hyperuricemia or gout. DETAILED DESCRIPTION

[0031] the term

[0032] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0033] Before describing the present disclosure in detail below, it should be understood that the present disclosure is not limited to the specific methodologies, protocols and reagents described herein, as these may vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of the present disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present disclosure belongs.

[0034] Certain embodiments disclosed herein include numerical ranges, and some aspects related to the present disclosure can be described in terms of ranges. Unless otherwise stated, it should be understood that numerical ranges or the use of range descriptions are only for the purpose of brevity and convenience, and should not be considered as strict limitations on the scope of the present disclosure. Therefore, the description using ranges should be considered to specifically disclose all possible subranges and all possible specific numerical points within the range, as these subranges and numerical points have been clearly written out in this article. For example, the description of a range from 1 to 6 should be considered to specifically disclose subranges from 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as specific numerical points within these ranges, such as 1, 2, 3, 4, 5, 6. Regardless of the width of the numerical value, the above principles apply equally. When describing in terms of ranges, the range includes the endpoints of the range.

[0035] When referring to a measurable value such as an amount, a temporal duration, etc., the term "about" is meant to include variations of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% of the specified value.

[0036] As used herein, "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during transcription of a xanthine dehydrogenase gene, including mRNAs that are RNA processing products of the primary transcript. The target portion of the sequence is at least long enough to serve as a substrate for iRNA-guided cleavage at or near the nucleotide sequence portion of the mRNA molecule formed during transcription of the XDH gene. In one embodiment, the target sequence is within the protein coding region of XDH.

[0037] The target sequence can be about 18 to 35 nucleotides in length, for example, about 18 to 30 nucleotides in length. For example, the target sequence can be about 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 30, 20 to 29, 20 to 28, 20 to 27, 20 to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, or 21 to 22 nucleotides in length. In some embodiments, the target sequence is about 19 to about 30 nucleotides in length. In other embodiments, the target sequence is about 19 to about 25 nucleotides in length. In other embodiments, the target sequence is about 19 to about 23 nucleotides in length. In some embodiments, the target sequence is about 19 to about 21 nucleotides in length. Ranges and lengths between the ranges and lengths recited above are also considered part of the present invention.

[0038] As used interchangeably herein, the terms "iRNA," "RNAi agent," "iRNA agent," "RNA interfering agent" refer to agents containing RNA, as that term is defined herein, that mediate the targeted cleavage of RNA transcripts through the RNA-induced silencing complex (RISC) pathway. iRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). For example, an "iRNA" for use in the compositions, uses, and methods of the present disclosure can be double-stranded RNA, and is referred to herein as a "double-stranded RNA agent," "double-stranded RNA (dsRNA) molecule," "dsRNA agent," or "dsRNA." The term "dsRNA" refers to a complex of ribonucleic acid molecules having a duplex structure comprising two antiparallel and substantially complementary nucleic acid strands that are referred to as having a "sense" orientation and an "antisense" orientation relative to the target RNA.

[0039] The terms "silencing," "reducing," "inhibiting," "suppressing," or "downregulating," and other similar terms are used interchangeably and encompass any level of inhibition.

[0040] Generally speaking, the majority of nucleotides in each strand of a dsRNA molecule are ribonucleotides, but a single strand or both strands may also include one or more non-ribonucleotides, such as deoxyribonucleotides and / or modified nucleotides. In addition, as used herein, "RNAi agents" may include chemically modified ribonucleotides; RNAi agents may include substantial modifications on multiple nucleotides.

[0041] The term "modified nucleotide" refers to a nucleotide that independently has a sugar moiety, a modified internucleotide linkage, and / or a modified nucleobase. Thus, the term "modified nucleotide" includes substitutions, additions, or exclusions, such as functional groups or atoms, on internucleotide linkages, sugar moieties, or nucleobases. Modifications suitable for use in the formulations herein include all modifications disclosed herein or known in the art. For purposes of this specification and claims, "RNA agent" includes any of these modifications for siRNA-type molecules.

[0042] The term "xanthine dehydrogenase" is used interchangeably with the term "XDH" and refers to a well-known gene and polypeptide, also known in the art as xanthine dehydrogenase / oxidase, xanthine oxidoreductase, XAN1, XDHA, XOR, XO, EC 1.17.1.4, and EC 1.7.2.2. XDH belongs to the class of platinum-containing hydroxylases that participate in purine oxidative metabolism. Based on its ability to mechanically perform different functions, the encoded protein has been identified as a part-time protein. Xanthine dehydrogenase can be converted into xanthine oxidase by reversible sulfhydryl oxidation or irreversible proteolytic modification. As used herein, unless the context clearly indicates, xanthine dehydrogenase or XDH is understood to include both xanthine dehydrogenase and xanthine oxidase ("XO" or "XOR") forms of the protein. This protein is primarily expressed in the intestine and liver, but is also expressed in adipose tissue. Two transcript variants of the human isoform of this gene have been identified. The term "XDH" also refers to naturally occurring DNA sequence variations in the XDH gene. In certain embodiments, such naturally occurring variants are included within the XDH gene sequence.

[0043] The term "strand comprising a sequence" refers to an oligonucleotide comprising a chain of nucleotides described by reference to the sequence using standard nucleotide nomenclature.

[0044] Typically, "G," "C," "A," "T," and "U" each represent a nucleotide containing guanine, cytosine, adenine, thymine, and uracil as a base, respectively. However, it should be understood that the term "ribonucleotide" or "nucleotide" may also refer to a modified nucleotide. It is well known to those skilled in the art that guanine, cytosine, thymine, adenine, and uracil can be replaced by other moieties without substantially changing the base pairing properties of the oligonucleotide containing the nucleotide bearing the replacement moiety. For example, but not limited to, a nucleotide containing inosine as its base can base pair with a nucleotide containing adenine, cytosine, or guanine. Thus, a nucleotide containing uracil, guanine, or adenine in the nucleotide sequence of the dsRNA characterized by the present invention can be replaced with a nucleotide containing, for example, inosine. In another example, adenine and cytosine at any position in the oligonucleotide can be replaced with guanine and uracil, respectively, to form GU wobble base pairing with the target mRNA. Sequences containing such replacement moieties are suitable for use in the compositions and methods featured herein.

[0045] The terms "sequence" and "nucleotide sequence" refer to the sequence or order of nucleobases or nucleotides, described by consecutive letters using standard nomenclature. A nucleic acid molecule may comprise unmodified and / or modified nucleotides. A nucleotide sequence may comprise unmodified and / or modified nucleotides.

[0046] The terms "base," "nucleotide base," or "nucleobase" are heterocyclic pyrimidine or purine compounds that are components of nucleotides, including the primary purine bases adenine and guanine, and the primary pyrimidine bases cytosine, thymine, and uracil. Nucleobases can be further modified to include, but are not limited to, universal bases, hydrophobic bases, hybrid bases, bases with increased size, and fluorinated bases. The synthesis of such modified nucleobases, including phosphoramidite compounds containing modified nucleobases, is known in the art.

[0047] The term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes from a dsRNA. For example, a nucleotide overhang exists when the 3'-end of one strand of a dsRNA extends beyond the 5'-end of the other strand, or vice versa. For example, the overhang can be located on the sense strand, the antisense strand, or any combination thereof. Additionally, for example, the one or more nucleotides of the overhang can appear at the 5'-end, the 3'-end, or both ends of the antisense or sense strand of the dsRNA.

[0048] The term "blunt" or "blunt end" refers to a dsRNA with no unpaired nucleotides at its terminus, i.e., no nucleotide overhang. The dsRNA herein includes a dsRNA agent having a nucleotide overhang at one end (i.e., an agent having one overhang and one blunt end), a dsRNA agent having nucleotide overhangs at both ends, or a dsRNA agent having nucleotide blunt ends at both ends.

[0049] The term "nucleotide" has the same meaning as is generally understood in the art. Thus, the term "nucleotide" as used herein refers to a glycoside comprising a sugar moiety, a base moiety, and a covalent linking group (linking group), such as a phosphate or thiophosphate internucleoside linking group, and encompasses naturally occurring nucleotides, such as DNA or RNA, and non-naturally occurring nucleotides comprising modified sugar and / or base moieties, which are also referred to herein as nucleotide analogs. In this article, a single nucleotide may be referred to as a monomer or unit.

[0050] The term "antisense strand" or "AS strand" refers to a strand of an iRNA (e.g., dsRNA) that includes a region that is substantially complementary to a target sequence (e.g., XDH mRNA). Where the region of complementarity is not completely complementary to the target sequence, mismatches are most tolerated in the terminal regions, and if mismatches occur, they are typically within one or more regions of the termini, e.g., 6, 5, 4, 3, or 2 nucleotides of the 5' and / or 3' termini.

[0051] The term "sense strand" or "SS strand" refers to the strand of a dsRNA that contains a region that is substantially complementary to a region of the antisense strand.

[0052] As used herein, unless otherwise indicated, the term "complementary" is used to describe a first nucleobase or nucleotide sequence (e.g., an RNAi agent sense strand, antisense strand, or single-stranded antisense oligonucleotide) that is associated with a second nucleobase or nucleotide sequence (e.g., an RNAi agent), and refers to an oligonucleotide or polynucleotide comprising a first nucleotide sequence that hybridizes (forms base pair hydrogen bonds under mammalian physiological conditions or other suitable in vivo or in vitro conditions) and forms a duplex or double helical structure with an oligonucleotide comprising a second nucleotide sequence under certain standard conditions. One of ordinary skill in the art will be able to select a set of conditions that best suits the hybridization test. Complementary sequences include Watson-Crick base pairs or non-Watson-Crick base pairs, and include natural or modified nucleotides or nucleotide mimetics, at least to the extent that the above hybridization requirements are met. Sequence identity or complementarity is independent of modification. For example, a and Af as defined herein are complementary to U (or T) and are identical to A for purposes of determining identity or complementarity.

[0053] As used herein, "complementary" or "fully complementary" means that in a pair of hybridized nucleobase or nucleotide sequence molecules, all (100%) bases in the contiguous sequence of the first oligonucleotide will hybridize to the same number of bases in the contiguous sequence of the second oligonucleotide. The contiguous sequence may comprise all or part of the first or second nucleotide sequence.

[0054] As used herein, the term "linked" or "conjugated" when referring to a connection between two compounds or molecules means that the two compounds or molecules are connected by a covalent bond. Unless otherwise indicated, the terms "linked" and "conjugated" as used herein may refer to a connection between a first compound and a second compound, with or without any intervening atoms or groups of atoms.

[0055] As used herein, the term "individual" or "subject" refers to any animal, such as a mammal or marsupial. The individuals contemplated by the present disclosure include, but are not limited to, humans, non-human primates (e.g., cynomolgus or rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats, and any type of poultry.

[0056] As used herein, the terms "disease," "condition," or "disorder" refer to any change or disorder that damages or interferes with the normal function of a cell, tissue, or organ. For example, the term "disease" includes, but is not limited to, tumors, pathogen infection, autoimmune diseases, T-cell dysfunction, or immune tolerance defects (e.g., transplant rejection).

[0057] As used herein, the term "treatment" refers to clinical intervention aimed at altering the course of a disease in an individual or cell, and can be either preventative or interventional in the clinical pathological process. Therapeutic effects include, but are not limited to, preventing the onset or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of a disease, preventing metastasis, slowing the progression of a disease, improving or relieving the condition, and alleviating or improving the prognosis.

[0058] Example

[0059] The present invention will be further described below with reference to specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions such as those described in J. Sambrook et al., Molecular Cloning Laboratory Manual, 3rd edition, Science Press, 2002, or according to the conditions recommended by the manufacturer.

[0060] Example 1: Design and synthesis of oligonucleotide sequences

[0061] Experiment 1: siRNA design

[0062] An antisense strand is used to target a specific position of the xanthine dehydrogenase (XDH) gene transcript (NCBI GeneID: 7498, NM_000379.4), starting from the 5' end of the nucleic acid, and has 21 bases that can complement each other with the target gene.

[0063] The detailed list of unmodified XDH sense and antisense strand nucleotide sequences is shown in Table 1-1.

[0064] Table 1-1 Unmodified XDH sense and antisense strand nucleotide sequences

[0065] Table 1-2 shows the abbreviations used for one or more nucleotides in nucleic acid sequence representations. It will be understood that when these monomers are present in an oligonucleotide, they are linked to each other by 5'-3'-phosphodiester bonds.

[0066] Table 1-2 List of abbreviations

[0067] The detailed lists of the modified XDH sense and antisense strand nucleotide sequences are shown in Tables 1-3.

[0068] Table 1-3 Modified XDH sense and antisense strand nucleotide sequences

[0069] Experiment 2: siRNA synthesis

[0070] siRNA synthesis follows the conventional phosphoramidite solid-phase synthesis method. Nucleoside phosphoramidite monomers are linked one by one on a synthesizer, starting with a universal CPG support. 5-Ethylthio-1H-tetrazole (ETT) is used as an activator (0.6 M in acetonitrile), 0.22 M PADS dissolved in a 1:1 volume ratio of acetonitrile and collidine is used as a sulfurization reagent, and an iodine-pyridine / water solution is used as an oxidant. After solid-phase synthesis, the oligonucleotides are cleaved from the solid support and soaked in a 3:1 solution of 28% ammonia and ethanol at 50°C for 16 hours. The mixture is then centrifuged, and the supernatant is transferred to another centrifuge tube, concentrated, evaporated, and purified by C18 reversed-phase chromatography using a mobile phase of 0.1 M TEAA and acetonitrile. DMTr is removed using 3% trifluoroacetic acid. The target oligonucleotides are collected, lyophilized, identified as the desired product by LC-MS, and quantified by UV spectroscopy (260 nm).

[0071] The resulting single-stranded oligonucleotides were annealed according to complementary pairing in an equimolar ratio to obtain the double-stranded siRNAs shown in Tables 1 to 3. The double-stranded siRNAs were dissolved in 1× PBS and adjusted to the desired concentration for use in the experiment.

[0072] Experiment 3: Synthesis of siRNA conjugates

[0073] L96 was attached to the 3' end of the sense strand. The specific procedure was to attach L96 to a resin, use it as a starting point, and ligate nucleoside monomers one by one in the 3'-5' direction according to the nucleotide sequence. Each ligate nucleoside monomer involves four steps: deprotection, coupling, capping, and oxidation or sulfurization. The procedure is conventional in the art; see the "siRNA Synthesis" section for details. This yields a GalNAc(L96)-conjugated siRNA conjugate targeting XDH.

[0074] It should be noted that L96 is an exemplary ligand. Based on the existing technology related to ligands known in the art, those skilled in the art may also select other ligands other than L96 to conjugate with the modified siRNA in Tables 1-3, as long as the ligand has similar properties as L96 that can increase the delivery of iRNA preparations to the liver.

[0075] Example 2: Screening of modified siRNA conjugates targeting XDH using transfection in HEK-293T cells

[0076] HEK-293T cells were obtained from the ATCC cell bank and cultured in a 37°C, 5% CO2 incubator. HEK-293T cell culture medium contains cell culture medium (DMEM, Gibco-11965092; 10% serum, Gibco-10099-141; 100 units / ml penicillin and 100 μg / ml streptomycin, Gibco-15140122; 1% wt non-essential amino acid solution, Gibco-11140050; 1% (v / v) GlutaMAX, Gibco-35050061). The plasmid pcDNA-Flag-XDH-Hibit was prepared and cloned into the pcDNA3.1 vector containing the XDH CDS region and 3' UTR. A Hibit tag was inserted after the CDS region for subsequent detection.

[0077] In order to detect the knockdown effect of siRNA on XDH mRNA, since the expression level of XDH in HEK-293T cells was extremely low, 5 μg of pcDNA-Flag-XDH-Hibit plasmid was transfected into 6-well plate cells. After 6 h, the cells were digested and 1.5×10 4 Cells were seeded into 96-well plates at a density of 100 cells / well and directly transfected. Plasmid transfection was performed using Lipofectamine 2000 (Invitrogen, 1166801) as described. TM RNAiMAX (Invitrogen, 13778500) transfection reagent was used for siRNA conjugate transfection (50 nM, 1:3 dilution, 9 concentrations).

[0078] 48 hours after transfection, cells were lysed using the Nano-Glo HiBiT lytic Detection System (Promega, N3040) according to the instructions, and chemiluminescence was detected; or cells were lysed using the AllPrep DNA / RNA 96 kit (Qiagen, 80311) according to the instructions, and mRNA was extracted using TaqMan Fast TM XDH mRNA levels were quantified using a 1-step premix (Applied Biosystems, 4444434) and human GAPDH and human XDH Taqman probes. XDH mRNA expression was normalized using GAPDH mRNA expression. Untransfected siRNA cells served as blank controls.

[0079] Table 2 summarizes the results obtained, respectively detecting HiBiT expression and mRNA expression-indicating siRNA knockdown efficiency of XDH.

[0080] Table 2 Effects of siRNA on XDH exogenous expression system and mRNA in transfection system

[0081] Example 3: In vitro screening assay

[0082] Experiment 3.1: In vitro screening using siRNA conjugates targeting XDH in a HEK-293T-XDH stably expressing cell line

[0083] HEK-293T cells were obtained from the ATCC cell bank and cultured in a 37°C, 5% CO2 incubator. HEK-293T cell culture medium contains cell culture medium (DMEM, Gibco-11965092; 10% serum, Gibco-10099-141; 100 units / ml penicillin and 100 μg / ml streptomycin, Gibco-15140122; 1% non-essential amino acid solution, Gibco-11140050; 1% GlutaMAX, Gibco-35050061).

[0084] XDH-HibiT was constructed into lentivirus (lentiviral vector was derived from GeneCare), and after infecting HEK-293T, Puromycine pressure screening was used to select single clones to construct HEK-293T-XDH-puro cell line for subsequent screening. In order to detect the knockdown effect of siRNA on the XDH exogenous expression system, the cells were digested and plated at 1.5×10 4 Cells were seeded into 96-well plates at a density of 100 cells / well and directly transfected using Lipofectamine as described. TMRNAiMAX (Invitrogen, 13778500) transfection reagent was used for siRNA conjugate transfection (300 pM, 3 nM).

[0085] 48 hours after transfection, cells were lysed using the Nano-Glo HiBiT lytic Detection System (Promega, N3040) according to the manufacturer's instructions, and chemiluminescence was measured. Sequence QLAD-000247 was used as a positive control, and untransfected siRNA cells were used as a blank control. Both positive and blank controls were set up in each 96-well plate. The calculation formula was as follows: Average relative residual expression (Avg.%) = (percentage of chemiluminescence expression of the target protein by the siRNA / percentage of chemiluminescence expression of the target protein by the positive siRNA) × 100%.

[0086] Table 3-1 summarizes the results obtained, detecting the knockdown efficiency of XDH by HiBiT expression indicator siRNA.

[0087] Table 3-1 Effects of siRNA in transfection system on XDH exogenous expression system

[0088] STDEV is the standard deviation.

[0089] From Table 3-1, sequences with better efficacy were selected, such as QLAD-000047, QLAD-000048, QLAD-000049, QLAD-000050, QLAD-000053, QLAD-000055, QLAD-000056, QLAD-000061, QLAD-000062, QLAD-000063, QLAD-000064, QLAD-000065, QLAD-000069, QLAD-000071, QLAD-000073, QLAD-000074, QLAD-000075, QLAD-000078, and QLAD-000079. 080, QLAD-000082, QLAD-000087, QLAD-000089, QLAD-000090, QLAD-000091, QLAD-000093, QLAD-0000118, QLAD-000120, QLAD-000126, QLAD-000130, QLAD-000131, QLAD-000133, QLAD-000135, QLAD-000138, QLAD-000149, QLAD-000156, QLAD-000157, QLAD-000159, QLAD-000169, etc., for further in vitro experiments.

[0090] 1.5×10 4 HEK-293T-XDH-puro cells were seeded into 96-well plates at a density of 10 cells / well and directly transfected using Lipofectamine® as described. TM siRNA transfection (3 nM, 100 pM) was performed using RNAiMAX (Invitrogen, 13778500) transfection reagent.

[0091] 48 hours after transfection, cells were lysed using the Nano-Glo HiBiT lytic Detection System (Promega, N3040) according to the instructions and luminescence was detected; or cells were lysed using the AllPrep DNA / RNA 96 kit (Qiagen, 80311) according to the instructions and mRNA was extracted and detected using TaqMan Fast TMXDH mRNA levels were quantified using a 1-step premix (Applied Biosystems, 4444434) and human GAPDH and human XDH Taqman probes. XDH mRNA expression was normalized using GAPDH mRNA expression. Sequence QLAD-000247 was used as a positive control, and untransfected siRNA-transfected cells were used as a blank control. Both positive and blank controls were set up in each 96-well plate. The calculation formula is as follows: Average relative residual information (Avg.%) = (percentage of target gene mRNA expression remaining by siRNA / percentage of target gene mRNA expression remaining by positive siRNA) × 100%.

[0092] Tables 3-2 and 3-3 summarize the results obtained, respectively. HiBiT expression was used to detect the knockdown efficiency of siRNA against XDH, HiBit was used to detect the knockdown effect of siRNA against the target at the protein level, and qPCR was used to detect the knockdown effect of siRNA against the target at the mRNA level. The results of these two methods were used to jointly select the dominant sequences. The residual mRNA content of XDH was detected to indicate the knockdown efficiency of siRNA against XDH.

[0093] Table 3-2 Effects of siRNA on XDH protein expression in a stable transfection system (HiBiT)

[0094] Table 3-3 Effects of siRNA on XDH mRNA expression in a stable transfection system (qPCR)

[0095] Experiment 3.2: In vitro screening using naked siRNA targeting XDH in HEK-293T-XDH stable expression cell line

[0096] HEK-293T cells were obtained from the ATCC cell bank and cultured in a 37°C, 5% CO2 incubator. HEK-293T cell culture medium contained cell culture medium (DMEM, Gibco-11965092; 10% serum, Gibco-10099-141; 100 units / ml penicillin and 100 μg / ml streptomycin, Gibco-15140122; 1% non-essential amino acid solution, Gibco-11140050; 1% GlutaMAX, Gibco-35050061).

[0097] XDH was constructed into lentivirus (lentiviral vector was derived from GeneCare), and after infecting HEK-293T, Puromycine pressure screening was used to select single clones to construct HEK-293T-XDH-puro cell line for subsequent screening. In order to detect the knockdown effect of siRNA on the XDH exogenous expression system, the cells were digested and cultured at 1.5×10 4 Cells were seeded into 96-well plates at a density of 100 cells / well and directly transfected using Lipofectamine as described. TM RNAiMAX (Invitrogen, 13778500) transfection reagent was used for siRNA conjugate transfection (300 pM, 3 nM).

[0098] Table 3-4 Duplex naked sequence

[0099] 48 hours after transfection, cells were lysed using the Nano-Glo HiBiT lytic Detection System (Promega, N3040) according to the manufacturer's instructions, and chemiluminescence was measured. Sequence QLAD-000247 was used as a positive control, and untransfected siRNA cells were used as a blank control. Both positive and blank controls were set up for each 96-well plate. The calculation formula was as follows: Average Relative Residual Information % (Avg.%) = (Residual Percentage of chemiluminescence expression of target protein by siRNA / Residual Percentage of chemiluminescence expression of target protein by positive siRNA) × 100%. The results are summarized in Tables 3-5 and 3-6, respectively.

[0100] Table 3-5 Detection of XDH mRNA expression indicators siRNA knockdown efficiency of XDH

[0101] Table 3-6 Detection of XDH knockdown efficiency by HiBiT expression indicator siRNA

[0102] Example 4: Inhibitory activity of siRNA conjugates in C57 / B6 mice

[0103] Multiple siRNA conjugates targeting XDH were dissolved in DPBS (Thermo, 14190144) to a 3.2 mg / ml siRNA storage solution. Six- to eight-week-old C57 / B6 female mice were used, with four mice per group, and siRNA drugs were administered subcutaneously in the back of the neck at a concentration of 3 mg / kg in a volume of 10 mL / kg. The blank control group received the same dose of PBS. Taking the administration time point as day 0, on day 4, 50 mg of liver tissue from the test group and the blank group was taken and frozen at -80°C in liquid nitrogen.

[0104] Thermofisher Kingfisher and its supporting reagents (Thermofisher, 5400930) were used for mRNA extraction and TaqMan Fast TM The 1-step premix (Applied Biosystems, 4444434) and human GAPDH and human XDH Taqman probes were used to quantify XDH mRNA levels. The expression of XDH mRNA was normalized using the expression of GAPDH mRNA. The results of the PBS group were normalized, and the relative expression of the target gene was calculated using 2 -ΔΔCT The calculation formula is as follows:

[0105] ΔCT = average Ct value of target gene (XDH) - average Ct value of reference gene (GAPDH);

[0106] ΔΔCT=ΔCT(drug-added group)-ΔCT(PBS control group);

[0107] mRNA relative expression = 2 -ΔΔCT The results are shown in Table 4-1.

[0108] Table 4-1 siRNA inhibitory activity in mice

[0109] As shown in Table 4-1, QLAD-000008, QLAD-000010, QLAD-000011, QLAD-000012, QLAD-000015, QLAD-000016, QLAD-000020, QLAD-000023, and QLAD-000025 have good inhibitory activity. Therefore, 0.3 mg / kg, 1 mg / kg, and 3 mg / kg were subsequently selected to screen the above compounds in vivo, and Table 4-2 was obtained.

[0110] Table 4-2 Inhibitory activity of different doses of siRNA in mice

[0111] Example 5: Inhibitory activity of multiple siRNA conjugates targeting XDH in transgenic mice

[0112] XDH transgenic mice were purchased from Jicui Pharmaceutical. These mice were homozygous transgenic mice constructed by inserting the full-length CDS and 3'UTR region of the human XDH gene (NM_000379.4) into the first exon of the mouse. Heterozygous transgenic mice were obtained by crossing homozygous transgenic mice with wild-type mice and used in this experiment.

[0113] Multiple XDH-targeting siRNA conjugates were dissolved in DPBS (Thermo, 14190144) to a 3.2 mg / ml siRNA storage solution. Four female mice, each with heterozygous hXDH, were administered siRNA via subcutaneous injection in the nape of the neck at a concentration of 2 mg / kg in a volume of 10 mL / kg. A blank control group received the same dose of PBS. On day 4, 50 mg of liver tissue from both the test and blank groups was collected and snap-frozen in liquid nitrogen for storage at -80°C.

[0114] The experimental method in Example 4 was used to extract mRNA, and the inhibitory effect of siRNA on the target gene hXDH was detected to obtain Table 5-1.

[0115] Table 5-1 siRNA inhibitory activity in transgenic mice

[0116] As shown in Table 5-1, QLAD-000048, QLAD000049, QLAD000050, QLAD000056, QLAD000063, QLAD-000071, QLAD-000075, QLAD-00087, QLAD-000120, QLAD-000138, QLAD-000251, and QLAD-000266 showed strong inhibitory activity. Further in vivo screening of these compounds was performed using a subset of these groups at different concentrations of 0.2 mg / kg, 0.8 mg / kg, and 1.6 mg / kg, as shown in Table 5-2.

[0117] Table 5-2 Inhibitory activity of different doses of siRNA in transgenic mice

[0118] From Table 5-2, we can see that QLAD-000048, QLAD-000071, QLAD-000075, QLAD-000120, QLAD-0000251, etc. have very good inhibitory activity on target genes, and QLAD-000048, QLAD-000071, QLAD-000075, QLAD-0000120, QLAD-0000251 and others have very good inhibitory activity on the target gene, and can reach an inhibitory effect close to or even exceeding 70% at 1.6 mpk. In particular, QLAD-000048 can achieve an inhibitory effect close to 50% at 0.2 mpk. Some of these groups were selected together with QLAD-000015 and QLAD-000025, which had good inhibitory effects in the previous data, to further verify the inhibitory effect on the target gene in transgenic mice. The screening concentration was 2 mg / kg, and the experimental methods and tissue processing methods were the same as before, resulting in Table 5-3.

[0119] Table 5-3 siRNA inhibitory activity in transgenic mice

[0120] As can be seen from Table 5-3, QLAD-000015, QLAD-000075, and QLAD-000048 can all achieve a knockdown effect of nearly or even exceeding 70% on the target gene in a transgenic mouse heterozygous model when administered at 2 mpk. 2 mpk is used as a moderate screening dose, and these siRNAs have already had a good knockdown effect in mice. Increasing the drug dose is expected to achieve even better knockdown effects and silence the target gene.

[0121] Example 6: Evaluation of siRNA efficacy in human primary hepatocytes by free uptake assay

[0122] Primary human hepatocytes are very close to human cells and cannot be passaged. ASGPR is the ligand for GalNAc in the liver, and similar to human liver, human primary hepatocytes express numerous ASGPRs on their surface. We selected fully modified siRNAs for free uptake into human primary hepatocytes to test whether they could enter the liver cells and exert their inhibitory effects on their targets.

[0123] On day 0, cryopreserved primary human hepatocytes (PHHs) were revived and adjusted to an appropriate density before being plated into 96-well plates. Simultaneously with cell seeding, siRNA was added to the cells via free uptake, and control wells containing no compound were set up. Eight concentrations of the test siRNA were set up in triplicate, with 200 nM selected as the highest concentration, and a 1:3 gradient dilution was performed. 48 hours after free uptake, cells were harvested and RNA extracted. Target gene mRNA in the samples was detected by RT-PCR. GAPDH was also detected as an internal reference gene, as shown in Table 6.

[0124] The relative expression level of the target gene was calculated using 2 -ΔΔCT The calculation formula is as follows:

[0125] ΔCT = average Ct value of target gene - average Ct value of reference gene;

[0126] ΔΔCT=ΔCT(drug-added group)-ΔCT(DMSO control group);

[0127] mRNA relative expression = 2 -ΔΔCT

[0128] Table 6 siRNA efficacy evaluation in human primary hepatocytes by free uptake assay

[0129] As shown in Table 6, QLAD-000048, QLAD-000071, and QLAD-000075 can reach IC in the free uptake experiment of human primary hepatocytes. 50 The potency is <1nM, among which, the IC 50 The uptake rate is even lower than that of other sequences, which is consistent with previous in vivo experimental results. Through free uptake experiments, we verified in vitro that the synthesized siRNA can mimic the human body's in vivo behavior, binding to the ASGPR receptor and being internalized into hepatocytes without the need for transfection reagents, and the dosage is extremely low.

[0130] Example 7: Inhibitory activity of multiple chemically modified siRNA conjugates targeting XDH in C57 / B6 mice

[0131] Following the method in Example 4, siRNAs with different chemical modifications were subcutaneously injected into WT mice at a screening dose of 2 mg / kg in a 10 mL / kg dosing volume. A blank control group received the same dose of PBS. On day 4, 50 mg of liver tissue was collected from the test and blank groups, with the dosing time being day 0. The samples were snap-frozen in liquid nitrogen and stored at -80°C. mRNA was extracted using a Thermofisher Kingfisher, and the expression of the target gene mXDH was detected using the same method as in Example 4. The results from the PBS group were normalized to obtain Tables 7-1, 7-2, and 7-3.

[0132] Table 7-1 siRNA inhibitory activity in mice

[0133] Table 7-2 siRNA inhibitory activity in mice

[0134] Table 7-3 siRNA inhibitory activity in mice

[0135] The sequences in Tables 7-1, 7-2, and 7-3 are the sequences after chemical modification optimization. QLAD-000485, QLAD-000484, QLAD-000490, QLAD-000475, QLAD-000474, QLAD-000476, QLAD-000570, QLAD-000505, QLAD-000573, QLAD-000564, and QLAD-000497 can all maintain or even improve the activities of QLAD-000015, QLAD-000025, QLAD-000048, QLAD-000071, and QLAD-000075. After optimization with different modifications, the activities of some groups were greatly improved.

[0136] Example 8: Inhibitory activity of multiple siRNA conjugates targeting XDH with different chemical modifications in transgenic mice

[0137] Following the method described in Example 4, transgenic mice were subcutaneously injected with various chemically modified siRNAs at a screening dose of 2 mg / kg in a 10 mL / kg dosing volume. A blank control group received the same dose of PBS. On day 4, 50 mg of liver tissue was collected from both the test and blank groups, with dosing time designated as day 0. The samples were then snap-frozen in liquid nitrogen and stored at -80°C. mRNA was extracted using a Thermofisher Kingfisher, and expression of the target gene mXDH was detected using the same method as in Example 2. Results from the PBS group were normalized to obtain Tables 8-1 and 8-2.

[0138] Table 8-1 siRNA inhibitory activity in transgenic mice

[0139] Table 8-2 siRNA inhibitory activity in transgenic mice

[0140] The sequences in Tables 8-1 and 8-2 are chemically modified and optimized. QLAD-000554, QLAD-000543, QLAD-000555, QLAD-000511, QLAD-000508, and QLAD-000513 all maintain or even enhance the activity of QLAD-000015, QLAD-000025, QLAD-000048, QLAD-000071, and QLAD-000075. The activity of QLAD-000048 has already been shown to knock down the target by 70%. QLAD-000554 and QLAD000537 have significantly enhanced their biological activities after optimization with various modifications.

[0141] The sequences obtained in the above examples were rearranged and combined, and all sequences were tested uniformly in transgenic mice. According to the method in Example 4, different chemically modified siRNAs were injected subcutaneously into transgenic mice. The screening dose was 2 mg / kg and the administration volume was 10 mL / kg. The blank control group was given the same dose of PBS. Taking the administration time point as day 0, on day 4, 50 mg of liver tissue from the test group and the blank group was taken and frozen at -80°C with liquid nitrogen. mRNA was extracted using a Thermofisher Kingfisher, and the expression of the target gene mXDH was detected using the same method as in Example 2. The results of the PBS group were normalized to obtain Tables 8-3 and 8-4.

[0142] Table 8-3 siRNA inhibitory activity in homozygous transgenic mice

[0143] Table 8-4 Inhibitory activity of siRNA in heterozygous transgenic mice

[0144] The sequences in Tables 8-3 and 8-4 were chemically modified after optimization in Tables 8-1 and 8-2. QLAD-001028, QLAD-001030, QLAD-001039, QLAD-001041, QLAD-001043, QLAD-001046, QLAD-001048, QLAD-001050, QLAD-001056, QLAD-1057, and QLAD-1058 can all maintain or even improve the activity of QLAD-000015, QLAD-000025, QLAD-000048, QLAD-000071, and QLAD-000075, among which the activity of QLAD-000048 can already knock down the target by 70%. QLAD-001028, QLAD-001048, QLAD-001050, and QLAD-001055 were optimized with different modifications to maintain or improve sequence biological activity.

[0145] Example 9: siRNA inhibitory activity in cynomolgus monkeys

[0146] The screened siRNA conjugates were evaluated for inhibitory activity in cynomolgus monkeys.

[0147] During the experiment, liver biopsies were performed on all animals during the acclimation period (Day 6) and on Day 28 after dosing (7 mg / kg). After an overnight fast, the animals were anesthetized with an intramuscular injection of ketamine (5-10 mg / kg) and then transferred to the operating room. Hair was removed from the triangular area of ​​the animal's chest and abdomen (xiphoid process, costal angle to navel area), and the puncture site was disinfected with iodine and alcohol. Under ultrasound guidance, a 16- or 18-gauge biopsy needle was used to perform a liver biopsy, collecting 5-10 mg of liver tissue samples.

[0148] Thermofisher Kingfisher and its supporting reagents (Thermofisher, 5400930) were used for mRNA extraction and TaqMan Fast TM The 1-step premix (Applied Biosystems, 4444434) and cyno GAPDH and cyno XDH Taqman probes were used to quantify XDH mRNA levels. GAPDH mRNA expression was used to normalize XDH mRNA expression. The results of D-6 group cynomolgus monkeys were normalized, and the relative expression of the target gene was calculated using 2 -ΔΔCT The calculation formula is as follows:

[0149] ΔCT = average Ct value of target gene (XDH) - average Ct value of reference gene (GAPDH)

[0150] ΔΔCT = ΔCT (corresponding day of drug-added group) - ΔCT (Day-6 mRNA);

[0151] mRNA relative expression = 2 -ΔΔCT , we get Table 9-1

[0152] Table 9-1 Experimental results

[0153] The results in Table 9-1 show that on day 28, the knockdown of XDH mRNA in the liver by siRNA was >50%.

[0154] The embodiments of the present disclosure described above are merely exemplary, and any person skilled in the art will recognize or be able to determine the equivalents of numerous specific compounds, materials, and operations without requiring undue experimentation. All such equivalents are within the scope of the present disclosure and are encompassed by the claims.

Claims

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

2. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of xanthine dehydrogenase (XDH), wherein the dsRNA agent comprises a sense strand and an antisense strand, The sense strand comprises the structure shown in formula (i): 5'-Q1-X-Q2-3' formula (i) in, Q1 and Q2 are 0, 1 or 2 nucleotide sequences selected from A, U, C, and G nucleotide motifs; X is a sequence comprising positions 1 to n1 of any sense strand sequence shown in Table 1-1, wherein n1 is position 18 or 19; The antisense strand comprises a structure as shown in formula (ii): 5'-Q3-Y-Q4-3' formula (ii) Wherein, Q3 and Q4 are 0, 1 or 2 nucleotide sequences selected from A, U, C, and G nucleotide motifs; Y is a sequence comprising positions n2 to n3 of any antisense chain sequence shown in Table 1-1, wherein n2 is 1 or 2, and n3 is 18, 19, 20 or 21.

3. The dsRNA agent of claim 1 or 2, wherein the dsRNA agent comprises at least one modified nucleotide.

4. The dsRNA agent of claim 1 or 2, wherein all or substantially all nucleotides in the antisense strand are modified nucleotides, preferably, all nucleotides of the sense strand and all nucleotides of the antisense strand comprise modifications.

5. The dsRNA agent of claim 3 or 4, wherein the at least one modified nucleotide comprises: 2'-O-methyl nucleotides, 2'-fluoro nucleotides, 2'-deoxy nucleotides, locked nucleotides (LNA), open-loop nucleic acid nucleotides (UNA), glycol nucleic acid nucleotides (GNA), 2'-F-arabino nucleotides, 2'-methoxyethyl nucleotides, abasic nucleotides, ribitol, inverted nucleotides, inverted abasic nucleotides, inverted 2'-OMe nucleotides, inverted 2'-deoxy nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides and 3'-OMe nucleotides, nucleotides containing a 5'-phosphorothioate group, or a terminal nucleotide linked to a cholesterol derivative or a dodecanoic acid bisdecylamide group, a 2'-amino modified nucleotide, phosphoramidate, or a non-natural base containing nucleotide.

6. The dsRNA agent of any one of claims 3 to 5, further comprising an E-vinylphosphonate nucleotide at the 5' end of the sense strand and / or the antisense strand; or comprising at least one phosphorothioate internucleoside linkage in the sense strand and / or the antisense strand.

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

8. The dsRNA agent of any one of the preceding claims, wherein the length of the double-stranded region is selected from: ①19 to 30 nucleotide pairs; ②19 to 25 nucleotide pairs; ③19 to 21 nucleotide pairs; ④19 to 23 nucleotide pairs; ⑤21 to 23 nucleotide pairs; ⑥ The length of each chain is independently no more than 25 nucleotides; 7. Each strand is independently no more than 23 nucleotides in length; and ⑧The length of each chain is independently no more than 21 nucleotides.

9. A dsRNA agent as claimed in any one of the preceding claims, wherein The dsRNA agent has: ① Two blunt ends; or ② at least one strand contains a 3' overhang of at least 1 nucleotide; or ③At least one strand contains a 3' overhang of at least 2 nucleotides.

10. A dsRNA agent, comprising a sense strand and an antisense strand, wherein: ① The sense strand contains a nucleotide sequence X, and the antisense strand contains a nucleotide sequence Y, and the nucleotide sequence X and the nucleotide sequence Y are at least partially reverse-complementary to form a double-stranded region; and, ② Each nucleotide in the sense strand and the antisense strand is independently a fluorinated modified nucleotide or a non-fluorinated modified nucleotide; and, ③ The fluorinated modified nucleotides are located in the nucleotide sequence X and the nucleotide sequence Y; and, ④ In the direction from the 5' end to the 3' end, in the sense strand, the nucleotides at positions 7, 9, 10, and 14 of the nucleotide sequence X are fluorinated modified nucleotides, and the nucleotides at the remaining positions in the sense strand are non-fluorinated modified nucleotides; and, ⑤ In the direction from the 5' end to the 3' end, in the antisense strand, the nucleotides at positions 2, 5, 14, and 18 of the nucleotide sequence Y are fluorinated modified nucleotides, and the nucleotides at the remaining positions in the antisense strand are non-fluorinated modified nucleotides; and, ⑥ The dsRNA targets and inhibits xanthine dehydrogenase (XDH); and / or ⑦ The sense strand comprises a structure as shown in formula (i): 5'-Q1-X-Q2-3' formula (i), wherein Q1 and Q2 are 0, 1 or 2 nucleotide sequences selected from A, U, C, and G nucleotide motifs; X is a sequence comprising positions 1 to n1 of any sense strand sequence shown in Table 1-1, wherein n1 is position 18 or position 19; and the antisense strand comprises a structure as shown in formula (ii): 5'-Q3-Y-Q4-3' formula (ii), wherein Q3 and Q4 are 0, 1 or 2 nucleotide sequences selected from A, U, C, and G nucleotide motifs; Y is a sequence comprising positions n2 to n3 of any antisense strand sequence shown in Table 1-1, wherein n2 is 1 and n3 is position 18, 19, 20, or 21.

11. The dsRNA agent according to any one of claims 1 to 10, wherein I) the nucleotide sequence X is equal in length to the nucleotide sequence shown in SEQ ID NO: 805 and differs in no more than 3 nucleotides, and the nucleotide sequence Y is equal in length to the nucleotide sequence shown in SEQ ID NO: 806 and differs in no more than 3 nucleotides; or II) the nucleotide sequence X is equal in length to the nucleotide sequence shown in SEQ ID NO: 823 and differs in no more than 3 nucleotides, and the nucleotide sequence Y is equal in length to the nucleotide sequence shown in SEQ ID NO: 824 and differs in no more than 3 nucleotides; or III) the nucleotide sequence X is equal in length to the nucleotide sequence shown in SEQ ID NO: 841 and differs in no more than 3 nucleotides, and the nucleotide sequence Y is equal in length to the nucleotide sequence shown in SEQ ID NO: 842 and differs in no more than 3 nucleotides; or IV) the nucleotide sequence X is equal in length to the nucleotide sequence shown in SEQ ID NO: 843 and differs in no more than 3 nucleotides, and the nucleotide sequence Y is equal in length to the nucleotide sequence shown in SEQ ID NO: 844 and differs in no more than 3 nucleotides; or V) the nucleotide sequence X is equal in length to the nucleotide sequence shown in SEQ ID NO: 1691 and differs in no more than 3 nucleotides, and the nucleotide sequence Y is equal in length to the nucleotide sequence shown in SEQ ID NO: 1692 and differs in no more than 3 nucleotides; or VI) the nucleotide sequence X is equal in length to the nucleotide sequence shown in SEQ ID NO: 1709 and differs in no more than 3 nucleotides, and the nucleotide sequence Y is equal in length to the nucleotide sequence shown in SEQ ID NO: 1710 and differs in no more than 3 nucleotides; or VII) the nucleotide sequence X is equal in length to the nucleotide sequence shown in SEQ ID NO: 1727 and differs in no more than 3 nucleotides, and the nucleotide sequence Y is equal in length to the nucleotide sequence shown in SEQ ID NO: 1728 and differs in no more than 3 nucleotides; or VIII) the nucleotide sequence X is equal in length to the nucleotide sequence shown in SEQ ID NO: 1729, and differs in no more than 3 nucleotides, and the nucleotide sequence Y is equal in length to the nucleotide sequence shown in SEQ ID NO: 1730, and differs in no more than 3 nucleotides.

12. The dsRNA agent of any one of the preceding claims, further comprising a ligand.

13. The dsRNA agent of claim 12, wherein the ligand is conjugated to the sense strand, preferably, the sense strand is conjugated to the ligand attached at the 3' end.

14. The dsRNA agent of claim 12 or 13, wherein the ligand is an N-acetylgalactosamine (GalNAc) derivative.

15. The dsRNA agent of any one of claims 12-14, wherein the ligand is one or more GalNAc derivatives attached via a monovalent, divalent, trivalent or tetravalent branched linker.

16. A pharmaceutical composition comprising a dsRNA agent as claimed in any one of the preceding claims for use in inhibiting the expression of a gene encoding xanthine dehydrogenase (XDH), wherein the expression of XDH is reduced by at least 40% in an animal.

17. The pharmaceutical composition of claim 16, further comprising a pharmaceutically acceptable carrier, a buffered solution, a non-buffered solution, or an additional therapeutic agent.

18. The pharmaceutical composition of claim 16 or 17, wherein the pharmaceutical composition is packaged in a kit, container, package, dispenser, prefilled syringe or vial.

19. The pharmaceutical composition of any one of claims 16-18, wherein the pharmaceutical composition is formulated for subcutaneous administration or is formulated for intravenous (IV) administration.

20. A cell comprising the dsRNA agent of any preceding claim.

21. The cell of claim 20, wherein The cell is a mammalian cell, optionally a human cell.

22. A method for inhibiting the expression of a xanthine dehydrogenase (XDH) gene, the method comprising contacting a target cell with a dsRNA agent as described in any one of claims 1 to 16 or a pharmaceutical composition as described in any one of claims 16 to 19, thereby inhibiting the expression of the XDH gene in the target cell; Preferably, the method is in vivo or in vitro.

23. A method for treating or preventing a subject suffering from a condition that would benefit from reduced expression of xanthine dehydrogenase (XDH), the method comprising administering to the subject a therapeutically or prophylactically effective amount of a dsRNA agent as described in any one of claims 1-15 or a pharmaceutical composition as described in any one of claims 16-19, thereby treating or preventing the subject suffering from a condition that would benefit from reduced expression of XDH; or, use of a dsRNA agent as described in any one of claims 1-15 or a pharmaceutical composition as described in any one of claims 16-19 in the preparation of a medicament for treating and / or preventing a condition that would benefit from reduced expression of XDH.

24. The method of claim 22 or 23, wherein: ① The cell is in a subject, preferably a human; and / or ② The subject suffers from a xanthine dehydrogenase (XDH)-related disorder; and / or ③ the cell is in the subject and the dsRNA agent is subcutaneously administered to the subject; and / or ④ the cell is in a subject and the dsRNA is administered to the subject via IV; and / or ⑤ Contact of the cell with the dsRNA agent inhibits the expression of xanthine dehydrogenase by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95%.

25. The method of claim 23 or 24, wherein the condition is an XDH-related disease, preferably hyperuricemia or gout.