Kidney-targeting double-stranded oligonucleotide conjugate and use thereof

Abstract

Provided is a polypeptide molecule suitable for kidney targeting. The polypeptide molecule is covalently linked to an active substance (such as an oligonucleotide) by means of a linking group to obtain an oligonucleotide conjugate. The oligonucleotide conjugate has an affinity for a cell receptor present on a kidney target cell and can selectively and effectively reduce or inhibit the expression of a kidney target gene in a subject (such as a human or animal). The provided oligonucleotide conjugate or pharmaceutical composition can effectively treat and / or prevent pathological conditions or diseases caused by abnormal expression of a specific gene in kidney tissue cells.

Description

Kidney-targeted double-stranded oligonucleotide conjugates and uses thereof

[0001] Cross-reference to related applications

[0002] The present disclosure claims priority to the Chinese patent application No. 202410536410.1, filed on April 30, 2024, entitled “Kidney-targeted double-stranded oligonucleotide conjugates and uses thereof”, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0003] The present disclosure is in the field of oligonucleotide pharmaceuticals and specifically relates to compounds and methods for the in vivo delivery of RNAi agents, e.g., double-stranded oligonucleotide molecules, to extrahepatic cells, and in particular to compounds and methods for the delivery of oligonucleotide molecules to kidney cells for the inhibition of the expression of genes expressed thereby. BACKGROUND

[0004] The kidney plays an important role in the transport and excretion of different substances in the body and controls the intermediate metabolism of a variety of substances in the body. At present, there are at least one hundred million people worldwide suffering from various chronic kidney diseases. Despite the existence of various drugs and treatment methods, such as immunosuppressants, anti-inflammatory agents or diuretics, there is still room for improvement in the targeted delivery of drugs for the treatment of kidney diseases to the kidney and the persistence of the effect.

[0005] Oligonucleotide-based drugs, such as double-stranded RNAi agents, have excellent therapeutic potential, but the extrahepatic delivery thereof has long been a challenge, in particular, there has not been a satisfactory implementation of the highest possible affinity and selectivity for the kidney. Therefore, there is still a need for new conjugate molecules or delivery carrier molecules to effectively direct oligonucleotide-containing drug molecules to extrahepatic, in particular, kidney cells. SUMMARY

[0006] The present disclosure relates to polypeptide molecules suitable for kidney targeting, which can serve as carrier molecules for the delivery of oligonucleotides. The present disclosure also relates to conjugate molecules comprising said polypeptides and at least one active substance, such as an oligonucleotide, covalently bound via a linker, and to methods for the preparation of said conjugates. Furthermore, the present disclosure relates to the use of said peptides and conjugates for kidney targeting, and to pharmaceutical compositions comprising said peptides or conjugates.

[0007] The present disclosure found that peptides of a particular amino acid sequence composition and their oligonucleotide conjugates covalently linked to at least one targeting ligand have selectivity for kidney cells and affinity to cell receptors present on kidney target cells. The conjugates of the present disclosure can selectively and effectively reduce or inhibit the expression of kidney target genes in a subject, such as a human or an animal.

[0008] The present disclosure also describes a pharmaceutical composition comprising the conjugate, the conjugate comprising an oligonucleotide molecule capable of inhibiting the expression of a target gene, the composition further comprising at least one pharmaceutically acceptable carrier or excipient. The pharmaceutical composition is capable of selectively and effectively reducing or inhibiting the expression of a target gene in a kidney cell in vivo.

[0009] In a first aspect of the present disclosure, the present disclosure provides an oligonucleotide conjugate for inhibiting the expression of a gene in a kidney cell, the oligonucleotide conjugate comprising:

[0010] (a) a single-stranded or double-stranded oligonucleotide molecule;

[0011] (b) a targeting ligand, the targeting ligand having affinity for a receptor present on the surface of a kidney cell;

[0012] (c) a linker group, the oligonucleotide molecule being covalently linked to the targeting ligand through the linker group.

[0013] In some embodiments of the present disclosure, the single-stranded or double-stranded oligonucleotide molecule has the following features:

[0014] (i) comprises an antisense strand of 17-35 nucleotides, wherein at least 15 nucleotides are complementary or substantially complementary to the mRNA sequence of a gene in a kidney cell;

[0015] (ii) a sense strand of 15-35 nucleotides in length, which is complementary or partially complementary to the antisense strand.

[0016] In some embodiments of the present disclosure, the targeting ligand is a polypeptide.

[0017] In some embodiments of the present disclosure, the receptor is a known or unknown endocytic receptor.

[0018] In some embodiments of the present disclosure, the receptor is selected from the group consisting of a megalin receptor or a receptor for a lina peptide.

[0019] In some embodiments of the present disclosure, the linker group is selected from a substituted or unsubstituted aliphatic chain, a 3-6 membered heterocyclic ring, a C6-C 10 aromatic ring, a (PEG) n group containing a disulfide bond, an amide group or a triazole group, or a combination of the above groups; wherein n is selected from an integer of 1-20, preferably an integer of 1-10.

[0020] The oligonucleotide conjugate for inhibiting the expression of a gene in a kidney cell as described above, wherein the targeting ligand linked by the linker is a ligand unit, in the present disclosure, the ligand unit is selected from any of the structures shown in C1) to C5):

[0021] C1) La-KKEEE-KKEEE-KKEEE-K-Laa;

[0022] C2) La-CKKEEE-KKEEE-KKEEE-K-Laa;

[0023] C3) La-CLPVASC-Laa; preferably a cyclic peptide

[0024] C4) La-CYFQNCPRG-Laa; preferably a cyclic peptide

[0025] C5) Lb-KIDRI-Laa;

[0026] In formulae C1) - C4), any one of Laand Laais selected from a linker, the other is a terminal blocking group, the linker is a linking group, and comprises at least one group of triazole group or PEG unit or acyl group.

[0027] In some embodiments of the present disclosure, Lais a linker, the Linker structure comprises -PEG2-CH2CH2CO- or triazole-PEG2-.

[0028] In formula C5), Lbis a linking group comprising a 6-10 membered aromatic ring or heteroaromatic ring, and Laais independently selected from an amino group, or an alkyl-substituted amine group such as -NHCH3.

[0029] In some embodiments of the present disclosure, at least one ligand unit is combined with an oligonucleotide molecule to form an oligonucleotide conjugate.

[0030] In some embodiments of the present disclosure, the oligonucleotide molecule inhibits the expression of mRNA of a gene in human kidney cells.

[0031] In a second aspect of the present disclosure, the present disclosure provides a polypeptide capable of delivering an oligonucleotide to kidney cells, the polypeptide comprising any one of the amino acid sequence segments shown in B1) - B5):

[0032] B1) -KKEEE-KKEEE-KKEEE-K-;

[0033] B2) -CKKEEE-KKEEE-KKEEE-K-;

[0034] B3)

[0035] B4)

[0036] B5) -KIDRI-.

[0037] In a third aspect of the present disclosure, the present disclosure provides a ligand unit molecule capable of delivering an oligonucleotide to a kidney cell, said ligand unit molecule is selected from any one of the compounds shown in E1) to E8):

[0038] E1) N3-PEG2-CH2CH2CO-KKEEE-KKEEE-KKEEE-K-NH2;

[0039] E2) Ac-CKKEEE-KKEEE-KKEEE-K-NH2;

[0040] E3) N3-(PEG)2-CH2CH2-CO-CLPVASC-NH2 (cyclopeptide);

[0041] E4) N3-(PEG)2-CH2CH2-CO-CYFQNCPRG-NH2;

[0042] E5)

[0043] E6)

[0044] E7)

[0045] E8)

[0046] In a fourth aspect of the present disclosure, the present disclosure provides a use of the polypeptide of the second aspect of the present disclosure or the ligand unit molecule of the third aspect of the present disclosure, said use is for delivering an oligonucleotide molecule to a kidney cell.

[0047] In a fifth aspect of the present disclosure, the present disclosure provides a composition comprising the oligonucleotide conjugate of the first aspect of the present disclosure or the polypeptide of the second aspect of the present disclosure or the ligand unit molecule of the third aspect of the present disclosure, or a pharmaceutically acceptable salt thereof.

[0048] In a sixth aspect of the present disclosure, the present disclosure provides a pharmaceutical composition comprising the oligonucleotide conjugate of the first aspect of the present disclosure, or a pharmaceutically acceptable salt thereof.

[0049] In some optional embodiments of the present disclosure, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers or excipients.

[0050] In a seventh aspect of the present disclosure, the present disclosure provides a method for preparing the oligonucleotide conjugate of the first aspect of the present disclosure, said method comprises the following steps:

[0051] (i) synthesizing the sense strand;

[0052] (ii) synthesizing the antisense strand;

[0053] (iii) annealing the sense strand and the antisense strand;

[0054] (iv) conjugating the ligand unit molecule to the sense strand or the antisense strand before or after annealing the sense strand and the antisense strand.

[0055] In an eighth aspect of the present disclosure, the present disclosure provides a method of treating a kidney-related disease or disorder, the method comprising administering to a subject the oligonucleotide conjugate of the first aspect of the present disclosure, the composition of the fifth aspect of the present disclosure, or the pharmaceutical composition of the sixth aspect of the present disclosure.

[0056] In some alternative embodiments of the present disclosure, the kidney-related disease or disorder is chronic kidney disease.

[0057] In some alternative embodiments of the present disclosure, the kidney-related disease or disorder is selected from the group consisting of hypertension, hypertension-related kidney damage, hyperglycemia, diabetes, diabetic nephropathy, hyperuricemia, gout, hyperuricemia kidney damage, hepatitis B virus-related kidney damage, myeloma kidney disease, chronic renal failure, glomerulonephritis, renal vascular disease, C3 glomerulopathy, lupus nephritis, IgA nephropathy, polycystic kidney disease, membranous nephropathy, atypical hemolytic nephropathy, uremic syndrome, or systemic lupus erythematosus (SLE)-related kidney disease.

[0058] In a ninth aspect of the present disclosure, the present disclosure provides a method of delivering one or more oligonucleotide molecules to a cell in vivo, the method comprising administering to a subject the target ligand-containing oligonucleotide conjugate of the first aspect of the present disclosure, the composition of the fifth aspect of the present disclosure, or the pharmaceutical composition of the sixth aspect of the present disclosure.

[0059] In a tenth aspect of the present disclosure, the present disclosure provides use of the oligonucleotide conjugate of the first aspect of the present disclosure, the composition of the fifth aspect of the present disclosure, or the pharmaceutical composition of the sixth aspect of the present disclosure in the manufacture of a medicament for treating a kidney-related disease or disorder.

[0060] In some alternative embodiments of the present disclosure, the kidney-related disease or disorder is chronic kidney disease.

[0061] In some alternative embodiments of the present disclosure, the kidney-related disease or disorder is selected from the group consisting of hypertension, hypertension-related renal impairment, hyperglycemia, diabetes, diabetic nephropathy, hyperuricemia, gout, hyperuricemia-related renal impairment, hepatitis B virus-related renal impairment, myeloma kidney, chronic renal failure, glomerulonephritis, renal vascular disease, C3 glomerulopathy, lupus nephritis, IgA nephropathy, polycystic kidney disease, membranous nephropathy, atypical hemolytic uremic syndrome, uremic syndrome, or systemic lupus erythematosus (SLE)-related nephropathy.

[0062] The present disclosure provides a polypeptide molecule suitable for kidney targeting, which is covalently linked to an active substance (e.g., an oligonucleotide) via a linker to obtain an oligonucleotide conjugate, which has affinity to a cell receptor present on a target cell of the kidney and can selectively and effectively reduce or inhibit the expression of a target gene in the kidney of a subject (e.g., a human or an animal). The oligonucleotide conjugate or pharmaceutical composition provided by the present disclosure can effectively treat and / or prevent a pathological condition or disease caused by abnormal expression of a specific gene in a kidney tissue cell. BRIEF DESCRIPTION OF DRAWINGS

[0063] FIG. 1 is the inhibitory activity of a target gene in the kidney of a mouse after administration of the siRNA conjugate described in Example 1.

[0064] FIG. 2 is the inhibitory activity of a target gene in the kidney of a mouse after administration of the siRNA conjugate described in Example 2.

[0065] FIG. 3 is the inhibitory activity of a target gene in the heart, liver, and duodenum of a mouse after administration of the siRNA conjugate described in Example 2.

[0066] FIG. 4 is the inhibitory activity of a target gene in the kidney of a mouse after administration of the siRNA conjugate described in Example 3.

[0067] FIG. 5 is the inhibitory activity of a target gene in the kidney of a mouse after administration of the siRNA conjugate described in Example 4.

[0068] FIG. 6 is the inhibitory activity of a target gene in the heart, liver, and duodenum of a mouse after administration of the siRNA conjugate described in Example 4.

[0069] FIG. 7 is the inhibitory activity of a target gene in the kidney of a mouse after administration of the siRNA conjugate described in Example 5.

[0070] FIG. 8 is the inhibitory activity of a target gene in the cortex of the kidney of a mouse after administration of the siRNA conjugate described in Example 6.

[0071] FIG. 9 is the inhibitory activity of a target gene in the medulla of the kidney of a mouse after administration of the siRNA conjugate described in Example 6.

[0072] Figure 10 is the inhibitory activity of target genes in mouse kidney after administration of siRNA conjugate described in Example 7.

[0073] Figure 11 is the inhibitory activity of target genes in mouse kidney after administration of siRNA conjugate described in Example 8.

[0074] Figure 12 is the inhibitory activity of target gene ALDH2 in mouse after administration of siRNA conjugate R381002, R381004 in Example 9.

[0075] Figure 13 is the inhibitory activity of target gene URAT1 in cynomolgus monkey after administration of siRNA conjugate R391005 in Example 10. DETAILED DESCRIPTION

[0076] The technical solutions in the embodiments of the present application will be described below in a clear and complete manner, and those skilled in the art can refer to the content herein to appropriately improve process parameters for implementation.

[0077] Terminology

[0078] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, suitable methods and materials are described below. The publications, patent applications, patents, and other references noted herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0079] In the present disclosure, the term "comprising" or "including" is an open-ended expression, which is used in the present disclosure to mean "including, but not limited to", and is used interchangeably with the same meaning, to mean that the present disclosure includes the indicated content, but does not exclude other aspects.

[0080] In the present disclosure, the term "optionally", "optional" or "optional" generally means that the event or condition described subsequently can but does not necessarily occur, and the description includes both cases where the event or condition occurs and cases where the event or condition does not occur.

[0081] In the present disclosure, the term "small interfering RNA (siRNA)" is a double-stranded RNA of 17 to 25 nucleotides in length, comprising a sense strand and an antisense strand. siRNA mediates the RISC pathway of RNA transcript targeting cleavage by forming a silencing complex (RISC). Specifically, siRNA directs the specific degradation of mRNA sequences through a known RNA interference (RNAi) process, inhibiting the translation of mRNA into amino acids and conversion into proteins.

[0082] In the present disclosure, the terms "sequence" and "nucleotide sequence" refer to a series of nucleobases or nucleotides. As used in the present disclosure, a "base," "nucleotide base," or "nucleobase" is a pyrimidine or purine compound that is a component of a nucleotide and includes the purine bases adenine and guanine, as well as the pyrimidine bases cytosine, thymine, and uracil. Nucleobases can be further modified. The synthesis of modified nucleobases, including phosphoramidite compounds of modified nucleobases, is known in the art.

[0083] In the present disclosure, the term "double-stranded oligonucleotide" refers to a double-stranded structure formed by two oligonucleotides, including a sense strand and an antisense strand, through partial or complete base pairing between them, and the length of the sense strand and the antisense strand can be the same or different, as long as there is at least a region of partial base pairing to form a duplex region. An oligonucleotide with a double-stranded structure is within the scope of the double-stranded oligonucleotide of the present disclosure. In the present disclosure, the nucleotides in the double-stranded oligonucleotide can be modified or unmodified nucleotides, and when referring to modified nucleotides, the modifications referred to in the present disclosure do not specifically refer to the modification site, unless otherwise specified. In the present disclosure, the double-stranded oligonucleotide can be modified in addition to the modification of the nucleotides, and the linkage between the nucleotides can also be modified. A double-stranded oligonucleotide containing a modified linkage between the modified nucleotides is also within the scope of the double-stranded oligonucleotide of the present disclosure. In the present disclosure, the double-stranded oligonucleotide can further contain a compound molecule or a modifier acceptable in the art to improve the properties of the double-stranded oligonucleotide, such as a ligand to form a conjugate.

[0084] In the present disclosure, the term "antisense strand (or guide strand)" includes a region that is substantially complementary to a target sequence. The term "sense strand (or passenger strand)" refers to an RNAi strand that contains a sequence that is substantially complementary to the antisense strand. The term "substantially complementary" means completely complementary or at least partially complementary, e.g., the antisense strand is completely complementary or at least partially complementary to the target sequence. In the case of partial complementarity, mismatches can exist within the interior of the molecule or within the terminal regions, with the most tolerated mismatches existing within the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5'- and / or 3' terminus of the iRNA. It is noted that "at least partially substantially complementary" of an antisense strand to an mRNA means that the antisense strand has a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest.

[0085] In the present disclosure, and unless otherwise indicated, the term "complementary" when used to describe a first nucleobase or nucleotide sequence (e.g., an RNAi agent sense / antisense strand or a targeted mRNA) relative to a second nucleobase or nucleotide sequence refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize (form base pair hydrogen bonds under mammalian physiological conditions (or in vitro analogous conditions)) and form a duplex or double helix structure under certain standard conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence.

[0086] In the present disclosure, "completely complementary" means that in a pair of hybridized nucleobase or nucleotide sequence molecules, all (100%) of the bases in a contiguous sequence of a first oligonucleotide will hybridize with the same number of bases in a contiguous sequence of a second oligonucleotide. The contiguous sequence can comprise all or a portion of the first or second nucleotide sequence. "Partially complementary" means that in a pair of hybridized nucleobase or nucleotide sequence molecules, a substantial portion, e.g., at least 70% (but not all) of the bases in a contiguous sequence of a first oligonucleotide will hybridize with the same number of bases in a contiguous sequence of a second oligonucleotide. The contiguous sequence can comprise all or a portion of the first or second nucleotide sequence.

[0087] In the present disclosure, "substantially complementary" means that in a pair of hybridized nucleobase or nucleotide sequence molecules, a substantial portion, e.g., at least 85% or 3 nucleotide differences (but not all) of the bases in a contiguous sequence of a first oligonucleotide will hybridize with the same number of bases in a contiguous sequence of a second oligonucleotide. The contiguous sequence can comprise all or a portion of the first or second nucleotide sequence.

[0088] In the present disclosure, the terms "complementary," "completely complementary," "partially complementary," and "substantially complementary" are used in reference to nucleobase or nucleotide matches between the sense and antisense strands of an RNAi agent, or between the antisense strand of an RNAi agent and the sequence of a target mRNA.

[0089] In the present disclosure, the term "nucleotide difference" and the term "nucleotide base difference" and the term "difference in nucleotide sequence" can be used interchangeably. It means that the base type of the nucleotide at the same or corresponding position has been changed compared to the original nucleotide sequence. For example, when one nucleotide base in the original nucleotide sequence is A, and the nucleotide base at the same or corresponding position is changed to U, C, G, or dT, dC, dG, etc., it is considered that there is a difference in the nucleotide sequence at that position. It should be noted here that in the case where the nucleotide at the same or corresponding position is only different in terms of whether there is a modification or the type of modification compared to the original nucleotide sequence, it is not considered that there is a difference in the nucleotide sequence at that position.

[0090] In the present disclosure, the term "ligand" or "conjugate group" refers to an atom or group of atoms that binds to an oligonucleotide or other oligomer. Generally, conjugate groups modify one or more properties of the compound to which they are attached, including, but not limited to, pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake, charge, and / or clearance properties. The term "linked" as used herein when referring to the linkage between two molecules means that the two molecules are connected directly or indirectly by a covalent bond, or that the two molecules are associated via a non-covalent bond (e.g., a hydrogen bond or an ionic bond).

[0091] In the present disclosure, the term "targeting ligand" refers to a polypeptide that has affinity for a receptor present on the surface of a kidney cell; the term "ligand unit" refers to a targeting ligand linked by a linker; the term "ligand unit molecule" refers to a compound that is reacted with an oligonucleotide in a molecular form.

[0092] In the present disclosure, the term "linked" or "conjugated" when referring to the linkage between two compounds or molecules means that the two molecules are connected by a covalent bond or are associated via a non-covalent bond (e.g., a hydrogen bond or an ionic bond). Unless otherwise specified, the terms "linked" and "conjugated" as used in the present disclosure can refer to the linkage between a first compound and a second compound, with or without any intervening atoms or groups of atoms.

[0093] In the present disclosure, a linking group is one or more atoms that link one molecule or part of a molecule to a second molecule or second part of a molecule. A linking group can comprise any number of atoms or functional groups. In some embodiments, a linking group is used only to link two biologically active molecules.

[0094] Unless otherwise specified, the symbols as used in the present disclosure mean that any group or groups can be attached thereto, which is consistent with the scope of the invention as described in the present disclosure.

[0095] According to common knowledge in the art, a peptide is a compound which is produced by the linkage of two or more amino acids by amide bonds. Here, the individual amino acids are linked in a certain order (sequence) into a chain. An amino acid is a compound which carries at least one amino group and at least one carboxyl group. Both natural (in vivo protein- generating amino acids), unnatural amino acids or prepared amino acids which can exist in organisms are included.

[0096] In the peptides of the present disclosure, the amino acid units can be present in D- or L-form, except where specifically noted.

[0097] As used herein, the term "standard amino acid" refers to the following twenty amino acids: alanine, arginine, asparagine, aspartic acid (aspartate), cysteine, glutamine, glutamic acid (glutamate), glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0098] As used herein, the term "non-standard amino acid" refers to an amino acid other than a "standard amino acid" as defined herein. "Non-standard amino acids" include, but are not limited to, N-formylmethionine, hydroxyproline, selenomethionine, isovaline, citrulline (Cit), ornithine, a-methyl-aspartate (aMeD), a-methyl-leucine (aMeL), N-methylalanine, N-methyl-glycine (NMe G), N-methyl-leucine (NMe L), O-cyclohexyl-alanine (Cha), N-ethylalanine, N,N-epsilon-dimethyllysine (K(Me)2), dimethylarginine (R(Me)2), n-alkylated L-a amino acids, and other amino acid analogs or mimetics that function in a similar manner to naturally occurring amino acids.

[0099] As used herein and as understood by one of skill in the art, a polyethylene glycol (PEG) unit refers to a repeating unit of the formula (CH2CH2O). It will be understood that in the chemical structures disclosed herein, a PEG unit can be depicted as (CH2CH2O), (OCH2CH2), or (CH2OCH2). It will further be understood that the number indicating the number of repeating PEG units can be placed on either side of the parentheses indicating the PEG unit. It will further be understood that a terminal PEG unit can be capped with an atom (e.g., a hydrogen atom) or some other moiety.

[0100] In the present disclosure, the term "cyclic peptide" indicates that two cysteines in a polypeptide chain are linked by a disulfide bond such that an intramolecular ring is formed to form a cyclic polypeptide chain. In the present disclosure, the disulfide bond between the two cysteines in the "cyclic peptide" is indicated by " ".

[0101] In the present disclosure, the term "pharmaceutical composition" or "composition" can refer to use in the treatment of a disease, as well as use in in vitro culture experiments with cells. When used in the treatment of a disease, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any one of the methods well known in the pharmaceutical art. All methods include the step of bringing the active ingredient into association with the carrier that constitutes one or more accessory ingredients. Typically, a composition is prepared by uniformly and intimately bringing the active siRNA into association with a liquid carrier, a finely divided solid carrier, or both.

[0102] In the present disclosure, the term "pharmaceutically acceptable" means that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients constituting a formulation and / or the mammal being treated with it. Preferably, "pharmaceutically acceptable" means approved or approvable by a regulatory agency of the Federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0103] In the present disclosure, the term "pharmaceutically acceptable carrier or adjuvant" can include any and all solvents, dispersion media, diluents, or other liquid vehicles, and the like, as is well known in the art of pharmaceutical formulation. Except insofar as any conventional carrier or diluent is incompatible with the siRNA of the present disclosure, its use is contemplated to be within the scope of the present disclosure. The use of such carriers or diluents is well known in the art of pharmaceutical formulation.

[0104] In the present disclosure, the terms "treatment," "treat," or "treating" can be used interchangeably herein. These terms refer to methods of obtaining beneficial or desired results, including but not limited to therapeutic benefit. By "therapeutic benefit" is meant eradication or amelioration of the underlying disorder being treated. Herein, therapeutic benefit is achieved by eradicating or ameliorating one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject can still be afflicted with the disorder.

[0105] In the present disclosure, the terms "prevention," "prevent," or "preventing" can be used interchangeably and refer to methods of obtaining beneficial or desired results, including but not limited to prophylactic benefit. To achieve a "prophylactic benefit," a conjugate, RNAi agent, or composition can be administered to a subject at risk of developing a particular disease, or to a subject reporting one or more physiological symptoms of a disease, even though the subject can not yet be diagnosed with the disease.

[0106] In the present disclosure, the term "administration" generally refers to the introduction or delivery of a pharmaceutical preparation of the present disclosure into the body of a subject by any route of introduction or delivery. Any method known to those skilled in the art for contacting a cell, organ or tissue with the drug can be employed. The administration can include, without limitation, intravenous, intra-arterial, intranasal, intra-abdominal, intramuscular, subcutaneous or oral. The daily dose can be divided into one, two or more doses of suitable form for administration at one, two or more times during a certain period of time.

[0107] As used in the present disclosure, the term "modulating gene expression" means that the expression of a gene, or the level of an RNA molecule or equivalent RNA molecule encoding one or more proteins or protein subunits, is up-regulated or down-regulated such that the expression, level or activity is greater than or less than that observed in the absence of the modulating agent. For example, the term "modulating" can mean "inhibiting", but the use of the term "modulating" is not limited to this definition.

[0108] In addition to any conventional excipients, the use of ranges which are incompatible with the siRNA of the present disclosure, for example any adverse biological effects produced or interactions with any other components of the pharmaceutically acceptable composition in a deleterious manner, are also within the scope of the present disclosure.

[0109] For the purposes of the present application, technical solutions and advantages, the following will be further described in detail in combination with embodiments.

[0110] Oligonucleotide conjugate:

[0111] In a first aspect of the present disclosure, the present disclosure provides an oligonucleotide conjugate for inhibiting gene expression in kidney cells, the oligonucleotide conjugate comprising:

[0112] (a) a single-stranded or double-stranded oligonucleotide molecule;

[0113] (b) a targeting ligand, the targeting ligand having affinity for a receptor present on the surface of a kidney cell;

[0114] (c) a linker group, the oligonucleotide molecule being covalently linked to the targeting ligand via the linker group.

[0115] In some embodiments of the present disclosure, the single-stranded or double-stranded oligonucleotide molecule has the following features:

[0116] (i) comprises an antisense strand of 17-35 nucleotides, wherein at least 15 nucleotides are complementary or substantially complementary to a mRNA sequence of a gene in a kidney cell;

[0117] (ii) a sense strand of 15-35 nucleotides in length, which is complementary or partially complementary to the antisense strand.

[0118] In some embodiments of the present disclosure, the targeting ligand is a polypeptide.

[0119] In some embodiments of the present disclosure, the polypeptide has at least 4 amino acid residues.

[0120] In some embodiments of the present disclosure, the receptor is a known or unknown endocytosis type receptor.

[0121] In some embodiments of the present disclosure, the receptor is selected from the group consisting of a megalin receptor or a lina peptide receptor.

[0122] In some embodiments of the present disclosure, the Linker group is connected to the N- or C-terminus of an amino acid on the polypeptide.

[0123] In some embodiments of the present disclosure, the Linker group is selected from the group consisting of a substituted or unsubstituted aliphatic chain, a 3-6 membered heterocyclic ring, a C6-C 10 aromatic ring, a (PEG) n group containing disulfide bond, amide group or triazole group, or a combination thereof, wherein n is an integer selected from 1-20, preferably 1-10.

[0124] In some embodiments of the present disclosure, the Linker group comprises any of the following substituents or any combination thereof:

[0125] disulfide bond, amide group, triazole group, -NH-, -C(O)-,

[0126] wherein a, b, c or d is an integer selected from 0-10, preferably 1-8.

[0127] In some embodiments of the present disclosure, the Linker group is selected from any of the following substituents:

[0128] * represents the connection point to the oligonucleotide molecule or to the targeting ligand, or the point of connection between the substituents.

[0129] In some embodiments of the present disclosure, the targeting ligand comprises any of the polypeptide fragments shown in A1) to A6):

[0130] A1) -X1(KKEEE) n -K m -, wherein n = 1-5, m = 0 or 1 or 2, X1is any L-alpha amino acid or L-beta amino acid except K, E;

[0131] A2) -CLPVASC-, preferably in the form of a cyclic peptide

[0132] A3) -CYFQNC-; preferably in cyclic peptide form

[0133] A4) -KIDRI-;

[0134] A5) -IDRI- (Ile-Asp-Arg-Ile-);

[0135] A6) -dXa-Ser-dXb-X2-dXc-Gly-Xd-Ile-Asp-Arg(Ak)-Ile-; wherein X2 is selected from any unnatural amino acid; dXa, dXb, dXc are selected from any D-amino acid; Arg(Ak) is selected from arginine or alkylated modified arginine; Xd is selected from Hyp or Pro.

[0136] In some embodiments of the present disclosure, the targeting ligand is selected from a polypeptide comprising any of the amino acid sequence segments shown in B1) - B6):

[0137] B1) -KKEEE-KKEEE-KKEEE-K-;

[0138] B2) -CKKEEE-KKEEE-KKEEE-K;

[0139] B3) -CLPVASC-, preferably in cyclic peptide form

[0140] B4) -CYFQNCPRG-, preferably in cyclic peptide form

[0141] B5) -KIDRI-;

[0142] B6) -DPhe-Ser-DHyp-Cha-DAla-Gly-Hyp-Ile-Asp-Arg(Me)-Ile-.

[0143] Ligand unit

[0144] The oligonucleotide conjugate for inhibiting gene expression in kidney cells as described before, wherein the targeting ligand linked by a linker is a ligand unit, in the present disclosure, the ligand unit is selected from any of the structures shown in C1) - C6):

[0145] C1) La-KKEEE-KKEEE-KKEEE-K-Laa;

[0146] C2) La-CKKEEE-KKEEE-KKEEE-K-Laa;

[0147] C3) La-CLPVASC-Laa; preferably a cyclic peptide

[0148] C4) La-CYFQNCPRG-Laa; preferably a cyclic peptide

[0149] C5) Lb-KIDRI-Laa;

[0150] C6) La-DPhe-Ser-DHyp-Cha-DAla-Gly-Hyp-Ile-Asp-Arg(Me)-Ile-Laa.

[0151] In formulas C1) - C4 and C6), either La and Laa is selected from a linker, the other is a terminal blocking group, the linker is a linking group and comprises at least one of a triazole group or a PEG unit or an acyl group.

[0152] In some embodiments of the disclosure, La is a linker, the Linker structure comprises -PEG2-CH2CH2CO- or triazole-PEG2-.

[0153] In formula C5), Lb is selected from a linking group comprising a 6-10 membered aromatic or heteroaromatic ring; Laa is independently selected from an amino group, or an alkyl substituted amine group such as -NHCH3.

[0154] In some embodiments of the disclosure, the Ligand Unit is selected from any one of the structures shown in formulas D1) - D9), or a pharmaceutically acceptable salt thereof:

[0155] D1) -La-KKEEE-KKEEE-KKEEE-K-Lc;

[0156] D2) -La-CKKEEE-KKEEE-KKEEE-K-Lc;

[0157] D3) -La-CLPVASC-Lc; preferably in cyclic peptide form

[0158] D4) -La-CYFQNCPRG-Lc; preferably in cyclic peptide form

[0159] D5)

[0160] D6)

[0161] D7)

[0162] D8)

[0163] D9)-La-DPhe-Ser-DHyp-Cha-DAla-Gly-Hyp-Ile-Asp-Arg(Me)-Ile-Lc.

[0164] wherein La is a linker independently selected from any bond, -NH-, an amido group or a linking group comprising at least one PEG unit.

[0165] In some embodiments of the disclosure, La is a linking group comprising at least 2 PEG units.

[0166] In some embodiments of the disclosure, La is selected from an amido group or -triazolyl-(PEG)n-, for example n is 1-3, m is 0 or 1, and Z is -CO- (carbonyl) or -NH-.

[0167] wherein Lc is a terminal blocking group; optionally, it is a C-terminal blocking group, said Lc is selected from an amino group, or an alkyl-substituted amine group, preferably -NHCH3.

[0168] In some embodiments of the disclosure, at least one ligand unit is conjugated to the oligonucleotide molecule to form an oligonucleotide conjugate.

[0169] In some embodiments of the disclosure, 1-4 ligand units are conjugated to the oligonucleotide molecule to form an oligonucleotide conjugate, including specifically 1, 2, 3 or 4 ligand units conjugated to the oligonucleotide molecule.

[0170] In some embodiments of the disclosure, the oligonucleotide molecule inhibits the expression of mRNA of a gene in a human kidney cell.

[0171] Polypeptides targeting kidney cells

[0172] In a second aspect of the disclosure, the disclosure provides a polypeptide capable of delivering an oligonucleotide to a kidney cell, said polypeptide comprising any one of the amino acid sequence segments shown in B1) - B6):

[0173] B1) -KKEEE-KKEEE-KKEEE-K-;

[0174] B2) -CKKEEE-KKEEE-KKEEE-K-;

[0175] B3)

[0176] B4)

[0177] B5)-KIDRI-;

[0178] B6)-DPhe-Ser-DHyp-Cha-DAla-Gly-Hyp-Ile-Asp-Arg(Me)-Ile-.

[0179] Ligand unit molecules targeting kidney cells

[0180] In a third aspect, this disclosure provides a ligand unit molecule capable of delivering oligonucleotides to kidney cells, said ligand unit molecule being selected from any of the compounds shown in E1)-E9):

[0181] E1)N3-PEG2-CH2CH2CO-KKEEE-KKEEE-KKEEE-K-NH2;

[0182] E2)Ac-CKKEEE-KKEEE-KKEEE-K-NH2;

[0183] E3)N3-(PEG)2-CH2CH2-CO-CLPVASC-NH2;

[0184] Its structural formula is

[0185] E4)N3-(PEG)2-CH2CH2-CO-CYFQNCPRG-NH2;

[0186] Its structural formula is or

[0187] E5)

[0188] E6)

[0189] E7)

[0190] E8)

[0191] E9) N3-(PEG)2-CH2CH2CO-DPhe-Ser-DHyp-Cha-DAla-Gly-Hyp-Ile-Asp-Arg(Me)-Ile-CONHCH3.

[0192] Uses of peptide and ligand unit molecules

[0193] In a fourth aspect of this disclosure, this disclosure provides the use of the polypeptide described in the second aspect of this disclosure or the ligand unit molecule described in the third aspect of this disclosure for delivering oligonucleotide molecules to kidney cells.

[0194] Composition

[0195] In a fifth aspect of the present disclosure, the present disclosure provides a composition comprising the oligonucleotide conjugate of the first aspect of the present disclosure, or the polypeptide of the second aspect of the present disclosure, or the ligand unit molecule of the third aspect of the present disclosure, or a pharmaceutically acceptable salt thereof.

[0196] Pharmaceutical composition

[0197] In a sixth aspect of the present disclosure, the present disclosure provides a pharmaceutical composition comprising the oligonucleotide conjugate of the first aspect of the present disclosure, or a pharmaceutically acceptable salt thereof.

[0198] In some alternative embodiments of the present disclosure, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers or excipients.

[0199] The pharmaceutical composition of the present disclosure includes formulations suitable for parenteral administration, which can conveniently be presented in unit dosage form and can be prepared by any methods well known in the art of pharmacy. The amount of active ingredient, i.e., siRNA, that is combined with the carrier material to produce a single dose will generally be that amount of the siRNA which produces a therapeutic effect. Generally, this amount will range from about 1% to about 99% of the active ingredient in unit dosage form.

[0200] Method for preparing the oligonucleotide conjugate

[0201] In a seventh aspect of the present disclosure, the present disclosure provides a method for preparing the oligonucleotide conjugate of the first aspect of the present disclosure, the method comprising the steps of:

[0202] (i) synthesizing the sense strand;

[0203] (ii) synthesizing the antisense strand;

[0204] (iii) annealing the sense strand and the antisense strand;

[0205] (iv) conjugating the ligand unit molecule to the sense strand or the antisense strand before or after annealing the sense strand and the antisense strand.

[0206] Method for treating a disease

[0207] In an eighth aspect of the present disclosure, the present disclosure provides a method for treating a kidney-related disease or disorder, the method comprising administering to a subject the oligonucleotide conjugate of the first aspect of the present disclosure, the composition of the fifth aspect of the present disclosure, or the pharmaceutical composition of the sixth aspect of the present disclosure.

[0208] In some alternative embodiments of the present disclosure, the kidney-related disease or disorder is chronic kidney disease.

[0209] In some alternative embodiments of the present disclosure, the kidney-related disease or disorder is selected from the group consisting of hypertension, hypertension-related kidney damage, hyperglycemia, diabetes, diabetic nephropathy, hyperuricemia, gout, hyperuricemia-related kidney damage, hepatitis B virus-related kidney damage, myeloma kidney disease, chronic renal failure, glomerulonephritis, renal vascular disease, C3 glomerulopathy, lupus nephritis, IgA nephropathy, polycystic kidney disease, membranous nephropathy, atypical hemolytic uremic syndrome, uremic syndrome, or systemic lupus erythematosus (SLE)-related kidney disease.

[0210] Methods of oligonucleotide molecule delivery

[0211] In a ninth aspect of the present disclosure, the present disclosure provides a method of delivering one or more oligonucleotide molecules to a cell in vivo, the method comprising administering to a subject the oligonucleotide conjugate comprising a targeting ligand of the first aspect of the present disclosure, the composition of the fifth aspect of the present disclosure, or the pharmaceutical composition of the sixth aspect of the present disclosure.

[0212] In some alternative embodiments of the present disclosure, the targeting ligand has affinity for a cell surface receptor.

[0213] In some alternative embodiments of the present disclosure, the receptor is selected from the group consisting of a Megalin receptor.

[0214] In some alternative embodiments of the present disclosure, the cell is selected from the group consisting of a kidney cell or a kidney epithelial cell.

[0215] In some alternative embodiments of the present disclosure, the subject is a human.

[0216] Pharmaceutical uses

[0217] In a tenth aspect of the present disclosure, the present disclosure provides use of the oligonucleotide conjugate of the first aspect of the present disclosure, the composition of the fifth aspect of the present disclosure, or the pharmaceutical composition of the sixth aspect of the present disclosure in the manufacture of a medicament for treating a kidney-related disease or disorder.

[0218] In some alternative embodiments of the present disclosure, the kidney-related disease or disorder is chronic kidney disease.

[0219] In some alternative embodiments of the present disclosure, the kidney-related disease or disorder is selected from the group consisting of hypertension, hypertension-related kidney damage, hyperglycemia, diabetes, diabetic nephropathy, hyperuricemia, gout, hyperuricemia-related kidney damage, hepatitis B virus-related kidney damage, myeloma kidney disease, chronic renal failure, glomerulonephritis, renal vascular disease, C3 glomerulopathy, lupus nephritis, IgA nephropathy, polycystic kidney disease, membranous nephropathy, atypical hemolytic uremic syndrome, uremic syndrome, or systemic lupus erythematosus (SLE)-related kidney disease.

[0220] In order to make the purpose, technical solutions and advantages of the present disclosure clearer, the present disclosure will be further described in detail below with embodiments.

[0221] Synthesis of ligand unit molecule:

[0222] Preparation Example 1: Synthesis of LD100

[0223] Fmoc solid-phase polypeptide synthesis method is used to synthesize from right to left, i.e. from C-terminal to N-terminal.

[0224] S1: Take Fmoc-Linker-MBHA Resin (degree of substitution about 0.5 mmol / g, total 1.0 mmol reaction sites) 2.0 g, and swell in DMF for 20 minutes;

[0225] S2: Add 3 times the resin volume of 20% Pip / DMF solution, and blow nitrogen for 30 minutes, and then dry to obtain H2N-Linker-MBHA Resin;

[0226] S3: Wash the resin in the previous step with 2 times the resin volume of DMF for 5 times;

[0227] S4: Take 3.0 mmol of Fmoc-Lys(Boc)-OH amino acid, 6.0 mmol of DIPEA, 2.85 mmol of HBTU, and an appropriate amount of solvent DMF, and react for 30 minutes to obtain Fmoc-Lys(Boc)-Liner-MBHA Resin;

[0228] S5: Wash the resin in the previous step with 2 times the resin volume of DMF for 3 times;

[0229] S6: Add 3 times the resin volume of 20% Pip / DMF solution, and blow nitrogen for 30 minutes, and then dry to obtain H2N-Lys(Boc)-Liner-MBHA Resin;

[0230] S7: Wash the resin in the previous step with 2 times the resin volume of DMF for 5 times;

[0231] S8: Repeat steps 4, 5, 6, and 7 to obtain Lys(Boc)-Lys(Boc)-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Lys(Boc)-Lys(Boc)-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Lys(Boc)-Lys(Boc)-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Lys(Boc)-Liner-MBHA Resin.

[0232] S9: Take 3.0 mmol of N3-PEG2-CH2CH2COOH raw material, 6.0 mmol of DIPEA, 2.85 mmol of HBTU, and an appropriate amount of solvent DMF, and react for 30 minutes. N3-PEG2-CH2CH2COOH-Lys(Boc)-Lys(Boc)-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Lys(Boc)-Lys(Boc)-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Lys(Boc)-Lys(Boc)-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Lys(Boc)-Lys(Boc)-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Lys(Boc)-Liner-MBHA Resin is obtained.

[0233] S10: Wash the resin with methanol for 3 times, and dry the resin (in preparation for cutting);

[0234] S11: Cutting: 6 times the resin volume of cutting solution (trifluoroacetic acid: anisole: 1,2-ethanedithiol: phenol: water = 87.5%: 5%: 2.5%: 2.5%: 2.5%), shake bed for 2 hours, filter out the resin, precipitate the filtrate with anhydrous ether, and wash the precipitate with anhydrous ether for 3 times, finally put the precipitate in a vacuum drying oven, dry at room temperature for 24 hours, and obtain the crude product N3-PEG2-CH2CH2CO-Lys-Lys-Glu-Glu-Glu-Lys-Lys-Glu-Glu-Glu-Lys-Lys-Glu-Glu-Glu-Lys-NH2 (i.e. LD100 molecule in the table below).

[0235] S12: Use C18 preparation column to purify by HPLC to obtain LD100 with a purity of more than 95%.

[0236] In addition, the preparation of LD101, LD105 and LD106 refers to the synthesis and purification method of the steps in Example 1.

[0237] The ligand unit molecules prepared by the present disclosure are as follows:

[0238] LD100 N3-PEG2-CH2CH2CO-KKEEE-KKEEE-KKEEE-K-NH2

[0239] LD101 N3-PEG2-CH2CH2CO-SHSNTQTLAKAPEHTGC-NH2

[0240] LD103 Ac-CKKEEE-KKEEE-KKEEE-K-NH2

[0241] LD104 Ac-SHSNTQTLA-K(N3-PEG2-CH2CH2CO)-APEHTGC-NH2

[0242] LD105 N3-PEG2-CH2CH2CO-CLPVASC-NH2 (cyclopeptide)

[0243] LD106 N3-PEG2-CH2CH2CO-DPhe-Ser-DHyp-Cha-DAla-Gly-Hyp-lle-Asp-Arg(Me)-lleCONHCH3

[0244] LD109 N3-PEG2-CH2CH2CO-CYFQNCPRG-NH2

[0245] Synthesis of Linker:

[0246] Preparation Example 2: Preparation of compound LK005

[0247] In this preparation example, the synthesis route of compound LK005 is as shown below:

[0248] (2-1) Synthesis of compound LK005-3

[0249] Compound LK005-1 (10 g, 69.9 mmol, 2-[2-(propargyloxy)ethoxy]ethanamine, CAS No. 944561-44-8) was dissolved in DMF (100 mL), and compound LK005-2 (14.19 g, 69.9 mmol, BOC-4-aminobutyric acid, CAS No. 57294-38-9), benzotriazol- N,N,N',N'-tetramethyluronium hexafluorophosphate (66.4 g, 174.75 mmol, HATU for short, CAS No. 148893-10-1), N,N-diisopropylethylamine (27.1 g, 209.7 mmol, DIEA for short, CAS No. 7087-68-5) were added in turn, and stirred at 25 °C for 6 hours. After the reaction was completed, the reaction solution was diluted with ethyl acetate (400 mL), washed with saturated aqueous sodium chloride solution (5 x 150 mL), and the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (eluent: petroleum ether / ethyl acetate = 4 / 1, v / v) to obtain compound LK005-3 (20.5 g, yield 87.3%) as a light yellow oil. MS ESI (m / z) = 329.27 [M+H] + .

[0250] (2-2) Synthesis of compound LK005-4

[0251] Compound LK005-3 (20 g, 60.9 mmol) was dissolved in 4M hydrogen chloride solution in dioxane (200 mL) and stirred at 25 °C for 2 hours. After the reaction was completed, the reaction solution was concentrated to obtain compound LK005-4 crude product (15.4 g), which was directly used in the next step reaction without purification. MS ESI (m / z) = 229.23 [M+H] + .

[0252] (2-3) Synthesis of compound LK005-5

[0253] Compound LK005-4 (10 g, 37.9 mmol) was dissolved in DMF (100 mL), and pentanedioic acid monomethyl ester (6.09 g, 41.69 mmol, CAS No. 1501-27-5), HATU (36.4 g, 94.75 mmol), DIEA (24.49 g, 189.5 mmol) were added successively, and stirred at 25 °C for 6 hours. After the reaction was completed, the reaction solution was diluted with ethyl acetate (300 mL), washed with saturated aqueous sodium chloride solution (5 x 100 mL), and the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (eluent: petroleum ether / ethyl acetate = 3 / 1, v / v) to obtain compound LK005-5 (10.5 g, yield 77.9%) as a light yellow oil. MS ESI (m / z) = 357.27 [M+H] + .

[0254] (2-4) Synthesis of compound LK005-6

[0255] Compound LK005-5 (10 g, 28.1 mmol) was dissolved in tetrahydrofuran (30 mL) at room temperature, and 1M sodium hydroxide solution in tetrahydrofuran (28.1 mL, 28.1 mmol) was added, and stirred at 25 °C for 1 hour. After the reaction was completed, the reaction solution was adjusted to pH 2-3 with 1N hydrochloric acid, and concentrated to obtain compound LK005-6 crude product, which was directly used in the next step reaction without purification. MS ESI (m / z) = 343.17 [M+H] + .

[0256] (2-5) Synthesis of compound LK005

[0257] Compound LK005-6 (5 g, 14.58 mmol) was dissolved in DMF (50 mL), p-nitrophenol (4.05 g, 29.16 mmol, CAS number 100-02-7), N, N'-dicyclohexylcarbodiimide (6.02 g, 29.16 mmol, DCC, CAS number 538-75-0) were added successively, and stirred at 25 °C for 12 hours. After the reaction was completed, the reaction solution was diluted with ethyl acetate (100 mL), washed with saturated aqueous sodium chloride solution (5 x 50 mL), and the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (eluent: petroleum ether / ethyl acetate = 4 / 1, v / v) to obtain compound LK005 (3.1 g, yield 44.3%) as a yellow solid. MS ESI (m / z) = 464.12 [M+H] + .

[0258] Preparation Example 3: Preparation of compound NM041

[0259] In this preparation example, the synthetic route of compound NM041 is as follows:

[0260] (3-1) Synthesis of compound NM041-2

[0261] Compound NM041-1 (25.0 g, 1.0 eq, 1,3,4,6-tetraacetyloxy-alpha-D-glucopyranose, CAS number 4292-12-0) was dissolved in 250 ml of toluene, and the temperature was raised to 110 °C, and tributyltin hydride (17.7 g, 1.0 eq, CAS number 688-73-3) and azobisisobutyronitrile (1 g, 0.1 eq, CAS number 78-67-1) were added, and the reaction was refluxed at 110 °C for 2 hours. After the reaction was completed, the reaction solution was cooled to 25 °C, and ethyl acetate (100 ml) and potassium fluoride (10.59 g in 30 ml water, 3.0 eq) were added, and stirred at 25 °C for 2 hours, and the reaction solution was filtered and the organic phase was separated, and the aqueous phase was washed with 50 ml of ethyl acetate twice (50 ml x 2), and the organic phases were combined, and the organic phase was washed with 50 ml of saturated aqueous sodium chloride solution once (50 ml x 1), dried over anhydrous sodium sulfate, and filtered under suction, and concentrated to obtain compound NM041-2 (18.7 g, yield 87.9%) as a light yellow oil. MS ESI (m / z) = 333.1 [M+H] + .

[0262] (3-2) Synthesis of compound NM041-3

[0263] Compound NM041-2 (18.7 g, 1 eq) was dissolved in anhydrous methanol (50 ml), sodium methoxide (0.288 g, 0.1 eq) was added, and the reaction was stirred at 25 °C for 1 hour. After the reaction was completed, the pH of the reaction solution was adjusted to 6-7 with a 4 mol / L hydrogen chloride solution in 1,4 dioxane under ice bath, concentrated, and then concentrated with acetonitrile twice, followed by vacuum drying to obtain compound NM041-3 (9.4 g, yield 100%) as a white solid.

[0264] (3-3) Synthesis of compound NM041-4

[0265] Compound NM041-3 (9.4 g, 1.0 eq) was dissolved in 150 ml of acetonitrile, benzaldehyde dimethyl acetal (30 ml, 3 eq, CAS No. 1125-88-8) and DL-10-camphorsulfonic acid (1.5 g, 0.1 eq, CAS No. 5872-08-2) were added, respectively, and stirred at 25 °C for 5 hours, 3 ml of triethylamine was added, and stirred at 25 °C for 30 min. After the reaction was completed, the reaction solution was concentrated, 100 ml of water was added to the concentrated reaction solution, and then extracted with 100 ml of ethyl acetate twice (100 ml x 2), the organic phase was combined, dried with anhydrous sodium sulfate, and filtered, and the organic phase was concentrated and purified by column chromatography (eluent: ethyl acetate / petroleum ether = 57 / 43, v / v) to obtain compound NM041-4 (8.6 g, yield 59.5%) as a white solid. MS ESI (m / z) = 252.2 [M+H] + .

[0266] (3-4) Synthesis of compound NM041-5

[0267] Compound NM041-4 (4.3 g, 1.0 eq) was dissolved in 40 ml of DMF, sodium hydride (2.7 g, 4 eq) was added under ice bath and reacted for 30 minutes under ice bath, 3-bromopropynyl (8.1 g, 4 eq, CAS No. 106-96-7) was added, and stirred at 25 °C for 2 hours, and then quenched with 20 ml of water. After the reaction was completed, the reaction solution was extracted with 50 ml of ethyl acetate three times (50 ml x 3), the organic phase was combined, dried with 20 ml of saturated sodium chloride solution five times (20 ml x 5), and then dried with anhydrous sodium sulfate and filtered, and concentrated to obtain compound NM041-5 (yield 100%) as a brown oil. MS ESI (m / z) = 329.2 [M+H] + .

[0268] (3-5) Synthesis of compound NM041-6

[0269] Compound NM041-5 (5.6 g, 1.0 eq) was dissolved in 30 ml of dichloromethane (DCM), 300 ml of 70 mass% aqueous acetic acid solution was added, and the reaction was carried out at 70°C for 1 hour. After the reaction was completed, the reaction solution was directly concentrated to obtain compound NM041-6 (yield 100%) as a yellow oil. MS ESI (m / z) = 241 [M+H]+.

[0270] (3-6) Synthesis of compound NM041-7

[0271] Compound NM041-6 (5.6 g, 1.0 eq) was dissolved in 50 ml of pyridine, 4,4'- dimethoxytrityl chloride (10.2 g, 30.16 mmol, 1.3 eq, abbreviated as DMTrCl, CAS No. 40615-36-9) was added under ice bath, nitrogen was replaced for 3 times, and the reaction was stirred at 25°C for 3 hours, and 50 ml of methanol was added for quenching. After the reaction was completed, the reaction solution was concentrated, 50 ml of water was added, 50 ml of ethyl acetate was extracted 3 times (50 ml x 3), the organic phase was combined, dried with anhydrous sodium sulfate, and filtered under suction, concentrated, and purified by column chromatography (normal phase) (eluent: ethyl acetate / ethyl ether = 16 / 84, v / v) to obtain compound NM041-7 (5 g, yield 39.6%) as a light yellow solid. MS ESI (m / z) = 543.1 [M+H] + .

[0272] (3-7) Synthesis of compound NM041

[0273] Compound NM041-7 (2.0 g, 1.5 eq) was dissolved in 20 ml of anhydrous dichloromethane, 4,5-dicyanoimidazole (347.4 mg, 0.8 eq, CAS No. 1122-28-7) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.22 g, 1.1 eq, CAS No. 102691-36-1) were added, respectively, nitrogen was replaced for 3 times, and the reaction was stirred at 25°C for 2 hours. After the reaction was completed, 20 ml of saturated sodium bicarbonate solution was added to the reaction solution, extracted with 20 ml of dichloromethane 3 times (20 ml x 3), the organic phase was combined, dried with anhydrous sodium sulfate, and filtered, concentrated, and purified by column chromatography (reverse phase) (C18 column, eluent: acetonitrile / water = 72 / 28, v / v) to obtain compound NM041 (2 g, yield 73.09%) as a white powder. MS ESI (m / z): 743.2 [M+H] + .

[0274] 1H NMR (400 MHz, Acetonitrile-d3) δ 7.53 - 7.47 (m, 2H), 7.36 (tt, J = 9.6, 3.4 Hz, 6H), 7.28 - 7.22 (m, 1H), 6.94 - 6.86 (m, 4H), 4.45 (d, J = 2.4 Hz, 1H), 4.35 (dt, J = 6.1, 1.9 Hz, 2H), 4.18 (ddd, J = 11.2, 5.4, 2.6 Hz, 1H), 3.81 (s, 7H), 3.79 - 3.20 (m, 8H), 3.07 (dt, J = 10.5, 7.0 Hz, 1H), 2.79 (q, J = 2.6 Hz, 1H), 2.72 (dt, J = 5.1, 2.4 Hz, 1H), 2.69 - 2.63 (m, 1H), 2.48 - 2.34 (m, 1H), 2.17 (d, J = 1.0 Hz, 2H), 1.08 (dd, J = 6.8, 4.1 Hz, 10H), 0.90 (d, J = 6.8 Hz, 2H).

[0275] Preparation Example 4: Synthesis of compound NM064

[0276] In this preparation example, the synthesis route of compound NM064 is as follows:

[0277] (4-1) Synthesis of compound NM064-2

[0278] Compound NM064-1 (13 g, 1.0 eq, methyl B-D-glucopyranoside, CAS No. 709-50-2) was dissolved in 150 ml of acetonitrile, and benzaldehyde dimethyl acetal (30 ml, 3.0 eq) and DL-10-camphorsulfonic acid (1.5 g, 0.1 eq) were added, respectively, and stirred at 25°C for 5 hours, 3 ml of triethylamine was added, and stirred at 25°C for 30 min. After the reaction was completed, the reaction solution was concentrated, 100 ml of water was added, extracted with 100 ml of ethyl acetate twice (100 ml x 2), the organic phase was combined, dried with anhydrous sodium sulfate and filtered, concentrated, and purified by column normal phase purification (eluent: ethyl acetate / petroleum ether = 57 / 43, v / v) to obtain compound NM064-2 (11.5 g, yield 60.8%) as a white solid. MS ESI (m / z) = 283 [M+H] + .

[0279] (4-2) Synthesis of compound NM064-3

[0280] Compound NM064-2 (5 g, 1.0 eq) was dissolved in 40 ml of N,N- dimethylformamide, sodium hydride (2.7 g, 4 eq) was added under ice bath, and the reaction was performed for 30 minutes under ice bath, 3-bromopropynyl (8.1 g, 4 eq) was added, and the reaction was performed for 2 hours at 25°C, and 20 ml of water was added for quenching. After the reaction was completed, the reaction solution was extracted with 50 ml of ethyl acetate three times (50 ml x 3), the combined organic phase was washed with 20 ml of saturated sodium chloride solution five times (20 ml x 5), the organic phase was dried over anhydrous sodium sulfate and filtered, and concentrated to obtain compound NM064-3 (6.3 g, yield 100%) as a brown oil. MS ESI (m / z) = 359 [M+H] + .

[0281] (4-3) Synthesis of compound NM064-4

[0282] Compound NM064-3 (6.3 g, 17 mmol, 1.0 eq) was dissolved in 30 ml of dichloromethane, 300 ml of 70 mass% aqueous acetic acid was added, and the reaction was performed for 1 hour at 70°C. After the reaction was completed, the reaction solution was directly concentrated to obtain compound NM064-4 (4.78 g, yield 100%) as a yellow oil. MS ESI (m / z) = 271 [M+H] + .

[0283] (4-4) Synthesis of compound NM064-5

[0284] Compound NM064-4 (4.78 g, 17.7 mmol, 1.0 eq) was dissolved in 50 ml of pyridine, DMTrCl (7.8 g, 23.0 mmol, 1.3 eq) was added under ice bath, nitrogen was replaced three times, and the reaction was performed for 3 hours at 25°C, and 50 ml of methanol was added for quenching. After the reaction was completed, the reaction solution was concentrated, 50 ml of water was added, and the reaction was extracted with 50 ml of ethyl acetate three times (50 ml x 3), the combined organic phase was dried over anhydrous sodium sulfate and filtered, and concentrated, and purified by column chromatography (eluent: ethyl acetate / petroleum ether = 16 / 84, v / v) to obtain compound NM064-5 (6.7 g, yield 66.3%) as a yellow solid. MS ESI (m / z) = 573 [M+H] + .

[0285] (4-5) Synthesis of compound NM064

[0286] Compound NM064-5 (2.0 g, 1.0 eq) was dissolved in 20 ml of anhydrous dichloromethane, and DCI (330.4 mg, 0.8 eq, 4,5-dicyanoimidazole) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.16 g, 1.1 eq) were added, respectively, and nitrogen was replaced for 3 times, and stirred at 25°C for 2 hours. After the reaction was completed, 20 ml of saturated aqueous sodium bicarbonate solution was added to the reaction solution, and extracted with 20 ml of dichloromethane 3 times (20 ml x 3), and the organic phase was combined, dried with anhydrous sodium sulfate, and filtered, concentrated, and purified by column chromatography (eluent: acetonitrile / water = 72 / 28, v / v), and vacuum dried for 12 hours to obtain compound NM064 (2 g, yield 74.07%) as a white powder. MS ESI (m / z) = 774 [M+H] + .

[0287] 1 H NMR (400 MHz, DMSO-d6) δ 7.45 - 7.39 (d, J = 7.8 Hz, 2H), 7.36 - 7.18 (tt, J = 14.5, 8.5 Hz, 7H), 6.95 - 6.84 (d, J = 7.5 Hz, 4H), 5.01 - 4.96 (s, 1H), 4.43 - 4.28 (s, 4H), 3.81 - 3.70 (s, 8H), 3.65 - 3.35 (m, 12H), 3.28 - 3.18 (dt, J = 14.5, 7.2 Hz, 1H), 3.06 - 2.97 (t, J = 9.5 Hz, 1H), 2.75 - 2.68 (m, 1H), 1.07 - 0.95 (q, J = 7.4, 6.8 Hz, 10H), 0.84 - 0.78 (d, J = 6.6 Hz, 2H).

[0288] Synthesis of siRNA

[0289] Unless otherwise specified, the reagents, reagent consumables and instrument equipment used in the present disclosure are all from commercial sources. Among them, the main reagent consumables are shown in Table 1, and the main instrument equipment is shown in Table 2.

[0290] Table 1 Main reagent consumables

[0291] Table 2 Main instrument equipment

[0292] Preparation Example 5: Synthesis of siRNA

[0293] (5-1) Synthesis of sense strand (SS)

[0294] The nucleotide sequence was connected with nucleoside monomers one by one in the order of 3'-5' by the method of phosphoramidite nucleic acid solid-phase synthesis. Each connection of nucleoside monomers included four steps of deprotection, coupling, capping, oxidation or sulfurization. The synthesis conditions are given as follows:

[0295] The nucleoside monomers were prepared into acetonitrile solutions of nucleoside monomers with a concentration of 0.1 M.

[0296] The conditions of deprotection reaction of each step were the same. The conditions of deprotection reaction were as follows: temperature was 25°C, reaction time was 70 seconds, deprotection reagent was dichloroacetic acid in dichloromethane solution (3% by volume), and the molar ratio of dichloroacetic acid to 4,4'-dimethoxytrityl protecting group on the solid support was 5:1.

[0297] The conditions of coupling reaction of each step were the same. The conditions of coupling reaction were as follows: temperature was 25°C, the molar ratio of the nucleic acid sequence connected on the solid support to the nucleoside monomer was 1:10, the molar ratio of the nucleic acid sequence connected on the solid support to the coupling reagent was 1:65, reaction time was 600 seconds, the coupling reagent was 5-ethylthio-1H-tetrazole in acetonitrile solution with a concentration of 0.5 M, and the sulfur reagent was a mixture of acetonitrile / pyridine with a volume ratio of 1:1, in which the concentration of hydrogenated xanthine was 0.2 mol / L.

[0298] The conditions of capping reaction of each step were the same. The conditions of capping reaction were as follows: temperature was 25°C; reaction time was 2 minutes; the capping reagent solution was a mixture of Cap1 and Cap2 with a molar ratio of 1:1, Cap1 was N-methylimidazole in pyridine / acetonitrile mixed solution with a concentration of 20% by volume, the volume ratio of pyridine to acetonitrile was 3:5, and Cap2 was acetic anhydride in acetonitrile solution with a concentration of 20% by volume; the molar ratio of N-methylimidazole in Cap1 capping reagent, acetic anhydride in Cap2 capping reagent, and the nucleic acid sequence connected on the solid support was 1:1:1.

[0299] The conditions of oxidation reaction of each step were the same. The conditions of oxidation reaction were as follows: temperature was 25°C; reaction time was 3 seconds; the concentration of oxidation reagent was 0.05 M of iodine water, and the molar ratio of iodine to the nucleic acid sequence connected on the solid support in the coupling reaction was 30:1; the oxidation reaction was carried out in a water / pyridine mixed solvent (the volume ratio of water to pyridine was 1:9). The conditions of sulfurization reaction were as follows: temperature was 25°C; reaction time was 360 seconds; the concentration of sulfur reagent was 0.2 M of hydrogenated xanthine in pyridine solution, and the molar ratio of sulfur reagent to the nucleic acid sequence connected on the solid support in the coupling reaction was 4:1; the sulfurization reaction was carried out in a water / pyridine mixed solvent (the volume ratio of water to pyridine was 1:9).

[0300] After the last nucleoside monomer is connected, the nucleic acid sequence connected to the solid phase carrier is sequentially cleaved, deprotected, purified, desalted, and then lyophilized to obtain the sense strand, wherein:

[0301] The cleavage and deprotection conditions are as follows: the synthesized nucleotide sequence connected to the solid phase carrier is added to 25% ammonia by mass, the amount of ammonia is 0.5 ml / μmol, and the reaction is carried out at 55°C for 16 hours. The solvent is removed, and vacuum concentration is carried out to dryness. After ammonia treatment, the product is dissolved in 0.4 ml / μmol N-methylpyrrolidine, and then 0.3 ml / μmol triethylamine and 0.6 ml / μmol triethylamine trifluoromethanesulfonate are added to remove the 2'-O-TBDMS protection on the ribose.

[0302] The purification and desalting conditions are as follows: the nucleic acid is purified by gradient elution of NaCl using a preparative ion chromatography purification column (Source 15Q). Specifically, eluent 1 is 20 mM sodium phosphate (pH = 8.1), the solvent is a water / acetonitrile mixture (water and acetonitrile in a volume ratio of 9:1), eluent 2 is 1.5 M sodium chloride, 20 mM sodium phosphate (pH = 8.1), and the solvent is a water / acetonitrile mixture (water and acetonitrile in a volume ratio of 9:1); the elution gradient is eluent 1:eluent 2 = (100:0)-(50:50). After the product eluate is collected, desalting is carried out using a reverse phase chromatography purification column, and the desalting conditions include desalting using a dextran gel column, and the filler is dextran gel G25, and elution is carried out using deionized water.

[0303] Detection: purity detection is carried out using ion exchange chromatography (IEX-HPLC); molecular weight detection is carried out using liquid chromatography-mass spectrometry (LC-MS), and the measured value is compared with the theoretical value. If the measured value and the theoretical value are consistent, it indicates that the siRNA sense strand is obtained.

[0304] (5-2) Synthesis of antisense strand (AS)

[0305] The nucleoside monomers are sequentially connected in the order of 3'-5' by the method of phosphoramidite nucleic acid solid phase synthesis. Each connection of a nucleoside monomer includes four reactions of deprotection, coupling, capping, oxidation or sulfurization. The deprotection, coupling, capping, oxidation or sulfurization reaction conditions in the solid phase synthesis method of the antisense strand, the cleavage and deprotection conditions, and the purification and desalting conditions are the same as those in step (5-1) for synthesizing the sense strand.

[0306] Detection: purity detection is carried out using ion exchange chromatography (IEX-HPLC); molecular weight detection is carried out using liquid chromatography-mass spectrometry (LC-MS), and the measured value is compared with the theoretical value. If the measured value and the theoretical value are consistent, it indicates that the siRNA sense strand is obtained.

[0307] (5-3) Synthesis of siRNA

[0308] The sense strand synthesized in step (5-1) and the antisense strand synthesized in step (5-2) were mixed in an equimolar ratio, dissolved in water for injection and heated to 95°C, slowly cooled to room temperature and kept at room temperature for 10 minutes, and the sense strand and the antisense strand were allowed to form a double-stranded structure by hydrogen bonding, thereby obtaining siRNA having the sense strand and the antisense strand shown in Table 3.

[0309] In the synthesis of siRNA, the compound NM041, the compound NM064, and 5'-AMINO-MODIFIER C6-TFA phosphoramidite monomer (CAS No. 133975-85-6) were respectively regarded as one nucleoside monomer to participate in the synthesis of siRNA, and formed Linker structures (NM041), (NM064), and (NH2-C6), respectively.

[0310] wherein the structural formula of (NM041) is: If (NM041) is conjugated to the 5' end of the sense strand of siRNA, the structural formula of siRNA is: If (NM041) is conjugated to the 3' end of the sense strand of siRNA, the structural formula of siRNA is:

[0311] wherein the structural formula of (NM041) (NM041) is If (NM041) (NM041) is conjugated to the 5' end of the sense strand of siRNA, the structural formula of siRNA is: If (NM041) (NM041) is conjugated to the 3' end of the sense strand of siRNA, the structural formula of siRNA is:

[0312] The structural formula of (NM064) is: If (NM064) is conjugated to the 5' end of the sense strand of siRNA, the structural formula of siRNA is: If (NM064) is conjugated to the 3' end of the sense strand of siRNA, the structural formula of siRNA is:

[0313] The structural formula of (NM064) (NM064) is: If (NM064) (NM064) is conjugated to the 5' end of the sense strand of siRNA, the structural formula of siRNA is: If (NH2-C6) is conjugated to the 5' end of the sense strand of the siRNA, the structure of the siRNA is:

[0314] If (NH2-C6) is conjugated to the 5' end of the sense strand of the siRNA, the structure of the siRNA is: If (NH2-C6) is conjugated to the 5' end of the sense strand of the siRNA, the structure of the siRNA is: If (NH2-C6) is conjugated to the 5' end of the sense strand of the siRNA, the structure of the siRNA is:

[0315] Preparation Example 6: Preparation of siRNA conjugate

[0316] Example: Conjugate RZ891002 is formed by conjugating ligand LD100 to the sense strand of RX891001.

[0317] The specific conjugation process is as follows:

[0318] (1) Take 150 μL of H2O, 70 μL of 0.2 M carbonate buffer solution (pH = 9.2), and 70 μL of N,N-dimethylformamide (DMF) to dissolve the RX891001 SS group in the mixed solvent to obtain a RX891001 SS solution with a concentration of 1.0 eq;

[0319] (2) Take 5.0 eq of ligand compound dissolved in 70 μL of DMF, and then dissolve the ligand unit molecule solution in the RX891001 SS solution obtained in step (1) to obtain a reactant mixture.

[0320] (3) Take 30.0 eq of tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) and 6.0 eq of CuSO4·5H2O, and mix them according to the volume ratio THPTA: CuSO4·5H2O = 5: 1, then take 37 μL and add it to the reactant mixture obtained in step (2) above, and perform vortex shaking to obtain an intermediate product mixture, and measure the pH of the intermediate product mixture to be 8, then take 25.0 eq of sodium ascorbate and quickly add it to the intermediate product mixture, and perform vortex shaking treatment, and react at 40°C for 1 h to obtain a product mixture;

[0321] 3 μL of the product mixture was diluted with a mixed solution of DMF and H2O (volume ratio of DMF to H2O was 1:5), and then separated and purified by HPLC treatment. In the HPLC treatment, a C18 chromatographic column was used, the mobile phase was ammonium bicarbonate buffer solution, and gradient elution was used. The product after HPLC treatment was freeze-dried to obtain the conjugate product. The obtained conjugate was single-stranded, and was annealed to finally obtain siRNA double-strand.

[0322] The structural formula of the siRNA conjugate RZ891002 is as follows:

[0323] The azido groups in the two LD100 and the four alkyne groups in the siRNA terminal Linker structure (NM041) (NM041) were respectively converted into triazole groups by click chemistry reaction to realize the covalent connection of the two ligand compounds LD100 and siRNA.

[0324] The structural formula of the siRNA conjugate RZ899065 is as follows:

[0325] The azido groups in the four LD100 and the four alkyne groups in the siRNA terminal Linker structure (NM041) (NM041) were respectively converted into triazole groups by click chemistry reaction to realize the covalent connection of the four ligand compounds LD100 and siRNA.

[0326] Preparation Example 7: Preparation of siRNA conjugate

[0327] (7-1) Conjugation of (C6-NH2) in siRNA and linker group LK005 to obtain siRNA-LK005.

[0328] For details, see WO2019010274A1 Example 2, which is incorporated herein by reference in its entirety, and will not be repeated here.

[0329] The structural formula of siRNA-LK005 is as follows:

[0330] (7-2) Conjugation of siRNA-LK005 and ligand compound:

[0331] The conjugation connection process of the ligand compound and the siRNA-LK005 group is as follows:

[0332] (7-2-1) Take 150 μL of H2O, 70 μL of 0.2 M carbonate buffer solution (pH = 9.2), and 70 μL of N,N-dimethylformamide (DMF) to mix to obtain a mixed solvent; dissolve siRAGE-LK005 with the mixed solvent to obtain a siRNA-LK005 solution with a concentration of 1.0 eq.

[0333] (7-2-2) Take 6.0 eq of ligand compound to dissolve with 70 μL of DMF to obtain a ligand compound solution.

[0334] (7-2-3) Mix the siRAGE-LK005 solution of step (7-2-1) and the ligand compound solution of step (7-2-2) to obtain a reactant mixture, wherein the molar ratio of siRAGE-LK005 and ligand compound is 1:6.

[0335] (7-2-4) Take 10.0 eq of tris(3-hydroxypropyltriazolylmethyl)amine (CAS No. 760952-88-3, abbreviated as THPTA) and 3.0 eq of CuSO4·5H2O to mix according to the volume ratio THPTA:CuSO4·5H2O = 10:3, shake for 5 min at 40°C, take 37 μL to add to the reactant mixture obtained in step (7-2-3) above, and vortex to obtain an intermediate product mixture, which has a pH = 8. Then take 25.0 eq of sodium ascorbate to quickly add to the intermediate product mixture and vortex to treat, and react for 1 h at 40°C to obtain a product mixture.

[0336] (7-2-5) Take 3 μL of the product mixture, dilute it with a mixed solution of DMF and H2O (volume ratio of DMF to H2O = 1:5), and then separate and purify it by HPLC treatment. In the HPLC treatment, a C18 chromatographic column is used, the mobile phase is ammonium bicarbonate buffer solution, and gradient elution is used. The product after HPLC treatment is freeze-dried to obtain the conjugate product.

[0337] (7-2-6) After diluting each conjugate to a concentration of 0.2 mg / mL (calculated as siRNA) with ultrapure water (Milli-Q ultrapure water instrument, resistivity 18.2 MΩ*cm (25°C)), use a liquid chromatograph-mass spectrometer (LC-MS, Liquid ChromatogRLhy-Mass SP1ectrometry, purchased from Waters Company, model: LCT Premier) to detect the molecular weight. The measured value is consistent with the theoretical value, indicating that the synthesized conjugate is the target designed siRNA conjugate.

[0338] Example: The structure of conjugate RZ899047 is shown below:

[0339] In which the azido group in ligand compound LD100 and one alkyne group in siRNA-LK005 form a triazole group through click chemistry reaction to achieve covalent connection of siRNA-LK005 and ligand compound LD100.

[0340] The siRNA conjugates with sequence information shown in Table 4 were prepared according to the above method.

[0341] Table 3 Unmodified nucleotide sequence information for forming siRNA

[0342] Table 4 Sequence information of siRNA conjugates

[0343] Unless otherwise specified, the base composition and modification meanings described in the disclosure are as follows: capital letters A, U, G, C, T represent the base composition of nucleotides, lowercase letter m represents that the nucleotide adjacent to the left of the letter m is a 2'-O-methyl modified nucleotide; lowercase letter f represents that the nucleotide adjacent to the left of the letter f is a 2'-fluorinated modified nucleotide; (moe) represents that the nucleotide adjacent to the left of (moe) is a 2'-O-methoxyethyl modified nucleotide, and lowercase letter s represents that the two nucleotides adjacent to the left and right of the letter s are connected by a phosphorothioate bond. VP represents that the 5' end of the antisense strand in siRNA is modified as 5'-(E)-vinyl phosphonate (5'-(E)-VP) modification.

[0344] The structure of VPUm is

[0345] The structure of 2'-O-methyl modified nucleotide is

[0346] The structure of 2'-fluorinated modified nucleotide is

[0347] The structure of 2'-O-methoxyethyl modified nucleotide is

[0348] For example, NM064+LD100*2 represents that the azido group in two ligand unit molecules LD100 and the two alkyne groups in NM064 form triazole groups through click chemistry reaction, respectively, so as to conjugate and connect the ligand unit molecules LD100 to the oligonucleotide molecules.

[0349] Wherein, Base represents nucleobase A, U, G, C, T.

[0350] Biological detection experiment

[0351] Unless otherwise specified, the siRNA sequences used in the present disclosure are synthesized by Suzhou Xuanjing Biotechnology Co., Ltd. and Beijing Xuanjing Rui Pharmaceutical Technology Co., Ltd.; PCR primer synthesis is entrusted to Beijing Qikexing Biotechnology Co., Ltd.; experimental animals C57BL / 6J mice are purchased from Spafas (Beijing) Biotechnology Co., Ltd.

[0352] mRNA expression level detection:

[0353] From the above RNA later, add 1 mL Trizol solution to the tissue sample in the Tissuelyser II type automatic tissue homogenizer, crush for 120 s, centrifuge instantly, stand at room temperature for 10 min, add 200 μL chloroform, shake well and stand at room temperature for 3 min. 4℃, 12000rpm centrifugation for 10 min. 400 μL supernatant is added to 400 μL isopropanol centrifuge tube, mixed well, and placed at room temperature for 10 min. 4℃, 12000rpm centrifugation for 10 min, discard the supernatant. Add 1 mL 75% ethanol, invert the centrifuge tube, wash the precipitate. 4℃, 12000rpm centrifugation for 5 min, remove the supernatant, dry at room temperature, and extract the total RNA.

[0354] Take the above 1 μg total RNA, use the reverse transcription kit (Promega Company, Reverse Transcription System, A3500) and select Oligo(dT)15 reverse transcription primer, configure 20 μL reverse transcription system according to the method recorded in the reverse transcription kit instruction book and complete the reverse transcription reaction. After the reaction, 80 μL RNase-Free water is added to the reverse transcription system to obtain cDNA solution. Then use real-time fluorescent quantitative PCR kit (ABI Company, SYBR TM Select Master Mix, Catalog number: 4472908) to detect the expression amount of target gene mRNA in the tissue. In this real-time fluorescent quantitative PCR method, primers for target genes and primers for internal reference genes are used to detect target genes and internal reference genes, respectively. According to the method recorded in the real-time fluorescent quantitative PCR kit instruction book, 20 μL Real-time PCR reaction system is configured for each PCR detection hole, and each reaction system contains 5 μL cDNA solution obtained by the above reverse transcription reaction, 10 μL SYBR TMSelect Master Mix, 0.5 μL 10 μM upstream primer, 0.5 μL 10 μM downstream primer, 4 μL RNase-Free H2O. The prepared reaction system was placed on a real-time fluorescence quantitative PCR instrument (ABI Company, StepOnePlus TM / 7500), and Real-time PCR amplification was performed using a three-step method, and the amplification program was 95°C pre-denaturation for 10 min, then 95°C denaturation for 30 s, 60°C annealing for 30 s, 72°C extension for 30 s, and the processes of denaturation, annealing and extension were repeated for 40 cycles.

[0355] In the real-time fluorescence quantitative PCR method, the expression level and inhibition rate of the target gene mRNA in each test group were relatively quantified by the ΔΔCt method, and the calculation method was as follows:

[0356] ΔCt(test group) = Ct(target gene in test group) - Ct(internal reference gene in test group)

[0357] ΔCt(control group) = Ct(target gene in control group) - Ct(internal reference gene in control group)

[0358] ΔΔCt(test group) = ΔCt(test group) - ΔCt(control group average)

[0359] ΔΔCt(control group) = ΔCt(control group) - ΔCt(control group average)

[0360] Wherein, ΔCt(control group average) is the arithmetic mean of the ΔCt(control group) of each of the 5 mice killed at the same time point in the control group. Therefore, each sample of the test group and the control group corresponds to a ΔΔCt value.

[0361] Relative expression level of target gene mRNA in test group = 2 -ΔΔCt( ΔΔCt(test group) × 100%

[0362] The mRNA expression level of the target gene in the test group was normalized based on the control group, and the mRNA expression level of the target gene in the control group was defined as 100%.

[0363] Inhibition rate of target gene mRNA expression in test group (%) = 100% - relative expression level of target gene mRNA in test group

[0364] Unless otherwise stated, the in vivo activity experimental data are expressed as , and the experimental data were plotted and analyzed using GraphPad prism 8.0 software.

[0365] Example 1 Evaluation of the inhibitory activity of LD106 conjugate on target gene Urate Transporter 1 (URAT1) in mice

[0366] This example used the "Method for evaluating the inhibitory activity of target gene in mice" to evaluate the inhibitory activity of siRNA sequence RZ891004 conjugated at the 3' end of the sense strand of LD106 at the URAT1 target point and control siRNA sequence RX891001 without conjugation on the target gene URAT1 in mice.

[0367] 6-8 week old C57BL / 6J mice were randomly divided into 3 groups by weight, 5 mice in each group, and each group of mice was given the above siRNA sequence by subcutaneous injection in the abdomen. The volume of each mouse in the PBS control group was 5 mL / kg, and the dose of each mouse in the experimental group was 3 mg / kg (calculated as siRNA), and the volume was 5 mL / kg. The mice were given drugs for 3 consecutive days. The day of administration was recorded as day 0 (D0), and 5 mice from each group were sacrificed on day 6 (D8) after the last administration. The animals were subjected to gross dissection, and the renal cortex and medulla were collected and cut into several 2 mm 3 pieces with RNAlater. RNA extraction, Real-time PCR detection was as described above, and the difference in gene expression was calculated by the ΔΔCt method.

[0368] Table 5 Sequence list of primers used in Example 1

[0369] The results of Example 1 showed that the LD106 conjugated siRNA sequence RZ891004 had certain target gene inhibitory effect in the renal cortex and medulla, and showed better inhibitory effect in the renal cortex, while the siRNA sequence RX891001 without conjugation had no inhibitory activity in the kidney (Figure 1, Table 6).

[0370] Table 6 Inhibitory activity of target gene in mice after administration of siRNA conjugate described in this example

[0371] Example 2 Evaluation of the inhibitory activity of LD106 conjugate on target gene Superoxide dismutase 1 protein (SOD1) in mice

[0372] This example used the "Method for evaluating the inhibitory activity of target gene in mice" to evaluate the inhibitory activity of siRNA sequence RZ899104 conjugated at the 3' end of the sense strand of LD106 at the SOD1 target point and control siRNA sequence RZ899056 without vector on the target gene SOD1 in mice.

[0373] 6-8 week old C57BL / 6J mice were randomly divided into 3 groups by weight, 5 mice in each group, and each group of mice was given the above siRNA sequence by subcutaneous injection in the abdomen, wherein the volume of each mouse in the PBS control group was 5 mL / kg, the dose of each mouse in the experimental group was 3 mg / kg (calculated by siRNA), and the volume was 5 mL / kg; continuous administration for 3 days. The day of administration is recorded as day 0 (D0), and 5 mice in each group were sacrificed on day 14 (D16) after the last administration, and the animal was subjected to gross dissection, and the renal cortex, renal medulla, heart, liver, duodenum tissue was collected, cut into several 2 mm 3 The small pieces were stored in RNAlater. RNA extraction, Real-time PCR detection was as described above, and the gene expression difference was calculated by ΔΔCt method.

[0374] Table 7 Sequence list of primers used in Example 2

[0375] The results of Example 2 show that the LD106 conjugated siRNA sequence RZ899104 has better target gene inhibition effect in the renal cortex and medulla than the unconjugated siRNA sequence RZ899056, and the inhibition activity in the renal cortex can reach about 70%, and the heart, liver, duodenum and other tissues have no target gene inhibition effect, indicating that the kidney has specific targeting effect (Figures 2-3, Table 8).

[0376] Table 8 Inhibition activity of target genes in mice after administration of siRNA conjugates described in this example

[0377] Example 3 Evaluation of the inhibition activity of LD105 conjugate on target gene URAT1 in mice

[0378] This example uses the "in vivo target gene inhibition activity evaluation method" to evaluate the inhibition activity of the siRNA sequence RZ891003 conjugated with two clusters at the 3' end of the sense strand of the URAT1 target LD105 and the control siRNA sequence RX891001 without conjugation on the target gene URAT1 in mice.

[0379] The 6-8 week old C57BL / 6J mice were randomly divided into 3 groups according to the body weight, 5 mice in each group, and each group of mice was given the above siRNA sequence by subcutaneous injection in the abdomen, wherein the administration volume of each mouse in the PBS control group was 5 mL / kg, the administration dose of each mouse in the experimental group was 3 mg / kg (calculated as siRNA), and the administration volume was 5 mL / kg; the administration was continuously performed for 3 days. The day of administration was recorded as day 0 (D0), and 5 mice in each group were sacrificed on day 6 (D8) after the last administration, and the animals were subjected to gross anatomy, and the renal cortex and renal medulla were collected, cut into several 2 mm 3 The small pieces were preserved with RNAlater. The RNA extraction and Real-time PCR detection were performed as described above, the primers were shown in Table 5 of Example 1, and the gene expression difference was calculated by the ΔΔCt method.

[0380] The results of Example 3 showed that the LD105 conjugated siRNA sequence RZ891003 had certain target gene inhibition effect in the renal cortex and medulla, and showed better inhibition effect in the renal cortex, while the siRNA sequence RX891001 without conjugation had no inhibition activity in the kidney (Figure 4, Table 9).

[0381] Table 9 shows the inhibition activity of the target gene in the mouse body after the administration of the siRNA conjugate described in the present example

[0382] Example 4: Inhibition activity evaluation of LD105 conjugate on SOD1 in the mouse body

[0383] In the present example, the “target gene inhibition activity evaluation method in the mouse body” was used to evaluate the inhibition activity of the siRNA sequence RZ899103 conjugated with two clusters at the 3' end of the sense strand of the SOD1 target LD105 and the control sequence RZ899056 without carrier conjugation on the target gene SOD1 in the mouse body.

[0384] The 6-8 week old C57BL / 6J mice were randomly divided into 3 groups according to the body weight, 5 mice in each group, and each group of mice was given the above siRNA sequence by subcutaneous injection in the abdomen, wherein the administration volume of each mouse in the PBS control group was 5 mL / kg, the administration dose of each mouse in the experimental group was 3 mg / kg (calculated as siRNA), and the administration volume was 5 mL / kg; the administration was continuously performed for 3 days. The day of administration was recorded as day 0 (D0), and 5 mice in each group were sacrificed on day 14 (D16) after the last administration, and the animals were subjected to gross anatomy, and the renal cortex and renal medulla, heart, liver, and duodenum tissues were collected, cut into several 2 mm 3 The small pieces were preserved with RNAlater. The RNA extraction and Real-time PCR detection were performed as described above, the primers were shown in Table 7 of Example 2, and the gene expression difference was calculated by the ΔΔCt method.

[0385] The results of Example 4 show that the LD106 conjugated siRNA sequence RZ899103 exhibits better inhibitory effect in the kidney cortex than the unconjugated siRNA sequence RZ899056, while the heart, liver, duodenum and other tissues have no target gene inhibitory effect, indicating kidney-specific targeting effect (Figures 5-6, Table 10).

[0386] Table 10 Inhibitory activity of target genes in mice after administration of siRNA conjugates described in this example

[0387] Example 5 Evaluation of inhibitory activity of LD100 conjugates on target gene URAT1 in mice

[0388] This example uses the "Method for evaluating target gene inhibitory activity in mice" to evaluate the inhibitory activity of the siRNA sequence RZ891002 conjugated to two clusters at the 3' end of the sense strand of the URAT1 target LD100 and the siRNA sequence RX891001 without carrier conjugation on the target gene URAT1 in mice.

[0389] 6-8 week old C57BL / 6j mice were randomly divided into 3 groups according to body weight, 5 mice in each group, and each group of mice was administered with the above siRNA conjugates by subcutaneous injection in the abdomen, wherein the volume of each mouse in the PBS control group was 5 mL / kg, the dose of each mouse in the experimental group was 3 mg / kg (calculated as siRNA), and the volume was 5 mL / kg; continuous administration for 3 days. The day of administration is recorded as day 0 (D0), and 5 mice in each group are sacrificed on day 6 (D8) after the last administration, and the animals are subjected to gross dissection, and the renal cortex and medulla are collected and cut into several 2 mm 3 small pieces for storage in RNAlater. RNA extraction, Real-time PCR detection are as described above, primers are as shown in Table 5 of Example 1, and gene expression difference is calculated by ΔΔCt method.

[0390] The results of Example 5 show that the LD100 conjugated siRNA sequence RZ891002 has certain target gene inhibitory effect in the kidney cortex and medulla, and exhibits better inhibitory effect in the kidney cortex, while the unconjugated siRNA sequence RZ891001 has no inhibitory activity in the kidney (Figure 7, Table 11).

[0391] Table 11 Inhibitory activity of target genes in mice after administration of siRNA conjugates described in this example

[0392] Example 6 Evaluation of inhibitory activity of LD100 conjugates on target gene SOD1 in mice

[0393] The present example uses the "in vivo evaluation of target gene inhibition activity in mice" to evaluate the inhibition activity of siRNA sequences RZ899063, RZ899064, RZ899065 and RZ899056 on the target gene SOD1 in mice.

[0394] The 6-8 week old C57BL / 6j mice were randomly divided into 5 groups according to body weight, 10 mice in each group, and each group of mice was administered with the above siRNA conjugates by subcutaneous injection. The volume of each mouse in the PBS control group was 5 mL / kg, the dose of each siRNA conjugate in the experimental group was 3 mg / kg (calculated by siRNA), and the volume was 5 mL / kg. The administration was performed for 3 consecutive days. The day of administration was recorded as day 0 (D0), and 5 mice in each group were sacrificed on day 14 (D16) and day 21 (D23) after the last administration. The animals were subjected to gross autopsy, and the renal cortex and medulla tissues were collected and cut into several 2 mm 3 The small pieces were stored in RNAlater. The RNA extraction and Real-time PCR detection were as described above, the primers were as shown in Table 7 of Example 2, and the gene expression difference was calculated by the ΔΔCt method.

[0395] The results of Example 6 show that, compared with the non-conjugated siRNA sequence RZ899056, the LD100 conjugated siRNA sequences RZ899063, RZ899064 and RZ899065 have higher inhibition activity in the renal cortex on D16 and D23, and the siRNA sequence RZ899063 conjugated with two clusters at the 3' end of the LD100 sense strand shows better inhibition effect and longer pharmacodynamic effect time in the kidney (Figures 8-9, Table 12).

[0396] Table 12 Inhibition activity of target genes in mice after administration of siRNA conjugates described in the present example

[0397] Example 7 Inhibition activity evaluation of LD100 conjugates administered intravenously on target gene SOD1 in mice

[0398] The present example uses the "in vivo evaluation of target gene inhibition activity in mice" to evaluate the inhibition activity of siRNA sequences RZ899046, RZ899047, RZ899051 and RX899001 on the target gene SOD1 in mice.

[0399] The 6-8 week old C57BL / 6j mice were randomly divided into 5 groups according to body weight, 5 mice in each group, and each group of mice was given the siRNA conjugate by tail vein injection, wherein the volume of each mouse in the PBS control group was 5 mL / kg, the dose of each siRNA conjugate in the experimental group was 3 mg / kg (calculated by siRNA), and the volume was 5 mL / kg; the drug was administered continuously for 3 days. The day of administration was recorded as day 0 (D0), and 5 mice in each group were sacrificed on day 14 (D16) after the last administration. The animals were subjected to gross dissection, and the renal cortex and medulla tissues were collected and cut into several 2 mm 3 The small pieces were stored in RNAlater. RNA extraction and Real-time PCR detection were performed as described above, the primers were shown in Table 7 of Example 2, and the gene expression difference was calculated by the ΔΔCt method.

[0400] The results of Example 7 show that the siRNA sequences RZ899046, RZ899047 and RZ899051 conjugated with LD100 show better inhibition activity compared with the siRNA sequence RX899001 without carrier conjugation, and RZ899051 shows better inhibition activity in the whole kidney compared with RZ899046 and RZ899047 (Figure 10, Table 13).

[0401] Table 13 shows the inhibition activity of the siRNA conjugate on the target gene in mice

[0402] Example 8 Evaluation of the inhibition activity of LD101 conjugate on the target gene SOD1 in mice

[0403] This embodiment uses the "In vivo target gene inhibitory activity assessment method" to evaluate the inhibitory activity of the following siRNA sequences in mice against the target gene SOD1: RZ899054 (two clusters concatenated at the 3' end of the LD101 sense strand), RZ899074 (two clusters concatenated at the 3' end of the LD101 sense strand), RZ899075 (four clusters concatenated at the 3' end of the LD100 sense strand), and RZ899056 (a concatenation without these groups). The difference between RZ899054 and RZ899074 is that RZ899074 has a VP modification at the 5' end of the antisense strand.

[0404] Six- to eight-week-old C57BL / 6j mice were randomly divided into five groups of five mice each, based on body weight. Each group received the aforementioned siRNA conjugate via subcutaneous abdominal administration. In the PBS control group, the dose was 5 mL / kg per mouse, while in the siRNA conjugate experimental group, the dose was 3 mg / kg (based on siRNA) per mouse, administered at a volume of 5 mL / kg. Administration continued for three consecutive days, with the day of administration designated as day 0 (D0). On day 14 (D16) after the last administration, five mice from each group were sacrificed. Gross dissection was performed, and renal cortex and medulla tissue were collected and cut into several 2 mm sections. 3 Small fragments were preserved using RNAlater. RNA extraction and Real-time PCR detection were performed as described above, with primers shown in Table 7 of Example 2. Gene expression differences were calculated using the ΔΔCt method.

[0405] The results of Example 8 showed that, compared with the vector-free siRNA sequence RZ899056, the LD101-conjugated siRNA sequences RZ899054, RZ899074, and RZ899075 exhibited certain inhibitory activity in the renal cortex. Among them, RZ899054 and RZ899074 were superior, and their activities were basically equivalent (Figure 11, Table 14).

[0406] Table 14 shows the inhibitory activity of the target gene in mice after administration of the siRNA conjugate described in this example.

[0407] Example 9 evaluates the inhibitory activity of the LD100 conjugate on the target gene aldehyde dehydrogenase 2 family member (ALDH2) in mice.

[0408] This embodiment uses the "In vivo target gene inhibitory activity assessment method" to evaluate the inhibitory activity of the two clusters of siRNA sequences R381002 and R381004 conjugated at the 3' end of the LD100 positive strand on the target gene ALDH2 in mice.

[0409] 6-8 weeks old C57BL / 6J mice were randomly divided into 3 groups according to body weight, 5 mice in each group, and single abdominal subcutaneous injection was used for administration. The administration volume of each mouse in the PBS control group was 5 mL / kg (based on the weight of the mouse); the administration dose of each mouse in the test group was 20 mg (based on siRNA) / kg (based on the weight of the mouse), and the administration volume was 5 mL / kg (based on the weight of the mouse). The day of administration was recorded as day 0 (D0), and 5 mice in each group were sacrificed on day 14 (D14) after administration. The animals were subjected to gross anatomy, and the kidney cortex, kidney medulla, heart, liver, spleen, lung, brain and other tissues were collected and cut into several 2 mm 3 The small pieces were stored in RNAlater. Tissue RNA extraction, Real-time PCR detection, and gene expression difference calculation by ΔΔCt method were performed.

[0410] In this embodiment, the specific operation process of real-time fluorescent quantitative PCR is as follows: real-time fluorescent quantitative PCR is performed according to the method recorded in the instruction manual of TaqMan Fast Advanced Master Mix (ABI Company, Catalog number: 4444557), that is, 20 μL of real-time PCR reaction system is configured in each reaction well, and each reaction system contains 5 μL of cDNA solution obtained by reverse transcription, 10 μL of TaqMan Fast Advanced Master Mix, 0.4 μL of 10 μM upstream primer, 0.4 μL of 10 μM downstream primer, 0.2 μL of 10 μM probe, and 3 μL of RNase-Free H2O. The prepared reaction system is placed on a real-time fluorescent quantitative PCR instrument (ABI Company, StepOnePlus TM / 7500), and two-step Real-time PCR amplification is performed, and the amplification program is 50°C pre-denaturation for 2 min, 95°C pre-denaturation for 20 s, then 95°C denaturation for 3 s, 60°C annealing for 30 s, and repeating the denaturation and annealing process for 40 cycles.

[0411] Table 15: Sequence list of primers used in Example 9:

[0412] The results of Example 9 (Figure 12, Table 16) show that the LD100 conjugated siRNA sequences R381002 and R381004 exhibit better target gene inhibition effect in the kidney cortex and medulla, and R381004 has better inhibition activity in the kidney cortex than R381002, which can reach about 70%, and the target gene has no inhibition effect in the heart, liver, lung, spleen, brain and other tissues, indicating kidney-specific targeting effect.

[0413] Table 16 Inhibitory activity of siRNA conjugates R381002 and R381004 on target gene ALDH2 in mice

[0414] Example 10 evaluates the inhibitory activity of LD100 conjugates on target gene Urate Transporter 1 (URAT1) in cynomolgus monkeys

[0415] This example evaluates the inhibitory activity of siRNA sequence R391005 conjugated at the 3' end of the sense strand with two clusters of LD100 on target gene URAT1 in cynomolgus monkeys using the "Method for evaluating the inhibitory activity of siRNA on target genes in cynomolgus monkeys".

[0416] Male cynomolgus monkeys aged 2-4 weeks were randomly divided into 2 groups according to body weight, including 1 experimental group and 1 blank control group (PBS control), 4 monkeys in each group, and the drugs were administered by subcutaneous injection on the back. The volume of drug administered to each monkey in the PBS control group was 1 mL / kg (based on the body weight of the cynomolgus monkey), and the dose of drug administered to each monkey in the experimental group was 15 mg (based on siRNA) / kg (based on the body weight of the cynomolgus monkey), and the volume of drug administered to each monkey in the experimental group was 1 mL / kg (based on the body weight of the cynomolgus monkey); the drug was administered once a week, and the administration was continued for two weeks, and the first administration day was recorded as day 0 (D0), and the second administration day was recorded as day 7 (D7). On day 14 (D14) after the first administration, the cynomolgus monkeys in each group were anesthetized and tissue biopsied, and about 10 mg of renal cortical tissue was removed by B-ultrasound assisted puncture and placed in RNAlater for RNA extraction, Real-time PCR detection, and calculation of gene expression differences by the ΔΔCt method.

[0417] In this example, the specific operation process of real-time fluorescent quantitative PCR is as follows: real-time fluorescent quantitative PCR is performed according to the method recorded in the TaqMan Fast Advanced Master Mix (ABI Company, Catalog number: 4444557) instruction book, that is, 20 μL of the real-time PCR reaction system is configured for each reaction well, and each reaction system contains 5 μL of the cDNA solution obtained by the reverse transcription reaction, 10 μL of TaqMan Fast Advanced Master Mix, 0.4 μL of 10 μM upstream primer, 0.4 μL of 10 μM downstream primer, 0.2 μL of 10 μM probe, and 3 μL of RNase-Free H2O. The prepared reaction system is placed in a real-time fluorescent quantitative PCR instrument (ABI Company, StepOnePlus TMThe Real-time PCR amplification was performed using a two-step method on the 7500 Fast Real-Time PCR System. The amplification program was 50°C pre-denaturation for 2 min, 95°C pre-denaturation for 20 s, then 95°C denaturation for 3 s, 60°C annealing for 30 s, repeating the denaturation and annealing process for 40 cycles.

[0418] Table 17 Sequence list of primers used in Example 10

[0419] The results of Example 10 (Figure 13, Table 18) show that the LD100 conjugated siRNA sequence R391005 exhibits better target gene inhibition effect in the kidney cortex, which can reach about 63%.

[0420] Table 18 Inhibition activity of target gene URAT1 in cynomolgus monkeys after administration of siRNA conjugate R391005

[0421] The above detailed description merely illustrates the content of the present application and does not represent the limitation of the content of the present application. Various modifications and improvements can be made by those of ordinary skill in the art without departing from the spirit and essence of the present disclosure, and these modifications and improvements are also considered to be within the scope of protection of the present disclosure.

Claims

1. An oligonucleotide conjugate for inhibiting gene expression in kidney cells, comprising: (a) a single- or double-stranded oligonucleotide molecule having the following characteristics: (i) an antisense strand comprising 17-35 nucleotides, wherein, at least 15 nucleotides are complementary or substantially complementary to the mRNA sequence of a gene in kidney cells; (ii) a sense strand of 15-35 nucleotides in length, which is complementary or partially complementary to the antisense strand; (b) a targeting ligand having affinity for a receptor present on the surface of a kidney cell, wherein the targeting ligand is a polypeptide; optionally, the polypeptide has at least 4 amino acid residues; and, (c) a linker group, wherein the oligonucleotide molecule is covalently linked to the targeting ligand via the linker group.

2. The oligonucleotide conjugate according to claim 1, characterized in that, The receptor is a known or unknown endocytic receptor; optionally, the receptor is selected from the group consisting of a megalin receptor or a lina peptide receptor.

3. The oligonucleotide conjugate of claim 1, wherein, The linker is attached to the N- or C-terminus of an amino acid on the polypeptide; Optionally, the Linker is selected from a substituted or unsubstituted aliphatic chain, a 3-6 membered heterocyclic ring, a C6-Ci2aromatic ring, a (PEG) 10 ring, a disulfide, amide or triazole containing group, or a combination thereof; wherein n is an integer selected from 1-20; and n ring, a disulfide, amide or triazole containing group, or a combination thereof; wherein n is an integer selected from 1-20; and Optionally, the linker comprises any of the following substituents or any combination thereof: disulfide bonds, amido, triazolyl, -NH-, -C(O)-, wherein a, b, c or d are each independently selected from an integer from 0 to 10; * represents a point of attachment to the oligonucleotide molecule or to the targeting ligand, or a point of attachment of the substituents to each other.

4. The oligonucleotide conjugate of claim 1, wherein, The targeting ligand comprises any of the polypeptide fragments represented by A1) to A6): A1)-X1(KKEEE) n - K m - wherein n = 1-5, m = 0 or 1 or 2, X1is selected from any L-alpha amino acid or L-beta amino acid except K, E; A2) -CLPVASC, preferably in the form of a cyclic peptide A3) -CYFQNC-; preferably in the form of a cyclic peptide A4) -KIDRI-; A5) -IDRI- (Ile-Asp-Arg-Ile-); A6) -dXa-Ser-dXb-X2-dXc-Gly-Xd-Ile-Asp-Arg(Ak)-Ile-; wherein X2 is selected from any unnatural amino acid; dXa, dXb, dXc are selected from any D-amino acid; Arg(Ak) is selected from arginine or alkylated modified arginine; Xd is selected from Hyp or Pro; Preferably, the targeting ligand is selected from a polypeptide comprising any of the amino acid sequence segments represented by B1) to B5): B1) -KKEEE-KKEEE-KKEEE-K-; B2) -CKKEEE-KKEEE-KKEEE-K-; B3) -CLPVASC, preferably in the form of a cyclic peptide B4) -CYFQNCPRG-, preferably in cyclic peptide form B5) -KIDRI-.

5. The oligonucleotide conjugate according to claim 4, characterized in that, The targeting ligand attached to the linker is a ligand unit selected from any of the structures represented by C1) to C5): C1) La-KKEEE-KKEEE-KKEEE-K-Laa; C2) La-CKKEEE-KKEEE-KKEEE-K-Laa; C3) La-CLPVASC-Laa; preferably a cyclic peptide C4) La-CYFQNCPRG-Laa; C5) Lb-KIDRI-Laa; In formulae C1) to C4), either of La and Laa is selected from a linker, which is a linking group, and comprises at least one of a triazole group or a PEG unit or an acyl group, and the other is a terminal blocking group; Optionally, La is a linker, and the linker structure comprises -PEG2-CH2CH2CO- or triazole-PEG2-; In formula C5), Lb is selected from a linking group comprising a 6-10 membered aromatic or heteroaromatic ring; and Laa is independently selected from an amino group, or an alkyl-substituted amine group.

6. The oligonucleotide conjugate of claim 5, wherein, the ligand unit is selected from any one of the structures depicted in D1) - D8), or a pharmaceutically acceptable salt thereof: D1) -La-KKEEE-KKEEE-KKEEE-K-Lc; D2) -La-CKKEEE-KKEEE-KKEEE-K-Lc; D3)-La-CLPVASC-Lc; preferably in cyclic peptide form D4) -La-CYFQNCPRG-Lc; preferably a cyclic peptide D5) D6) D7) D8) wherein La is a linker independently selected from any bond, -NH-, an amido group, or a linking group comprising at least one PEG unit; optionally, La is a linking group comprising at least 2 PEG units; Optionally, La is selected from amido or -triazolyl-(PEG)n-; wherein n is 1-3; wherein Lc is a terminal blocking group.

7. The oligonucleotide conjugate according to any one of claims 1 to 6, characterized in that, conjugating the at least one ligand unit to an oligonucleotide molecule.

8. The oligonucleotide conjugate according to any one of claims 1 to 7, characterized in that, the oligonucleotide molecule inhibits the expression of mRNA of a gene in a kidney cell.

9. A polypeptide capable of delivering an oligonucleotide to a kidney cell, the polypeptide comprising any one of the amino acid sequence segments depicted in B1) - B5): B1) -KKEEE-KKEEE-KKEEE-K-; B2) -CKKEEE-KKEEE-KKEEE-K-; B3) B4) B5) -KIDRI-.

10. A ligand unit molecule capable of delivering an oligonucleotide to a kidney cell, selected from any one of the compounds depicted in E1) - E8): E1) N3-PEG2-CH2CH2CO-KKEEE-KKEEE-KKEEE-K-NH2; E2) Ac-CKKEEE-KKEEE-KKEEE-K-NH2; E3) N3-(PEG)2-CH2CH2-CO-CLPVASC-NH2; E4) N3-(PEG)2-CH2CH2-CO-CYFQNCPRG-NH2; E5) E6) E7) E8) 11. Use of the polypeptide of claim 9 or the ligand unit molecule of claim 10, characterized in that the use is for delivering an oligonucleotide molecule to a kidney cell.

12. A composition comprising the oligonucleotide conjugate of any one of claims 1-8, the polypeptide of claim 9, or the ligand unit molecule of claim 10, or a pharmaceutically acceptable salt thereof.

13. A pharmaceutical composition comprising the oligonucleotide conjugate of any one of claims 1-8, or a pharmaceutically acceptable salt thereof.

14. A method of making the oligonucleotide conjugate of any one of claims 1-8, the method comprising: (i) synthesizing the sense strand; (ii) synthesizing the antisense strand; (iii) annealing the sense strand and the antisense strand; (iv) conjugating the ligand unit molecule to the sense strand or the antisense strand before or after annealing the sense strand and the antisense strand.

15. A method of treating a kidney-related disease or disorder, the method comprising administering to a subject the oligonucleotide conjugate of any one of claims 1-8, the composition of claim 12, or the pharmaceutical composition of claim 13; optionally, the kidney-related disease or disorder is chronic kidney disease; Optionally, the kidney-related disease or disorder is selected from the group consisting of hypertension, hypertension-related renal impairment, hyperglycemia, diabetes, diabetic nephropathy, hyperuricemia, gout, hyperuricemia-related renal impairment, hepatitis B virus-related renal impairment, myeloma kidney, chronic renal failure, glomerulonephritis, renal vascular disease, C3 glomerulopathy, lupus nephritis, IgA nephropathy, polycystic kidney disease, membranous nephropathy, atypical hemolytic uremic syndrome, uremic syndrome, or systemic lupus erythematosus (SLE)-related nephropathy.

16. A method of delivering one or more oligonucleotide molecules to a cell in vivo, the method comprising administering to a subject the oligonucleotide conjugate comprising a targeting ligand having affinity for a cell surface receptor of any one of claims 1-8, the composition of claim 12, or the pharmaceutical composition of claim 13; Optionally, the receptor is selected from the group consisting of a Megalin receptor.

17. The method of claim 16, wherein, The cell is selected from the group consisting of a kidney cell or a kidney epithelial cell.

18. The method of claim 16, wherein, The subject is a human.

19. Use of the oligonucleotide conjugate comprising a targeting ligand of any one of claims 1-8, the composition of claim 12, or the pharmaceutical composition of claim 13 for the manufacture of a medicament for the treatment of a kidney-related disease or disorder.

Images