Selenium modified double stranded oligonucleotides, conjugates, compositions and uses thereof

Abstract

The invention relates to a selenium-modified double-stranded oligonucleotide, a conjugate, a composition and application thereof, and belongs to the technical field of nucleic acid drugs. The double-stranded oligonucleotide comprises a positive-sense strand and an antisense strand, each of the positive-sense strand and the antisense strand has 17-35 nucleotides, and the positive-sense strand and the antisense strand are at least partially reversely complementary to form a double-stranded region; characterized in that the sense strand and / or the antisense strand comprises at least one nucleotide modified by a 2 '-Se (CH2) nCH3 group. Selenium modification is introduced into the double-stranded oligonucleotide, and the modified double-stranded oligonucleotide can improve the biological activity and / or the safety of oligonucleotide drugs and can be used for treating and / or preventing diseases or symptoms related to disorder of the mRNA level expressed by a target gene.

Description

Technical Field

[0001] The present invention belongs to the technical field of nucleic acid drugs, and particularly relates to a double-stranded oligonucleotide molecule with selenium modification, a conjugate, a composition formed by the double-stranded oligonucleotide, and their uses. Background Art

[0002] Chemical modification of oligonucleotide drugs can improve the stability and safety of this type of drugs, avoid immune reactions, and is also one of the main means to improve the active effect.

[0003] Selenium is a biologically active element. In drug molecules, it can not only regulate the conformation of the molecule where it is located, but also enhance the molecular potency through potential intermolecular forces (such as Se-ChB interaction), thus playing an important role in the activity and structure of drug molecules. Considering that the Se-ChB interaction is similar to the H bond, there is an interaction between Se and O, S, N, halogen, and electron-rich aromatic rings. This interaction can be applied to drug design to improve the safety and biological activity of drug molecules. Summary of the Invention

[0004] The present disclosure provides selenium-modified double-stranded oligonucleotides, conjugates formed by the double-stranded oligonucleotides, pharmaceutical compositions, and their uses. The present disclosure introduces selenium modification into the double-stranded oligonucleotide. The modified double-stranded oligonucleotide can improve the biological activity and / or safety of oligonucleotide drugs and can be used for treating and / or preventing diseases or disorders related to the dysregulation of the mRNA level of target gene expression.

[0005] In a first aspect of the present disclosure, the present disclosure provides a double-stranded oligonucleotide, the double-stranded oligonucleotide comprising a sense strand and an antisense strand, each of the sense strand and the antisense strand having 17-35 nucleotides, and the sense strand and the antisense strand at least partially reverse complementarily forming a duplex region; the sense strand and / or the antisense strand comprise at least one nucleotide modified with 2'-Se(CH2) n CH3 group; wherein, n is an integer selected from 0 to 3.

[0006] In some embodiments of the present disclosure, the nucleotide modified with 2'-Se(CH2) n CH3 group is located on the antisense strand.

[0007] In some embodiments of the present disclosure, the nucleotide modified with 2'-Se(CH2) n CH3 group is located on the sense strand.

[0008] In some embodiments of the present disclosure, each nucleotide in the duplex of the double-stranded oligonucleotide is modified.

[0009] In some embodiments of the present disclosure, the 3'-end and / or 5'-end of the sense strand and the antisense strand comprise one or more overhang regions.

[0010] In a second aspect of the present disclosure, the present disclosure provides an oligonucleotide conjugate comprising the double-stranded oligonucleotide described in the first aspect of the present disclosure and one or more ligands capable of binding to a cell surface receptor.

[0011] In a third aspect of the present disclosure, the present disclosure provides a composition comprising the double-stranded oligonucleotide described in the first aspect of the present disclosure or the double-stranded oligonucleotide conjugate described in the second aspect of the present disclosure.

[0012] In a fourth aspect of the present disclosure, the present disclosure provides the use of the double-stranded oligonucleotide described in the first aspect of the present disclosure, the double-stranded oligonucleotide conjugate described in the second aspect of the present disclosure, or the composition described in the third aspect of the present disclosure in the preparation of a medicament for treating and / or preventing a disease or disorder associated with dysregulation of the mRNA level of a target gene expression.

[0013] In a fifth aspect of the present disclosure, the present disclosure provides a kit comprising the double-stranded oligonucleotide described in the first aspect of the present disclosure, the double-stranded oligonucleotide conjugate described in the second aspect of the present disclosure, or the composition described in the third aspect of the present disclosure.

[0014] The double-stranded oligonucleotide, double-stranded oligonucleotide conjugate, or composition provided by the present disclosure can effectively treat and / or prevent a disease or disorder associated with dysregulation of the mRNA level of a target gene expression. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 are the relative expression levels of the target gene on the HepG2 cell line after administration of the siRNA conjugates RZ002006, RZ002044, RZ002046, RZ002047, RZ002048, and RZ002049 in Example 1, respectively.

[0016] Figure 2 are the relative expression levels of the target gene on the HepG2 cell line after administration of the siRNA conjugates RZ002003, RZ002061, RZ002062, and RZ002063 in Example 1, respectively.

[0017] Figure 3 are the relative expression levels of the target gene on the HepG2 cell line after administration of the siRNA conjugates RZ002031, RZ002077, RZ002078, RZ002079, and RZ002080 in Example 1, respectively.

[0018] Figure 4 The relative expression levels of the target genes on the HepG2 cell line after administering siRNA conjugates RZ002011, RZ002093, RZ002095, and RZ002098 in Example 1, respectively.

[0019] Figure 5 The relative expression levels of the target genes on the Huh7 cell line after administering siRNA conjugates RZ003017, RZ003033, RZ003035, RZ003036, and RZ003037 in Example 2 at concentrations of 1 nM and 0.1 nM, respectively.

[0020] Figure 6 The relative expression levels of the target genes on the Huh7 cell line after administering siRNA conjugates RZ003020, RZ003047, RZ003048, and RZ003049 in Example 2 at concentrations of 1 nM and 0.1 nM, respectively.

[0021] Figure 7 The relative expression levels of the target genes on the Huh7 cell line after administering siRNA conjugates RZ003021, RZ003058, RZ003059, and RZ003061 in Example 2 at concentrations of 1 nM and 0.1 nM, respectively.

[0022] Figure 8 The relative expression levels of the target genes on primary mouse liver after administering siRNA conjugates RZ599062, RZ599063, RZ599064, RZ599065, RZ599066, RZ599067, RZ599068, RZ599069, and RZ599070 in Example 3, respectively.

[0023] Figure 9 The relative expression levels of the target genes in mice after administering siRNA conjugates RZ002001, RZ502001, and RZ502002 in Example 4, respectively.

[0024] Figure 10 The relative expression levels of the target genes in mice after administering siRNA conjugates RZ597002, RZ597003, and RZ597005 in Example 5, respectively.

[0025] Figure 11 The ALT test results in the blood of female mice after administering siRNA conjugates RZ597002, RZ597003, and RZ597005 in Example 6, respectively.

[0026] Figure 12These are the results of detecting AST in the blood of female mice after administration of siRNA conjugates RZ597002, RZ597003, and RZ597005 in Example 6, respectively.

[0027] Figure 13 These are the results of detecting blood pressure in hREN×hAGT hypertensive mice after administration of siRNA conjugates RZ003063 and RZ003067 in Example 7, respectively.

[0028] Figure 14 These are the relative expression levels of C3 mRNA in the liver tissues of cynomolgus monkeys after administration of siRNA conjugates RZ002104 and RZ002109 in Example 8, respectively.

[0029] Figure 15 These are the relative expression levels of C3 protein in the sera of cynomolgus monkeys after administration of siRNA conjugates RZ002104 and RZ002109 in Example 8, respectively. Detailed implementation manners

[0030] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only partial embodiments of the present invention, rather than all. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0031] Glossary of Terms

[0032] In the present disclosure, the term "comprising" or "including" is an open expression, that is, it includes the content specified in the present disclosure, but does not exclude other aspects.

[0033] In the present disclosure, the terms "optionally", "optional" or "option" generally mean that the subsequent event or condition may or may not occur, and this description includes the case where the event or condition occurs and the case where the event or condition does not occur.

[0034] In the present disclosure, the term "double-stranded oligonucleotide" refers to a double-stranded structure formed by two oligonucleotides through partial or complete base complementary pairing. The two oligonucleotides include a sense strand and an antisense strand, and the lengths of the sense strand and the antisense strand may be the same or different, as long as there is at least a partial base complementary pairing region to form a duplex region. The oligonucleotide having a double-stranded structure belongs to the double-stranded oligonucleotide described in the present disclosure. The nucleotides constituting the double-stranded oligonucleotide in the present disclosure may be modified or unmodified nucleotides. When referring to modified nucleotides, unless otherwise specified, the modifications described in the present disclosure do not specifically refer to the modification sites. In the present disclosure, in addition to the modification of nucleotides in the double-stranded oligonucleotide, the linking bonds between nucleotides may also be modified, and the double-stranded oligonucleotide containing the linking bonds between modified nucleotides also belongs to the double-stranded oligonucleotide described in the present invention. In the present disclosure, in addition to the nucleotide part, the double-stranded oligonucleotide may further contain acceptable compound molecules or modifiers in the art to improve the properties of the double-stranded oligonucleotide, such as forming a conjugate by linking a ligand.

[0035] In the present disclosure, the term "ligand" or "conjugating group" refers to an atom or atomic group that binds to an oligonucleotide or other oligomer. Generally, the conjugating group modifies one or more properties of the compound to which it is attached, including but not limited to pharmacodynamic, pharmacokinetic, binding, absorption, cellular distribution, cellular uptake, charge, and / or clearance properties. When referring to the connection between two molecules, the term "link" used herein means that the two molecules are directly or indirectly connected by a covalent bond, or the two molecules are associated by a non-covalent bond (e.g., hydrogen bond or ionic bond).

[0036] In the present disclosure, the term "pharmaceutical composition" or "composition" may refer to the treatment of diseases and can also be used in in vitro cell culture experiments. When used for the treatment of diseases, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any of the methods well known in the pharmaceutical field. All methods include the step of combining the active ingredient with excipients constituting one or more accessory components. Generally, the composition is prepared by uniformly and sufficiently combining the active siRNA with liquid excipients, finely divided solid excipients, or both.

[0037] In the present disclosure, the term "pharmaceutically acceptable" means that a substance or composition must be chemically and / or toxicologically compatible with other components of the formulation and / or the mammal being treated therewith. Preferably, the "pharmaceutically acceptable" as described in the present disclosure refers to those approved by a federal regulatory agency or a national government or listed in the United States Pharmacopeia or other generally recognized pharmacopeias for use in animals, particularly in humans.

[0038] In the present disclosure, the term "pharmaceutically acceptable carrier or excipient" may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for a particular target dosage form. Their use is also contemplated in the present disclosure, except to the extent that any conventional excipient is incompatible with the siRNA of the present disclosure, such as any adverse biological effects produced or interactions with any other component of the pharmaceutically acceptable composition in a harmful manner.

[0039] In the present disclosure, the term "Small interfering RNA (siRNA)" refers to a double-stranded RNA that is 17 to 25 nucleotides in length and contains a sense strand and an antisense strand. siRNA mediates the targeted cleavage of RNA transcripts in the RISC pathway by forming the RNA-induced silencing complex (RISC). Specifically, siRNA directs the specific degradation of mRNA sequences through the well-known RNA interference (RNAi) process, inhibiting the translation of mRNA into amino acids and the conversion into proteins.

[0040] 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 trailing strand)" refers to an iRNA strand that contains a sequence that is substantially complementary to that in the antisense strand. The term "substantially complementary" means completely complementary or at least partially complementary, for example, the antisense strand is completely complementary or at least partially complementary to the target sequence. In the case of partial complementarity, mismatches may exist within the internal or terminal regions of the molecule, where the most tolerated mismatches exist within the terminal regions, such as within 5, 4, 3, or 2 nucleotides at the 5'- and / or 3'-ends of the iRNA. It should be noted that "at least partially substantially complementary" between the antisense strand and the mRNA means that the antisense strand has a polynucleotide that is substantially complementary to a continuous portion of the mRNA of interest.

[0041] In the present disclosure, "substantially reverse complementary" means that there are no more than 3 base mismatches between the two nucleotide sequences involved.

[0042] In the present disclosure, the terms "treat", "alleviate" or "improve" may be used interchangeably herein. These terms refer to methods of obtaining a beneficial or desired result, including but not limited to a therapeutic benefit. "Therapeutic benefit" means eradicating or improving the underlying disorder being treated. Here, a therapeutic benefit is obtained by eradicating or improving one or more physiological symptoms associated with the underlying disorder, resulting in an observed improvement in the subject, although the subject may still be afflicted with the underlying disorder.

[0043] In the present disclosure, the terms "prevent" and "prevention" are used interchangeably and refer to methods of obtaining a beneficial or desired result, including but not limited to prophylactic benefits. To obtain a "prophylactic benefit", a conjugate, an RNAi reagent, or a composition may 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 if a diagnosis of the disease has not yet been made.

[0044] In the present disclosure, the term "administer" generally refers to introducing a pharmaceutical formulation 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 bringing a cell, an organ, or a tissue into contact with the drug may be employed. The administration may include, but is not limited to, intravenous, intra-arterial, intranasal, intraperitoneal, intramuscular, subcutaneous, or oral. The daily dose may be divided into one, two, or more appropriate forms of doses for administration at one, two, or more times during a certain period of time.

[0045] As used in the present disclosure, the term "regulate gene expression" means that the expression of a gene, or the level of an RNA molecule or an 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 a modulator. For example, the term "regulate" may mean "inhibit", but the use of the word "regulate" is not limited to this definition.

[0046] In addition to any conventional excipients, the scope of those that are incompatible with the siRNA of the present disclosure, such as any adverse biological effects produced or interactions with any other components of a pharmaceutically acceptable composition in a harmful manner, their uses are also within the scope contemplated by the present disclosure.

[0047] Double-stranded oligonucleotide

[0048] In a first aspect of the present disclosure, the present disclosure provides a double-stranded oligonucleotide comprising a sense strand and an antisense strand, each of the sense strand and the antisense strand having 17-35 nucleotides, and the sense strand and the antisense strand being at least partially reverse complementary to form a duplex region; the sense strand and / or the antisense strand comprises at least one nucleotide modified with a 2'-Se(CH2) n CH3 group.

[0049] In some alternative embodiments of the present disclosure, n is an integer selected from 0 to 3.

[0050] Further, in some alternative embodiments of the present disclosure, n is selected from 0 or 1.

[0051] In some alternative embodiments of the present disclosure, the duplex region comprises 17-23 pairs of nucleotides; further, it comprises 19-21 pairs of nucleotides.

[0052] In some alternative embodiments of the present disclosure, the antisense strand of the double-stranded region is reverse complementary or substantially reverse complementary to the sense strand nucleotide sequence; the substantially reverse complementary means that the number of nucleotide mismatches in the double-stranded region does not exceed three.

[0053] In some alternative embodiments of the present disclosure, the nucleotide modified with 2'-Se(CH2) n CH3 group is located on the antisense strand; specifically, in the direction from the 5'-end to the 3'-end, it is located at at least one of the remaining positions other than the 1st to 2nd positions of the antisense strand.

[0054] In some alternative embodiments of the present disclosure, the nucleotide modified with 2'-Se(CH2) n CH3 group is located on the sense strand.

[0055] In some alternative embodiments of the present disclosure, each nucleotide in the duplex of the double-stranded oligonucleotide is modified; in the direction from the 5'-end to the 3'-end, at least two nucleotides among the 7th to 10th positions in the sense strand are nucleotides modified with 2'-fluoro, and the nucleotides at the remaining positions are nucleotides modified with 2'-O-methyl and / or 2'-Se(CH2) n CH3 group; and, the antisense strand contains nucleotides modified with 2'-O-methyl, and at least one selected from 2'-Se(CH2) n CH3, 2'-O-MOE modified nucleotides;

[0056] In some alternative embodiments of the present disclosure, at least three nucleotides among the 7th to 10th positions in the sense strand are nucleotides modified with 2'-fluoro.

[0057] In some alternative embodiments of the present disclosure, there is a 2'-deoxy modified nucleotide among the 7th to 10th positions in the sense strand.

[0058] In some alternative embodiments of the present disclosure, each nucleotide in the duplex of the double-stranded oligonucleotide has any one of the following modification methods 1)-4):

[0059] 1) In the direction from the 5'-end to the 3'-end, the 7th to 9th positions in the sense strand are nucleotides modified with 2'-fluoro, and the nucleotides at the remaining positions are nucleotides modified with 2'-O-methyl; the 2nd, 6th, 14th, and 16th positions of the antisense strand are nucleotides modified with 2'-fluoro, and there is a nucleotide modified with 2'-Se(CH2) n CH3 modification among the 3rd to 19th positions of the nucleotides, and the nucleotides at the remaining positions are nucleotides modified with 2'-O-methyl;

[0060] 2) In the 5'-terminal to 3'-terminal direction, the 7th and 9th positions in the sense strand are 2'-fluoro-modified nucleotides, the 8th position is a 2'-deoxy-modified nucleotide, and the nucleotides at the remaining positions are 2'-O-methyl-modified nucleotides; the 2nd, 6th, 14th, and 16th positions in the antisense strand are 2'-fluoro-modified nucleotides, and there is one 2'-Se(CH2) n CH3-modified nucleotide among the nucleotides at the 4th - 7th positions, and the nucleotides at the remaining positions are 2'-O-methyl-modified nucleotides;

[0061] 3) In the 5'-terminal to 3'-terminal direction, the 7th - 10th positions in the sense strand are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl-modified nucleotides; the 2nd, 6th, 9th, 14th, and 16th positions in the antisense strand are 2'-fluoro-modified nucleotides, and there is at least one 2'-Se(CH2) n CH3-modified nucleotide among the nucleotides at the 3rd - 8th positions, and the nucleotides at the remaining positions are 2'-O-methyl-modified nucleotides;

[0062] Optionally, the 2'-Se(CH2) n CH3-modified nucleotide in the antisense strand is present at the 3rd position, or the 4th position, or the 5th position, or the 7th position;

[0063] 4) In the 5'-terminal to 3'-terminal direction, the sense strand contains at least two 2'-Se(CH2) n CH3-modified nucleotides, and the 7th - 10th positions are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl-modified nucleotides; at least four of the 2nd, 6th, 9th, 14th, and 16th positions in the antisense strand are 2'-fluoro-modified nucleotides, the 15th position is a 2'-O-MOE-modified nucleotide, and the nucleotides at the remaining positions are 2'-O-methyl-modified nucleotides;

[0064] Optionally, in the sense strand, in the 5'-terminal to 3'-terminal direction, the positions of the 2'-Se(CH2) n CH3-modified nucleotides are selected from the following: the 4th - 6th positions, or the 11th - 13th positions, or the 18th - 19th positions, or the 15th - 19th positions.

[0065] In some alternative embodiments of the present disclosure, the 3'-terminal and / or 5'-terminal of the sense strand and the antisense strand comprise one or more overhang regions, optionally, the overhang region contains 1 - 3 nucleotides;

[0066] In some alternative embodiments of the present disclosure, the sense strand and the antisense strand contain 1 - 3 phosphorothioate groups;

[0067] In some alternative embodiments of the present disclosure, the thiophosphate group is present at at least one of the following positions:

[0068] 1) between the 1st and 3rd nucleotides at the 5'-end of the sense strand;

[0069] 2) between the 0th and 3rd nucleotides at the 3'-end of the sense strand;

[0070] 3) between the 1st and 3rd nucleotides at the 5'-end of the antisense strand;

[0071] 4) between the 1st and 3rd nucleotides at the 3'-end of the antisense strand.

[0072] Oligonucleotide conjugate

[0073] In a second aspect of the present disclosure, the present disclosure provides an oligonucleotide conjugate, which comprises the double-stranded oligonucleotide described in the first aspect of the present disclosure.

[0074] Further, the oligonucleotide conjugate further comprises one or more ligands capable of binding to a cell surface receptor.

[0075] The ligand described in the present disclosure refers to a targeting ligand, and at least one of the targeting ligands can bind to one or more cell receptors, cell channels, and / or cell transporters capable of promoting endocytosis.

[0076] In some alternative embodiments of the present disclosure, the ligand is selected from asialoglycoprotein receptor ligands containing galactosamine derivatives;

[0077] In some alternative embodiments of the present disclosure, the galactosamine derivative is selected from at least one of N-acetylgalactosamine (GalNAc), galactose, galactosamine, N-formylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine, N-isobutyrylgalactosamine, macrocycles, folic acid molecules, fatty acids, bile acids, cholesterol and its derivatives.

[0078] Further, the galactosamine derivative is selected from galactosamine, N-formylgalactosamine, N-acetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine or N-isobutyrylgalactosamine.

[0079] In some alternative embodiments of the present disclosure, the number of the ligands is one, and it is conjugated to the 3'-end of the sense strand of the double-stranded oligonucleotide.

[0080] Composition

[0081] In a third aspect of the present disclosure, the present disclosure provides a composition, which comprises the double-stranded oligonucleotide described in the first aspect of the present disclosure or the double-stranded oligonucleotide conjugate described in the second aspect of the present disclosure.

[0082] In some alternative embodiments of the present disclosure, the composition further comprises one or more pharmaceutically acceptable carriers or excipients.

[0083] The pharmaceutically acceptable carriers or excipients described in the present disclosure are well-known to those skilled in the art and can be selected and used by conventional methods, meaning non-toxic materials that do not interfere with the biological activity and effectiveness of the active ingredient.

[0084] Use

[0085] In a fourth aspect of the present disclosure, the present disclosure provides the use of the double-stranded oligonucleotide described in the first aspect of the present disclosure, the double-stranded oligonucleotide conjugate described in the second aspect of the present disclosure, or the composition described in the third aspect of the present disclosure in the preparation of a medicament for treating and / or preventing a disease or disorder associated with dysregulation of the mRNA level of a target gene expression.

[0086] Kit

[0087] In a fifth aspect of the present disclosure, the present disclosure provides a kit, which comprises the double-stranded oligonucleotide described in the first aspect of the present disclosure, the double-stranded oligonucleotide conjugate described in the second aspect of the present disclosure, or the composition described in the third aspect of the present disclosure.

[0088] Method for regulating gene expression in target cells

[0089] In a sixth aspect of the present disclosure, the present disclosure provides a method for reducing gene expression in a target cell, the method comprising: contacting the target cell with the double-stranded oligonucleotide described in the first aspect of the present disclosure, the double-stranded oligonucleotide conjugate described in the second aspect of the present disclosure, or the composition described in the third aspect of the present disclosure.

[0090] Method for reducing the expression of a target gene in a subject

[0091] In a seventh aspect of the present disclosure, the present disclosure provides a method for reducing the expression of a target gene in a subject, the method comprising: administering to the subject the double-stranded oligonucleotide described in the first aspect of the present disclosure, the double-stranded oligonucleotide conjugate described in the second aspect of the present disclosure, or the composition described in the third aspect of the present disclosure.

[0092] The dual oligonucleotides, double-stranded oligonucleotide conjugates or compositions thereof of the present disclosure can be administered in a formulation, and the formulation can be administered as a pharmaceutically acceptable solution, which can conventionally contain pharmaceutically acceptable concentrations of salts, buffers, preservatives, compatible carriers, excipients and optionally other therapeutic ingredients. According to the method of the present disclosure, it can be administered as a pharmaceutical composition. Generally, the pharmaceutical composition contains the double-stranded oligonucleotides, double-stranded oligonucleotide conjugates described in the present disclosure and a pharmaceutically acceptable carrier or excipient.

[0093] Method for preventing and / or treating diseases

[0094] In the eighth aspect of the present disclosure, the present disclosure provides a method for preventing and / or treating a disease or disorder associated with the dysregulation of the mRNA level of a target gene in a target cell, the method comprising: administering to a subject a pharmaceutically acceptable dose of the double-stranded oligonucleotide described in the first aspect of the present disclosure, the double-stranded oligonucleotide conjugate described in the second aspect of the present disclosure, or the composition described in the third aspect of the present disclosure.

[0095] In some embodiments of the present disclosure, the subject is selected from humans. Specific examples

[0097] Unless otherwise specified, the reagent ratios described in the examples of the present disclosure are calculated by volume ratio (v / v).

[0098] Unless otherwise specified, the reagents, reagent consumables and instruments used in the present disclosure are all commercially available. Among them, the main reagent consumables are shown in Table 1, and the main instruments are shown in Table 2.

[0099] Table 1 Main reagent consumables

[0100]

[0101] Table 2 Main instruments

[0102]

[0103]

[0104] Nucleoside analog

[0105] Synthesis of compound NM011 in Preparation Example 1

[0106]

[0107] In this preparation example, the synthesis route of compound NM011 is as follows:

[0108]

[0109] (1-1) Synthesis of Compound NM011-02

[0110] Add NM011-01 (20 g, 82 mmol, 1.0 eq, CAS No. 58-96-8, uridine), diphenyl carbonate (19.3 g, 90 mmol, 1.1 eq, CAS No. 102-09-0), sodium bicarbonate (720 mg, 3.6%, 8.8 eq), and N,N-dimethylformamide (200 mL, abbreviated as DMF) into a 500 mL four-necked flask. Replace the air with nitrogen three times, heat up to 120 °C and stir at 120 °C for 4 h. Monitor the reaction by LCMS until it is completed. Filter the reaction system by suction, wash the filter cake with H2O (100 mL), and dry it under vacuum to obtain Compound NM011-02 in the form of a white solid (11.5 g, yield 62.1%). MS ESI (m / z) = 227.0 [M+H] + .

[0111] (1-2) Synthesis of Compound NM011-03

[0112] Add NM011-02 (11.2 g, 49.5 mmol, 1.0 eq) and pyridine (110 mL) into a 250 mL reactor. Cool down to 0 °C and add 4,4'-dimethoxytriphenylmethyl chloride (16.8 g, 49.5 mmol, 1.0 eq, CAS No. 40615-36-9, abbreviated as DMTrCl) in three batches at 0 °C. React at 25 °C for 12 h. Monitor the reaction by LCMS until it is completed. Add 400 mL of saturated sodium bicarbonate aqueous solution into the reaction system, extract with dichloromethane three times (400 mL each time), combine the organic phases, wash the organic phase once with saturated sodium chloride aqueous solution, dry it with anhydrous sodium sulfate, filter, concentrate, and purify by normal-phase column chromatography (eluent: dichloromethane / methanol = 10 / 1 (the eluent contains 1% by volume of triethylamine), volume ratio v / v) to obtain Compound NM011-03 (18.3 g, yield 69.8%). MS ESI (m / z) = 529.0 [M+H] + .

[0113] (1-3) Synthesis of Compound NM011-04

[0114] Sodium borohydride (6.9 g, 181.8 mmol, 6.0 eq) was added to 230 ml of anhydrous tetrahydrofuran. Nitrogen was displaced 7 - 8 times. Dimethyldiselenide (11.5 g, 61.17 mmol, 2.0 eq, CAS No. 7101 - 31 - 7) was added dropwise. After the addition of dimethyldiselenide was completed, anhydrous ethanol (16 ml) was added. The reaction was carried out at 25 °C for 1 h under a nitrogen atmosphere. Then, a solution of NM1 - 03 (16 g, 30.3 mmol, 1.0 eq) in tetrahydrofuran (320 ml) was added dropwise. After that, the reaction was carried out at 25 °C for 2 h under a nitrogen atmosphere, and the reaction was monitored by LCMS until completion. 65 ml of purified water was added to the reaction system, and the pH of the reaction system was adjusted to 7 with an aqueous solution of 20 vol% acetic acid. The mixture was concentrated, 500 ml of purified water was added, and the mixture was extracted three times with ethyl acetate (500 ml each time). The organic phase was combined; the organic phase was washed once with a saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered and concentrated, and purified by normal-phase column chromatography to obtain compound NM011 - 04 (14.5 g, yield 76.7%). MS ESI (m / z) = 625.0 [M + H] + .

[0115] (1 - 4) Synthesis of compound NM011

[0116] NM - 011 - 04 (7 g, 11.2 mmol, 1.0 eq) and 4,5 - dicyanoimidazole (1.72 g, 14.6 mmol, 1.3 eq) were dissolved in 70 ml of dichloromethane. Nitrogen was displaced three times. A solution of bis(diisopropylamino)(2 - cyanoethoxy)phosphine (5.1 g, 16.8 mmol, 1.5 eq, CAS No. 102691 - 36 - 1) in dichloromethane was added dropwise at 0 °C. The reaction was carried out at 25 °C for 2 h under a nitrogen atmosphere, and the reaction was monitored by LCMS until completion. 300 ml of saturated sodium bicarbonate aqueous solution was added to the reaction system, and the mixture was extracted twice with dichloromethane (300 ml each time). The organic phases were combined; the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, and purified by normal-phase column chromatography (eluent: heptane / ethyl acetate = 1 / 1 (the eluent contained 1 vol% triethylamine), v / v) to obtain compound NM011 as a white foamy solid (4 g, yield 43.3%). MS ESI (m / z) = 824.0 [M + H] + .

[0117] 11H NMR (400 MHz, Chloroform-d) δ 9.53 - 8.74 (s, 1H), 7.97 - 7.74 (d, J = 8.2 Hz, 1H), 7.43 - 7.26 (m, 9H), 6.97 - 6.76 (m, 4H), 6.42 - 6.28 (d, J = 6.9 Hz, 1H), 5.46 - 5.15 (dd, J = 22.1, 8.1 Hz, 1H), 4.87 - 4.43 (dddd, J = 17.3, 9.5, 5.7, 3.4 Hz, 1H), 4.39 - 4.19 (dq, J = 23.4, 2.8 Hz, 1H), 4.08 - 3.91 (m, 1H), 3.88 - 3.77 (d, J = 2.3 Hz, 6H), 3.74 - 3.39 (m, 6H), 2.15 - 2.04 (d, J = 15.7 Hz, 3H), 2.03 - 1.99 (s, 1H), 1.30 - 1.16 (m, 10H), 1.10 - 0.99 (d, J = 6.8 Hz, 3H).

[0118] Synthesis of compound NM055 in Preparation Example 2

[0119]

[0120] In this preparation example, the synthesis route of compound NM055 is as follows:

[0121]

[0122] (2 - 1) Synthesis of compound NM055 - 02

[0123] Dissolve compound NM055 - 01 (10 g, 37.44 mmol, 1 eq, CAS No. 58 - 61 - 7, adenosine) in pyridine (100 mL), add 1,3 - dichloro - 1,1,3,3 - tetraisopropyldisiloxane (14.2 g, 44.93 mmol, 1.2 eq, CAS No. 69304 - 37 - 6), displace with nitrogen three times, and stir at 0 °C for 6 hours in a nitrogen atmosphere. After the reaction is completed, concentrate the reaction solution to remove the solvent, add water (300 mL) for dilution, extract with ethyl acetate (3 × 200 mL), combine the organic phases, wash the organic phase with saturated ammonium chloride aqueous solution (2 × 100 mL) and saturated sodium chloride aqueous solution (2 × 100 mL), dry over anhydrous sodium sulfate, filter and concentrate, and purify by normal - phase column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 1, v / v) to obtain compound NM055 - 02 as a white solid (15.6 g, yield 81.8%). MS ESI (m / z) = 510.3 [M + H] + .

[0124] (2 - 2) Synthesis of compound NM055 - 03

[0125] At 25 °C, dissolve compound NM055-02 (15.6 g, 30.63 mmol, 1 eq) in dichloromethane (300 mL), add 4-dimethylaminopyridine (11.2 g, 91.89 mmol, 3 eq, CAS No. 1122-58-3, abbreviated as DMAP), cool to 0 °C in an ice bath, and add N-phenylbis(trifluoromethanesulfonyl)imide (13.13 g, 36.76 mmol, 1.2 eq, CAS No. 37595-74-7, PhNTf2) portionwise at 0 °C. Replace the air with nitrogen three times, and stir the reaction solution at 0 °C in a nitrogen atmosphere for 2 hours. After the reaction is complete, add water (300 mL) to the reaction solution to quench it, extract with dichloromethane (3 × 150 mL), and combine the organic phases; wash the organic phase with saturated aqueous sodium bicarbonate solution (2 × 200 mL) and saturated aqueous sodium chloride solution (2 × 100 mL), dry over anhydrous sodium sulfate, filter, concentrate, and purify by normal-phase column chromatography (eluent: petroleum ether / ethyl acetate = 3 / 1, v / v) to obtain compound NM055-03 (13.3 g, yield 67.72%) as a yellow solid. MS ESI (m / z) = 642.5 [M+H] + .

[0126] (2-3) Synthesis of compound NM055-04

[0127] At 25 °C, dissolve dimethyldiselenide (1.64 g, 8.72 mmol, 0.8 eq) in ethanol (14 mL), cool to 0 °C in an ice bath, add sodium borohydride (619 mg, 16.37 mmol, 1.5 eq) at 0 °C, and dropwise add a solution of compound NM055-03 (7 g, 10.91 mmol, 1 eq) in tetrahydrofuran (42 mL). Replace the air with nitrogen 3 times, heat to 70 °C, and stir the reaction solution at 70 °C in a nitrogen atmosphere for 3.5 hours. After the reaction is complete, add water (200 mL) to the reaction solution to dilute it, extract with ethyl acetate (3 × 100 mL), and combine the organic phases; wash the organic phase with saturated aqueous sodium bicarbonate solution (2 × 80 mL) and saturated aqueous sodium chloride solution (100 mL), dry over anhydrous sodium sulfate, filter, concentrate, and purify by normal-phase column chromatography (eluent: petroleum ether / ethyl acetate = 3 / 1, v / v) to obtain compound NM055-04 (3.2 g, yield 49.92%) as a yellow solid. MS ESI (m / z) = 588.4 [M+H] + .

[0128] (2-4) Synthesis of compound NM055-05

[0129] At 25 °C, compound NM055-04 (2.9 g, 4.94 mmol, 1 eq) was dissolved in dichloromethane (30 mL). N,N-Diisopropylethylamine (2.55 g, 19.76 mmol, 4 eq, CAS No. 7087-68-5, DIPEA) and 4-dimethylaminopyridine (301.7 mg, 2.47 mmol, 0.5 eq, CAS No. 1122-58-3, DMAP) were added. The temperature was lowered to 0 °C in an ice bath, and benzoyl chloride (2.08 g, 14.82 mmol, 3 eq, CAS No. 98-88-4) was added at 0 °C. The reaction mixture was purged with nitrogen three times and stirred at 25 °C for 2 hours under a nitrogen atmosphere. After completion of the reaction, the reaction mixture was washed with saturated aqueous sodium bicarbonate solution (2 × 50 mL) and saturated aqueous sodium chloride solution (2 × 100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. It was purified by normal-phase column chromatography (eluent: petroleum ether / ethyl acetate = 4 / 1, v / v) to obtain compound NM055-05 (3.1 g) as a yellow solid. MS ESI (m / z) = 691.9 [M+H] + .

[0130] (2-5) Synthesis of compound NM055-06

[0131] At 25 °C, compound NM055-05 (3.1 g, 4.48 mmol, 1 eq) was dissolved in tetrahydrofuran (30 mL). A solution of tetrabutylammonium fluoride (CAS No. 429-41-4) in tetrahydrofuran (21.9 mL, 19.92 mmol, 4 eq) was added, and the reaction mixture was stirred at 25 °C for 16 hours. After completion of the reaction, the solvent was removed by rotary evaporation, and it was purified by normal-phase column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 1, v / v) to obtain compound NM055-06 (1.3 g, yield 57.78%) as a yellow solid. MS ESI (m / z) = 450.3 [M+H] + .

[0132] (2-6) Synthesis of compound NM055-07

[0133] At 25 °C, compound NM055-06 (1.1 g, 2.44 mmol, 1 eq) was dissolved in pyridine (25 mL). The temperature was lowered to 0 °C in an ice bath, and DMTrCl (1.41 g, 4.15 mmol, 1.7 eq) was added portionwise at 0 °C. The reaction mixture was stirred at 0 °C for 3 hours. After completion of the reaction, methanol was added to quench the reaction. The solvent was removed by rotary evaporation, and the residue was diluted with ethyl acetate (50 mL). The mixture was washed with aqueous sodium bicarbonate solution (2 × 20 mL) and saturated sodium chloride solution (2 × 10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. Purification by normal-phase column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 1, v / v) gave compound NM055-07 as a white solid (1.62 g, yield 88.04%). MS ESI (m / z) = 751.1 [M+H] + .

[0134] (2-7) Synthesis of compound NM055

[0135] Compound NM055-07 (1.5 g, 2.00 mmol, 1 eq), which had been dried three times with acetonitrile (3 × 20 mL), was dissolved in dichloromethane (15 mL). A solution of bis(diisopropylamino)(2-cyanoethoxy)phosphine (723.8 mg, 2.40 mmol, 1.2 eq, CAS No. 102691-36-1) in dichloromethane (15 mL), which had been dried three times with acetonitrile (3 × 20 mL), was added. 1H-Imidazole-4,5-dicarbonitrile (188.5 mg, 1.60 mmol, 0.8 eq, CAS No.: 1122-28-7, abbreviated as DCI) was added. The reaction mixture was purged with nitrogen three times and stirred at 25 °C in a nitrogen atmosphere for 1 hour. After completion of the reaction, the reaction mixture was diluted with saturated aqueous sodium bicarbonate solution (30 mL) and extracted with dichloromethane (3 × 20 mL). The organic phases were combined, washed with saturated sodium chloride solution (2 × 30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. Purification by reverse-phase column chromatography (eluent: water / acetonitrile = 7 / 93, v / v) gave compound NM055 as a white solid (1.17 g, yield 61.58%). MS ESI (m / z) = 952.3 [M+H] + .

[0136] 11H NMR (400 MHz, DMSO-d6) δ 11.26 (s, 1H), 8.71 (d, J = 2.4 Hz, 1H), 8.63 (d, J = 4.8 Hz, 1H), 8.05 (d, J = 7.4 Hz, 2H), 7.65 (t, J = 7.4 Hz, 1H), 7.56 (t, J = 7.6 Hz, 2H), 7.40 (t, J = 8.4 Hz, 2H), 7.25 (dtd, J = 15.0, 7.9, 7.0, 3.8 Hz, 7H), 6.89 - 6.80 (m, 4H), 6.40 (dd, J = 15.1, 8.3 Hz, 1H), 4.68 - 4.49 (m, 2H), 4.30 (s, 1H), 3.86 (dd, J = 7.3, 3.5 Hz, 1H), 3.73 (d, J = 2.5 Hz, 6H), 3.70 - 3.53 (m, 3H), 3.36 (dd, J = 11.0, 6.8 Hz, 1H), 3.29 (dd, J = 10.9, 5.0 Hz, 1H), 2.78 (t, J = 5.9 Hz, 1H), 2.59 (t, J = 5.9 Hz, 1H), 1.71 (s, 1H), 1.62 (s, 1H), 1.17 (t, J = 7.1 Hz, 10H), 1.06 (d, J = 6.7 Hz, 3H).

[0137] Synthesis of compound NM056 in Preparation Example 3

[0138]

[0139] In this preparation example, the synthetic route of compound NM056 is as follows:

[0140]

[0141] (3-1) Synthesis of compound NM056-02

[0142] Dissolve N2-isobutyrylguanosine monohydrate (10 g, 0.0283 mol, 1.0 eq, CAS No. 64350-24-9) dried three times with pyridine (3 × 100 ml) in pyridine (100 ml), add 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (10.7 g, 0.034 mol, 1.2 eq, CAS No. 69304-37-6), displace with nitrogen 3 times, stir the reaction solution at 25 °C in a nitrogen atmosphere for 3 hours, and monitor the completion of the reaction by HPLC. Concentrate the reaction solution, add purified water (200 ml), extract with ethyl acetate (200 ml) 3 times, combine the organic phases, dry the organic phase with anhydrous sodium sulfate, filter and concentrate, and purify by column chromatography to obtain compound NM056-02 (15 g, yield 88.9%). MS ESI (m / z) = 596.0 [M+H]+ .

[0143] (3-2) Synthesis of Compound NM056-03

[0144] Dissolve Compound NM056-02 (13 g, 0.0218 mol, 1.0 eq) and DMAP (8.0 g, 0.0655 mol, 3.0 eq) in dichloromethane (260 ml), cool to 0 °C, and add N-phenylbis(trifluoromethanesulfonyl)imide (9.36 g, 0.0262 mol, 1.2 eq, CAS No. 37595-74-7) portionwise at 0 °C. Replace the air with nitrogen three times, stir the reaction system at 0 °C under a nitrogen atmosphere for 2 hours, and monitor the end of the reaction by LCMS. Add water (200 ml) to the reaction solution, separate the layers to obtain the organic phase. The organic phase is dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain Compound NM056-03 (6 g, yield 37.8%). MS ESI (m / z) = 728.0 [M+H] + .

[0145] (3-3) Synthesis of Compound NM056-04

[0146] Dissolve Compound NM056-03 (2.5 g, 0.0034 mol, 1.0 eq) and potassium trifluoroacetate (2.6 g, 0.0171 mol, 5.0 eq, CAS No. 2923-16-2) in toluene (125 ml), add 18-crown-6 (1.8 g, 0.0068 mol, 2.0 eq, CAS No. 17455-13-9) and N,N-diisopropylethylamine (0.66 g, 0.0228 mol, 8.2 eq, CAS No. 7087-68-5, abbreviated as DIEA). Replace the air with nitrogen three times, heat to 80 °C, stir the reaction system at 80 °C under a nitrogen atmosphere for 16 hours, and monitor the end of the reaction by LCMS. Cool the reaction system, add purified water (200 ml), separate the layers to obtain the organic phase. The organic phase is dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain Compound NM056-04 (1.3 g, yield 63.4%). MS ESI (m / z) = 596.0 [M+H] + .

[0147] (3-4) Synthesis of Compound NM056-05

[0148] Dissolve compound NM056-04 (1.3 g, 0.0022 mol, 1.0 eq) and DMAP (0.8 g, 0.0067 mol, 3.0 eq) in dichloromethane (26 ml). Cool the reaction system to 0 °C, and add N-phenylbis(trifluoromethanesulfonyl)imide (0.94 g, 0.0026 mol, 1.2 eq, CAS No. 37595-74-7) portionwise at 0 °C. Replace the gas with nitrogen three times, stir the reaction system at 0 °C under a nitrogen atmosphere for 2 hours, and monitor the end of the reaction by LCMS. Add water (20 ml) to the reaction solution, separate the organic phase, dry the organic phase with anhydrous sodium sulfate, filter, concentrate, and purify by column chromatography to obtain compound NM056-05 (1.5 g, yield 94.3%). MS ESI (m / z) = 728.0 [M+H] + .

[0149] (3-5) Synthesis of compound NM056-06

[0150] At 0 °C, add a solution of sodium borohydride (127 mg, 0.0034 mol, 1.5 eq) in ethanol (3 ml) and a solution of compound NM056-05 (1.5 g, 0.002 mol, 1.0 eq) in tetrahydrofuran (9 ml, CAS No. 109-99-9, THF) to dimethyldiselenide (310 mg, 0.0017 mol, 0.85 eq). Replace the gas with nitrogen three times, heat to 70 °C, stir the reaction system at 70 °C under a nitrogen atmosphere for 3.5 hours, and monitor the end of the reaction by LCMS. Cool the reaction system to 25 °C, concentrate the reaction solution, add H2O (20 ml) and ethyl acetate (30 ml) for extraction, separate the organic phase, dry the organic phase with anhydrous sodium sulfate, filter, concentrate, and purify by column chromatography to obtain compound NM056-06 (0.8 g, yield 57.5%). MS ESI (m / z) = 673.0 [M+H] + .

[0151] (3-6) Synthesis of compound NM056-07

[0152] At 25 °C, dissolve compound NM056-06 (0.8 g, 0.0012 mol, 1.0 eq) in THF (8 ml), add a solution of tetrabutylammonium fluoride (1.43 ml, 0.0014 mol, 1.2 eq) in tetrahydrofuran. Replace the gas with nitrogen three times, stir the reaction system at 25 °C under a nitrogen atmosphere for 2 hours, and monitor the end of the reaction by LCMS. Add H2O (20 ml) and ethyl acetate (20 ml) to the reaction solution for extraction, separate the organic phase, dry the organic phase with anhydrous sodium sulfate, filter, and concentrate to obtain compound NM056-07 (500 mg, yield 97.4%). MS ESI (m / z) = 432.0 [M+H]+ .

[0153] (3-7) Synthesis of Compound NM056-08

[0154] At 25 °C, compound NM056-07 (500 mg, 1.16 mmol, 1.0 eq) was dissolved in pyridine (5 ml), cooled to 0 °C, and 4,4'-dimethoxytriphenylmethyl chloride (412 mg, 1.3 mmol, 1.1 eq) was added at 0 °C. The reaction system was purged with nitrogen three times and stirred at 0 °C in a nitrogen atmosphere for 2 hours. The reaction was monitored by LCMS until completion. The reaction solution was concentrated and purified by reverse-phase column chromatography to obtain compound NM056-08 (500 mg, yield 58.9%). MS ESI (m / z) = 734.0 [M+H] + .

[0155] (3-8) Synthesis of Compound NM056

[0156] At 25 °C, compound 8 (0.5 g, 0.0007 mol, 1.0 eq) was dissolved in dichloromethane (10 ml), and bis(diisopropylamino)(2-cyanoethoxy)phosphine (247 mg, 0.0008 mol, 1.1 eq) and 4,5-dicyanoimidazole (64 mg, 0.0006 mol, 0.8 eq) were added. The reaction system was purged with nitrogen three times and stirred at 25 °C in a nitrogen atmosphere for 3 hours. The reaction was monitored by LCMS until completion. An aqueous solution of 10% by mass sodium bicarbonate was added to the reaction solution, and the organic phase was separated. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reverse-phase column chromatography to obtain compound NM056 (460 mg, yield 72.3%). MS ESI (m / z) = 934.0 [M+H] + .

[0157] 11H NMR (400 MHz, DMSO-d6) δ 12.31 - 11.96 (s, 1H), 11.78 - 11.35 (s, 1H), 8.24 - 8.18 (d, J = 3.6 Hz, 1H), 7.41 - 7.32 (m, 2H), 7.31 - 7.18 (m, 7H), 6.88 - 6.78 (dt, J = 8.8, 6.9 Hz, 4H), 6.23 - 6.13 (dd, J = 12.6, 8.4 Hz, 1H), 4.44 - 4.19 (m, 3H), 3.85 - 3.79 (d, J = 6.6 Hz, 1H), 3.76 - 3.71 (d, J = 2.0 Hz, 6H), 3.69 - 3.52 (dddd, J = 24.3, 14.1, 8.0, 4.4 Hz, 3H), 3.42 - 3.33 (dd, J = 10.3, 7.0 Hz, 1H), 3.21 - 3.13 (dd, J = 10.2, 3.8 Hz, 1H), 2.80 - 2.72 (m, 2H), 2.60 - 2.53 (t, J = 5.9 Hz, 1H), 1.74 - 1.65 (d, J = 17.7 Hz, 3H), 1.18 - 1.10 (dd, J = 8.7, 6.7 Hz, 18H).

[0158] Synthesis of compound NM057 in Preparation Example 4

[0159]

[0160] In this preparation example, the synthetic route of compound NM057 is as follows:

[0161]

[0162] (4 - 1) Synthesis of compound NM057 - 02

[0163] At 25 °C, dissolve compound NM057 - 01 (10 g, 0.0412 mol, 1.0 eq, CAS No. 147 - 94 - 4, cytarabine) in pyridine (150 ml), add trimethylchlorosilane (26.6 g, 0.247 mol, 6.0 eq, CAS No. 75 - 77 - 4), and stir the reaction system at 25 °C for 1 hour; cool down to 0 °C, add benzoyl chloride (6.9 g, 0.0494 mol, 1.2 eq), displace with nitrogen 3 times, stir the reaction system at 0 °C in a nitrogen atmosphere for 6 hours, monitor the end of the reaction by HPLC, and add H2O to quench the reaction to the reaction system. Add ammonia water (80 ml) to the reaction solution and stir for 15 minutes, concentrate, filter, wash the filter cake with water, and dry it under vacuum to obtain compound NM057 - 02 (10 g, yield 69.8%). MS ESI (m / z) = 348.0 [M + H] + .

[0164] (4-2) Synthesis of Compound NM057-03

[0165] At 25 °C, compound NM057-02 (10 g, 0.0288 mol, 1.0 eq) was dissolved in pyridine (100 ml), and 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (10.7 g, 0.034 mol, 1.2 eq, CAS No. 69304-37-6) was added. After purging with nitrogen three times, the reaction system was stirred at 25 °C under a nitrogen atmosphere for 3 hours, and the reaction was monitored by HPLC until completion. The reaction solution was concentrated, purified water (200 ml) was added, and the mixture was extracted with ethyl acetate three times (200 ml each time). The combined organic phases were dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound NM057-03 (8 g, yield 47.2%). MS ESI (m / z) = 590.0 [M+H] + .

[0166] (4-3) Synthesis of Compound NM057-04

[0167] Compound NM057-03 (8 g, 0.0136 mol, 1.0 eq) and DMAP (4.97 g, 0.407 mol, 3.0 eq) were dissolved in dichloromethane (160 ml). The temperature was lowered to 0 °C, and N-phenylbis(trifluoromethanesulfonyl)imide (5.82 g, 0.0163 mol, 1.2 eq) was added portionwise at 0 °C. After purging with nitrogen three times, the reaction system was stirred at 0 °C under a nitrogen atmosphere for 2 hours, and the reaction was monitored by LCMS until completion. H2O (200 ml) was added to the reaction solution for extraction, and the organic phase was separated. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound NM057-04 (8.3 g, yield 84.8%). MS ESI (m / z) = 722.0 [M+H] + .

[0168] (4-4) Synthesis of Compound NM057-05

[0169] At 0 °C, a solution of sodium borohydride (0.65 g, 0.0172 mol, 1.5 eq) in ethanol (16 ml) and a solution of compound NM057-04 (8.3 g, 0.0115 mol, 1.0 eq) in THF (50 ml) were added to dimethyldiselenide (1.73 g, 0.0092 mol, 0.8 eq). The system was purged with nitrogen three times, then heated to 70 °C, and the reaction mixture was stirred at 70 °C under a nitrogen atmosphere for 3.5 hours. The reaction was monitored by LCMS until completion. The reaction system was cooled to 25 °C, the reaction solution was concentrated, H2O (800 ml) and ethyl acetate (800 ml) were added for extraction. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound NM057-05 (2.8 g, yield 36.5%). MS ESI (m / z) = 667.0 [M+H] + .

[0170] (4-5) Synthesis of compound NM057-06

[0171] At 25 °C, compound NM057-05 (2.8 g, 0.0042 mol, 1.0 eq) was dissolved in THF (28 ml), and a solution of tetrabutylammonium fluoride (5.0 ml, 0.005 mol, 1.2 eq) in tetrahydrofuran was added. The system was purged with nitrogen three times, and the reaction mixture was stirred at 25 °C under a nitrogen atmosphere for 2 hours. The reaction was monitored by LCMS until completion. H2O (50 ml) and ethyl acetate (50 ml) were added to the reaction solution for extraction. The organic phase was separated, dried over anhydrous sodium sulfate, filtered, concentrated, to obtain compound NM057-06 (1.78 g, yield 98%). MS ESI (m / z) = 425.0 [M+H] + .

[0172] (4-6) Synthesis of compound NM057-07

[0173] At 25 °C, compound NM057-06 (1.78 g, 0.0042 mol, 1.0 eq) was dissolved in pyridine (18 ml), cooled to 0 °C, and 4,4'-dimethoxytriphenylmethyl chloride (1.48 mg, 0.0044 mol, 1.05 eq) was added at 0 °C. The system was purged with nitrogen three times, and the reaction mixture was stirred at 0 °C under a nitrogen atmosphere for 2 hours. The reaction was monitored by LCMS until completion. The reaction solution was concentrated and purified by reverse-phase column chromatography to obtain compound NM057-07 (1.5 g, yield 49.2%). MS ESI (m / z) = 727.0 [M+H] + .

[0174] (4-7) Synthesis of compound NM057

[0175] At 25 °C, compound NM057-07 (1.5 g, 0.0021 mol, 1.0 eq) was dissolved in dichloromethane (30 ml), bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.94 g, 0.0031 mol, 1.5 eq) and 4,5-dicyanoimidazole (195 mg, 0.0017 mol, 0.8 eq) were added. After displacing the air with nitrogen three times, the reaction system was stirred at 25 °C under a nitrogen atmosphere for 3 hours, and the reaction was monitored by LCMS until completion. An aqueous solution of 10% by mass sodium bicarbonate was added to the reaction solution, and the organic phase was separated. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reverse-phase column chromatography to obtain NM057 (1.5 g, yield 78.9%). MS ESI (m / z) = 927.0 [M+H] + .

[0176] 1 H NMR (400 MHz, DMSO-d6) δ 11.40 - 11.24 (s, 1H), 8.31 - 8.23 (t, J = 7.5 Hz, 1H), 8.04 - 7.97 (m, 2H), 7.67 - 7.59 (d, J = 7.4 Hz, 1H), 7.56 - 7.48 (t, J = 7.7 Hz, 2H), 7.46 - 7.38 (t, J = 7.0 Hz, 2H), 7.38 - 7.19 (m, 8H), 6.95 - 6.87 (dd, J = 8.7, 4.6 Hz, 4H), 6.35 - 6.28 (m, 1H), 4.57 - 4.43 (ddt, J = 15.5, 10.2, 5.3 Hz, 1H), 4.30 - 4.18 (dq, J = 16.4, 4.2 Hz, 1H), 3.91 - 3.73 (m, 8H), 3.69 - 3.46 (m, 3H), 3.44 - 3.32 (d, J = 4.5 Hz, 2H), 2.80 - 2.73 (t, J = 5.9 Hz, 1H), 2.60 - 2.53 (d, J = 1.7 Hz, 1H), 2.09 - 2.06 (s, 2H), 2.05 - 2.02 (s, 1H), 2.00 - 1.95 (s, 1H), 1.22 - 1.07 (dd, J = 9.0, 6.7 Hz, 9H), 1.02 - 0.96 (d, J = 6.7 Hz, 3H).

[0177] Preparation Example 5: Preparation of Compound CR01008 and Compound CR01008Z

[0178] (5-1) Synthesis of compound CR01008

[0179] In this preparation example, the synthesis route of compound CR01008 is as follows:

[0180]

[0181] (5-1-1) Synthesis of Compound CR01008-02

[0182] Dissolve Compound CR01008-01 (trans-4-(Boc-amino)cyclohexanecarbaldehyde, 10.0 g, 1.0 eq) and aqueous formaldehyde solution (8.9 g, 37% by mass, 2.4 eq) in 33 ml of methanol. Dropwise add 13 ml of an aqueous KOH solution with a concentration of 45.3% by mass. After the addition is complete, stir the reaction at 25 °C for 30 minutes, then raise the temperature to 60 °C and reflux the reaction at 60 °C for 2 hours. After the reaction is completed, allow the reaction solution to cool to room temperature and then evaporate the reaction solution to dryness under reduced pressure to obtain a crude product in the form of a white solid. Add a small amount of water to the crude product for pulping, filter, and obtain Compound CR01008-02 in the form of a white solid (9 g, yield 78.9%). MS-ESI (m / z) = 260 [M+H] + .

[0183] (5-1-2) Synthesis of Compound CR01008-03

[0184] Dissolve Compound CR01008-02 (9 g, 1 eq) in 70 ml of 1,4-dioxane, add a 1,4-dioxane solution of hydrogen chloride (45 ml, 4 M), and stir the reaction at 25 °C for 1 hour. After the reaction is completed, evaporate the reaction solution to dryness under reduced pressure to obtain Compound CR01008-03 in the form of a white solid (6.8 g, yield 100%).

[0185] (5-1-3) Synthesis of Compound CR01008-05

[0186] Dissolve Compound CR01008-03 (1.8 g, 2.0 eq), Compound CR01008-04 (5-[[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)-2-tetrahydropyranyl]oxy]pentanoic acid, 2.1 g, 1.0 eq) and N,N-diisopropylethylamine (3.5 g, 6.0 eq, abbreviated as DIEA) in 15 ml of DMF, add HBTU (1.9 g, 1.1 eq), and stir the reaction at 25 °C for 3 hours under a nitrogen atmosphere. After the reaction is completed, evaporate the reaction solution to dryness under reduced pressure and purify by reverse-phase column chromatography (eluent: acetonitrile / water = 22 / 78, v / v) to obtain Compound 5 in the form of a white solid (1.78 g, yield 64.4%). MS-ESI (m / z) = 589 [M+H] + .

[0187] (5-1-4) Synthesis of Compound CR01008-06

[0188] Dissolve compound CR01008-05 (1.54 g, 1.0 eq) in 15 ml of pyridine. Cool the reaction system to 0 °C in an ice-water bath and add 4,4'-dimethoxytrityl chloride (1.32 g, 1.5 eq, abbreviated as DMTrCl, CAS No. 40615-36-9) at 0 °C. React at 25 °C for 3 hours. Add 15 ml of methanol to the reaction solution to quench the reaction. After the reaction is completed, evaporate the reaction solution to dryness under reduced pressure and purify by reverse-phase column chromatography (eluent: acetonitrile / water = 60 / 40, v / v) to obtain compound CR01008-06 in the form of a yellow solid (1 g, yield 42.7%). MS-ESI (m / z) = 891 [M+H] + .

[0189] (5-1-5) Synthesis of compound CR01008

[0190] Dissolve compound CR01008-06 (1.08 g, 1.0 eq) in 20 ml of anhydrous dichloromethane. Add DCI (115 mg, 0.8 eq) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (732 mg, 2.1 eq) respectively. Replace the nitrogen 3 times and stir and react at 25 °C for 2 hours. After the reaction is completed, add 20 ml of saturated sodium bicarbonate aqueous solution to the reaction solution, extract with 20 ml of dichloromethane 3 times (3×20 ml), combine the organic phases, evaporate the organic phases to dryness under reduced pressure, and purify by reverse-phase column chromatography (eluent: acetonitrile / water = 72 / 28, v / v) to obtain compound CR01008 in the form of a white powder (1 g, yield 76.0%). MS-ESI (m / z) = 1091 [M+Na] + .

[0191] 11H NMR (400 MHz, DMSO-d6) δ 1.05 (d, J = 6.7 Hz, 6H), 1.14 (d, J = 6.7 Hz, 6H), 1.37 - 1.17 (m, 5H), 1.60 - 1.40 (m, 6H), 1.68 - 1.62 (m, 1H), 1.80 (s, 3H), 1.80 (s, 3H), 1.92 (s, 3H), 2.02 (s, 5H), 2.13 (s, 3H), 2.71 (t, J = 5.9 Hz, 2H), 2.79 (d, J = 8.4 Hz, 1H), 2.87 (d, J = 8.4 Hz, 1H), 3.36 (s, 1H), 3.58 - 3.39 (m, 3H), 3.69 - 3.60 (m, 2H), 3.75 (s, 7H), 3.90 (dt, J = 11.2, 8.8 Hz, 1H), 4.05 (s, 3H), 4.51 (d, J = 8.4 Hz, 1H), 4.99 (dd, J = 11.3, 3.4 Hz, 1H), 5.24 (d, J = 3.4 Hz, 1H), 5.78 (s, 1H), 6.93 - 6.87 (m, 4H), 7.35 - 7.21 (m, 7H), 7.44 - 7.37 (m, 2H), 7.66 (d, J = 7.8 Hz, 1H), 7.84 (d, J = 9.2 Hz, 1H).

[0192] (5 - 2) Synthesis of Compound CR01008Z

[0193] In this preparation example, the synthetic route of compound CR01008Z is as follows:

[0194]

[0195] Among them, represents amino CPG, purchased from Beijing Coupling Technology Co., Ltd. CPG represents Controlled Pore Glass support.

[0196] (5 - 2 - 1) Synthesis of Compound CR01008 - 07

[0197] Dissolve compound CR01008 - 06 (500 mg) in 10 ml of dichloromethane, add succinic anhydride (112 mg), DMAP (6.8 mg) and triethylamine (226.2 mg), displace with nitrogen 3 times, stir and react at 25 °C for 16 hours, and purify by flash to obtain compound CR01008 - 07 (300 mg, yield 53.6%). MS - ESI (m / z) = 1013 [M + Na]+.

[0198] (5 - 2 - 2) Synthesis of Compound CR01008Z

[0199] Add compound CR01008-07 (50 mg), amino CPG (1.25 g, 80 μmol / g, 0.1 mmol, English name Aminoalkyl-CPG, model C3006-1000), benzotriazol-N,N,N',N'-tetramethyluronium hexafluorophosphate (27 mg, abbreviated as HBTU, CAS number 94790-37-1), and N,N-diisopropylethylamine (12 mg, abbreviated as DIEA, CAS number 7087-68-5) to a 20 ml sample vial, and react on a shaker for 16 hours. After the reaction is completed, filter the reaction solution to obtain a filter cake. Wash the filter cake with 10 ml of acetonitrile once (1×10 ml), and then dry it under vacuum. Add the dried filter cake, DMAP (3 mg), Cap1 (10 ml, 20 V), and Cap2 (1 ml, 20 V) to a 20 ml sample vial, and react on a shaker for 6 hours. After the reaction is completed, filter the reaction solution to obtain a filter cake. Wash the filter cake with 10 ml of acetonitrile once (1×10 ml), and then dry it under vacuum to obtain compound CR01008Z (1.03 g, loading amount 20-30 μmol / g).

[0200] Among them, Cap1 and Cap2 are capping reagents. Cap1 is a pyridine / acetonitrile mixed solution of 20% by volume of N-methylimidazole, and the volume ratio of pyridine to acetonitrile is 3:5; Cap2 is an acetonitrile solution of 20% by volume of acetic anhydride.

[0201] siRNA conjugate

[0202] Unless otherwise specified, the siRNA sequences used in this disclosure are all synthesized by Suzhou Beixin Biotechnology Co., Ltd.

[0203] Synthesis of siRNA conjugate conjugated with L96 in Preparation Example 6

[0204] Compound L96-PS is purchased from Kelaiying Pharmaceutical Group (Tianjin) Co., Ltd., and the loading amount is 120±12 μmol / g (the detection method is: UV / HPLC). The structural formula of compound L96-PS is shown as follows:

[0205]

[0206] Among them, PS represents a polystyrene resin solid-phase carrier.

[0207] (6-1) Synthesis of sense strand (SS)

[0208] A method by the phosphoramidite nucleic acid solid-phase synthesis method, starting the cycle with the compound L96-PS or the compound CR01008Z linked to a solid support, and successively linking nucleoside monomers one by one in the 3'-5' direction of the nucleotide sequence (during the synthesis process, the compound CR01018 is regarded as a nucleoside monomer). Each linking of a nucleoside monomer includes four reaction steps: deprotection, coupling, capping, and oxidation or sulfurization. The synthesis conditions are given as follows:

[0209] Prepare an acetonitrile solution of nucleoside monomers with a concentration of 0.1 M.

[0210] The conditions for each deprotection reaction are the same. The conditions for the deprotection reaction are: temperature is 25 °C, reaction time is 70 seconds, the deprotection reagent is a dichloromethane solution of dichloroacetic acid (3 vol%), and the molar ratio of dichloroacetic acid to the 4,4'-dimethoxytrityl protecting group on the solid support is 5:1.

[0211] The conditions for each coupling reaction are the same. The conditions for the coupling reaction are: temperature is 25 °C, the molar ratio of the nucleic acid sequence linked to the solid support to the nucleoside monomer is 1:10, the molar ratio of the nucleic acid sequence linked to the solid support and the coupling reagent is 1:65, reaction time is 600 seconds, the coupling reagent is an acetonitrile solution of 5-ethylthio-1H-tetrazole with a concentration of 0.5 M, and the sulfurizing reagent is an acetonitrile / pyridine mixed solution (volume ratio of acetonitrile to pyridine is 1:1) of hydrogen xanthate with a concentration of 0.2 M.

[0212] The conditions for each capping reaction are the same. The conditions for the capping reaction are: temperature is 25 °C; reaction time is 2 minutes; the capping reagent solution is a mixed solution of Cap1 and Cap2 with a molar ratio of 1:1, Cap1 is a pyridine / acetonitrile mixed solution of N-methylimidazole with a concentration of 20 vol%, the volume ratio of pyridine to acetonitrile is 3:5, Cap2 is an acetonitrile solution of acetic anhydride with a concentration of 20 vol%; the molar ratio of N-methylimidazole in Cap1 capping reagent, acetic anhydride in Cap2 capping reagent to the nucleic acid sequence linked to the solid support is 1:1:1.

[0213] The conditions for each oxidation reaction are the same. The conditions for the oxidation reaction are: temperature is 25 °C; reaction time is 3 seconds; the concentration of the oxidation reagent is 0.05 M iodine water, and the molar ratio of iodine to the nucleic acid sequence linked to the solid support in the coupling reaction is 30:1; the oxidation reaction is carried out in a water / pyridine mixed solvent (volume ratio of water to pyridine is 1:9). The conditions for the sulfurization reaction are: temperature is 25 °C; reaction time is 360 seconds; the concentration of the sulfurizing reagent is 0.2 M hydrogen xanthate pyridine solution, and the molar ratio of the sulfurizing reagent to the nucleic acid sequence linked to the solid support in the coupling reaction is 4:1; the sulfurization reaction is carried out in a water / pyridine mixed solvent (volume ratio of water to pyridine is 1:9).

[0214] After the last nucleotide monomer is connected, the nucleic acid sequence connected to the solid support is successively cleaved, deprotected, purified, desalted, and then lyophilized to obtain the sense strand, where:

[0215] The cleavage and deprotection conditions are as follows: The synthesized nucleotide sequence connected to the solid support is added to ammonia water with a concentration of 25% by mass, and the amount of ammonia water used is 0.5 ml / μmol. The reaction is carried out at 55 °C for 16 hours, the solvent is removed, and it is concentrated to dryness under vacuum. After the ammonia water treatment, relative to the amount of single-stranded nucleic acid, the product is dissolved in 0.4 ml / μmol of N-methylpyrrolidone, and then 0.3 ml / μmol of triethylamine and 0.6 ml / μmol of triethylamine trihydrofluoride are added to remove the 2'-O-TBDMS protection on the ribose.

[0216] The conditions for purification and desalting: 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), and the solvent is a water / acetonitrile mixed solution (the volume ratio of water to acetonitrile is 9:1); Eluent 2 is 1.5 M sodium chloride, 20 mM sodium phosphate (pH = 8.1), and the solvent is a water / acetonitrile mixed solution (the volume ratio of water to acetonitrile is 9:1); The eluate is Eluent 1:Eluent 2 = (100:0)-(50:50). After collecting and combining the product eluate, reverse chromatography purification columns are used for desalting. The desalting conditions include desalting using a Sephadex column, and the packing material is Sephadex G25, and it is eluted with deionized water.

[0217] Detection: Ion exchange chromatography (IEX-HPLC) is used for purity detection; a liquid chromatography-mass spectrometer (LC-MS, Liquid Chromatography-Mass Spectrometry, purchased from Waters, model: LCT Premier) is used for molecular weight detection. The measured value of the molecular weight is compared with the theoretical value. If the measured value and the theoretical value are consistent, it indicates that the sense strand of the siRNA is obtained.

[0218] In this step, three clusters of CR01008 are synthesized during the synthesis of the sense strand, denoted as (CR01008)×3 or (CR01008×3).

[0219] The structural formulas of the three clusters of CR01008 are as follows:

[0220]

[0221] (6-2) Synthesis of the antisense strand (AS)

[0222] The antisense strand was synthesized using a general solid-phase support. The conditions and steps for deprotection, coupling, capping, oxidation or sulfurization reactions, cleavage and deprotection, purification and desalting in the solid-phase synthesis method of the antisense strand were the same as those for synthesizing the sense strand in step (6-1).

[0223] Detection: Purity detection was performed using ion exchange chromatography (IEX-HPLC); molecular weight detection was performed using liquid chromatography-mass spectrometry (LC-MS). The measured value of the molecular weight was compared with the theoretical value. If the measured value and the theoretical value were consistent, it indicated that the antisense strand of siRNA was obtained.

[0224] (6-3) Synthesis of siRNA conjugate

[0225] The sense strand synthesized in step (6-1) and the antisense strand synthesized in step (6-2) were mixed in an equimolar ratio, dissolved in water for injection, and heated to 95 °C. Then, it was slowly cooled to room temperature and kept at room temperature for 10 minutes to allow the sense strand and the antisense strand to form a double-stranded structure through hydrogen bonds, thereby obtaining an siRNA conjugate with the sense strand and antisense strand shown in Table 3. Among them, the unmodified nucleotide sequence information for forming the siRNA conjugate is shown in Table 3a.

[0226] Among them, the structural formula of the siRNA conjugate with the L96 vector conjugated to the 3'-end of the sense strand is as follows:[[]]END]]

[0227]

[0228] Among them, represents siRNA. L96 is conjugated to the 3'-end of the sense strand of siRNA through a phosphodiester bond.

[0229] The structural formula of the siRNA conjugate with the (CR01008)×3 vector conjugated to the 3'-end of the sense strand is as follows:[[]]END]]

[0230] Among them, when the ligand is three clusters of CR01008, the structural formula of the siRNA conjugate is as follows:[[]]END]]

[0231]

[0232] Among them, represents siRNA. The (CR01018×3) vector is conjugated to the 3'-end of the sense strand of siRNA.

[0233] Table 3a Unmodified nucleotide sequence information for forming the siRNA conjugate

[0234]

[0235]

[0236]

[0237] Table 3 Sequence Information of siRNA Conjugates

[0238]

[0239]

[0240]

[0241]

[0242] Unless otherwise specified, the base composition and modification meanings described in each embodiment of the present disclosure are as follows: The capital letters A, U, G, C, and T represent the base composition of nucleotides. The lowercase letter m represents that the nucleotide adjacent to the left of the letter m is a nucleotide with a 2'-methyl modification (alias 2'-methoxy); the lowercase letter f represents that the nucleotide adjacent to the left of the letter f is a nucleotide with a 2'-fluoro modification; the lowercase letter d represents that the nucleotide adjacent to the left of the letter d is a nucleotide with a 2'-deoxy modification; (moe) represents that the nucleotide adjacent to the left of the combination identifier (moe) is a nucleotide with a 2'-O-methoxyethyl (alias 2'-O-MOE) modification; (NM011) represents that the nucleotide at this site is replaced by a nucleotide analog (NM011); (NM055) represents that the nucleotide at this site is replaced by a nucleotide analog (NM055); (NM056) represents that the nucleotide at this site is replaced by a nucleotide analog (NM056); (NM057) represents that the nucleotide at this site is replaced by a nucleotide analog (NM057); the lowercase letter s represents that a phosphorothioate diester bond is connected between the two nucleotides adjacent to the left and right of the letter s.

[0243] The structural formula of a 2'-O-methyl modified nucleotide is

[0244] The structural formula of a 2'-fluoro modified nucleotide is

[0245] The structural formula of a 2'-deoxy modified nucleotide is

[0246] The structural formula of a 2'-O-MOE modified nucleotide is

[0247] Among them, Base represents the base of the nucleotide, such as uracil U, thymine T, cytosine C, adenine A, or guanine G.

[0248] The structural formula of the nucleotide analog (NM011) is It is formed after the nucleoside analog NM011 participates in the synthesis of siRNA.

[0249] The structural formula of the nucleotide analogue (NM055) is It is formed after the nucleoside analogue NM055 participates in the synthesis of siRNA.

[0250] The structural formula of the nucleotide analogue (NM056) is It is formed after the nucleoside analogue NM056 participates in the synthesis of siRNA.

[0251] The structural formula of the nucleotide analogue (NM057) is It is formed after the nucleoside analogue NM057 participates in the synthesis of siRNA.

[0252] Table 4 Detection results of siRNA conjugates

[0253]

[0254]

[0255] It can be seen from the data in Table 4 that the present disclosure has siRNA conjugates with the sense strand and antisense strand in Table 3, and the siRNA conjugates can maintain a high purity.

[0256] Biological detection experiment

[0257] Unless otherwise specified, all PCR primers used in the present disclosure were synthesized by Beijing Tsingke Biotechnology Co., Ltd.

[0258] Unless otherwise specified, all experimental animals C57BL / 6J mice used in the present disclosure were purchased from Beijing Specific Pathogen Free Biotechnology Co., Ltd.; all experimental animals ICR female mice used in the present disclosure were purchased from Beijing Specific Pathogen Free Biotechnology Co., Ltd.; hREN×hAGT hypertensive mice were purchased from Cyagen Biosciences (Suzhou) Co., Ltd.; healthy cynomolgus monkeys were purchased from Guangzhou Blue Island Biotechnology Co., Ltd.

[0259] Evaluation method for the inhibitory activity of siRNA conjugates against target genes

[0260] Preparation of test samples:

[0261] After centrifuging each of the above siRNA conjugate test samples, an appropriate amount of PBS was added for dissolution according to the specifications of each tube to prepare a 20 μM stock solution, and then the stock solution was further serially diluted with PBS to prepare a working solution with a final test concentration 100 times that of the siRNA.

[0262] 96-well transfection and detection:

[0263] Digest the Huh7 or HepG2 cells that have grown to near confluence with trypsin, wash the cells to prepare a cell suspension, add 100 μL of the cell suspension to each well of a 96-well plate, with 12,000 cells per well, and place it in an incubator at 37 °C and 5% CO2 for culture. When the cells have adhered for 24 h, aspirate the DMEM medium in the 96-well plate, and add 80 μL of Opti-MEM TM medium to each well. Then, place the 96-well plate back into the incubator for continued culture; Disperse the working solution in 9 μL of Opti-MEM to form an siRNA mixture, and disperse 0.3 μL of RNAiMAX in 9.7 μL of Opti-MEM. Mix them with each of the aforementioned siRNA mixtures respectively to form a test group transfection complex. At the same time, set up a control group (Mock) transfection complex by dispersing 0.3 μL of RNAiMAX in 9.7 μL of Opti-MEM and then mixing it with 10 μL of Opti-MEM. Incubate the transfection complex at room temperature for 10 minutes, and then add the transfection complex to the 96-well plate, 20 μL per well. After culturing for 4 h, supplement each well with 100 μL of DMEM medium containing 20% FBS, and place the 96-well plate back into the incubator for continued culture for 24 h.

[0264] Take out the 96-well plate, and use an automatic nucleic acid extractor (purchased from Zhejiang Hanwei Technology Co., Ltd.) and a nucleic acid extraction kit (purchased from Zhejiang Hanwei Technology Co., Ltd., GO-MNTR-100) to extract total RNA according to the standard operating procedures for total RNA extraction.

[0265] Use a reverse transcription kit (Thermo Fisher Scientific, RevertAid First Strand cDNA Synthesis Kit, K1622) and select the Oligo(dT)18 reverse transcription primer. Configure a 20 μL reverse transcription system and complete the reverse transcription reaction according to the method described in the reverse transcription kit instructions. Then, use a real-time fluorescence quantitative PCR kit (Thermo Fisher Scientific, TaqMan Fast Advanced Master Mix, 4444557) to detect the expression level of the target gene mRNA in Huh7 or HepG2 cells on a fluorescence quantitative PCR instrument (Bio-Rad CFX Opus 384). In this real-time fluorescence quantitative PCR method, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene is used as an internal reference gene, and primers specific for the target gene and primers specific for the GAPDH internal reference gene are used to detect the target gene and the GAPDH internal reference gene respectively.

[0266] Example 1. Inhibitory activity evaluation of Se nucleic acid single-site modified siRNA conjugate on target gene complement component 3 (complement component 3, CC3) on HepG2 cells

[0267] In this example, an siRNA conjugate was used to evaluate the inhibitory activity of target genes on cell lines. The siRNA conjugates RZ002044 to RZ002049 with NM011 substitutions at different positions of the antisense strand were evaluated against the siRNA conjugate RZ002006 without NM011, the siRNA conjugates RZ002061 to RZ002065 with NM011 substitutions against the siRNA conjugate RZ002003 without NM011, the siRNA conjugates RZ002077 to RZ002080 with NM011 substitutions against the siRNA conjugate RZ002031 without NM011, and the siRNA conjugates RZ002093 to RZ002098 with NM011 substitutions against the siRNA conjugate RZ002011 without NM011 for their inhibitory activity on the target gene CC3 in the HepG2 cell line.

[0268] Table 5 Primer sequence information in Example 1

[0269]

[0270] The results of Example 1 showed that at concentrations of 5 nM and 1 nM, compared with the control conjugate RZ002006, the antisense strand 3-position modified conjugate RZ002044 and the antisense strand 7-position modified conjugate RZ002046 had comparable or better inhibitory activity; the antisense strand 8-position modified conjugate RZ002047, the antisense strand 11-position modified conjugate RZ002048, and the antisense strand 17-position modified conjugate RZ002049 had comparable inhibitory activity compared with the control conjugate RZ002003 ( Figure 1 , Table 6).

[0271] Compared with the control conjugate RZ002003, the antisense strand 4-position modified conjugate RZ002061 and the antisense strand 7-position modified conjugate RZ002062 had better inhibitory activity; the antisense strand 12-position modified conjugate RZ002063 had comparable inhibitory activity ( Figure 2 , Table 7).

[0272] Compared with the control conjugate RZ002031, the antisense strand 3-position modified conjugate RZ002077, the antisense strand 5-position modified conjugate RZ002078, and the antisense strand 7-position modified conjugate RZ002079 had better inhibitory activity; the antisense strand 15-position modified conjugate RZ002080 had comparable activity to the control conjugate RZ002031 at 5 nM and 0.5 nM ( Figure 3 , Table 8).

[0273] Compared with the control conjugate RZ002011, the antisense strand 3-position modified conjugate RZ002093, the antisense strand 7-position modified conjugate RZ002095, and the antisense strand 18-position modified conjugate RZ002098 have better inhibitory activity( Figure 4 , Table 9).

[0274] Based on the results of Example 1, it can be found that NM011 has little effect on the activity at most sites of the antisense strand. At some sites in the 3-9 positions of the antisense strand, comparable or better inhibitory activity can be achieved. For example, the antisense strand 3-position modified conjugate RZ002077, the antisense strand 3-position modified conjugate RZ002093, the antisense strand 4-position modified conjugate RZ002061, the antisense strand 7-position modified conjugate RZ002062, and the antisense strand 7-position modified conjugate RZ002095 sequences all have better inhibitory activity than the control conjugate; the antisense strand 5-position and 8-position modified conjugates have comparable inhibitory activity compared with the control conjugate. In addition, the activity is basically comparable after modification at positions 11, 12, 15, 17, and 18 compared with the control sequence.

[0275] Table 6 Inhibitory activity of the target gene on the HepG2 cell line after administration of siRNA conjugate

[0276]

[0277] Table 7 Inhibitory activity of the target gene on the HepG2 cell line after administration of siRNA conjugate

[0278]

[0279] Table 8 Inhibitory activity of the target gene on the HepG2 cell line after administration of siRNA conjugate

[0280]

[0281] Table 9 Inhibitory activity of the target gene on the HepG2 cell line after administration of siRNA conjugate

[0282]

[0283] Example 2. Inhibitory activity evaluation of Se nucleic acid single-site modified siRNA conjugate on target gene angiotensinogen (angiotensinogen, AGT) on Huh7 cells

[0284] In this example, a method for evaluating the inhibitory activity of siRNA conjugates on target genes in cell lines was used to evaluate the siRNA conjugates RZ003033 to RZ003037 in which the U base at different positions of the antisense strand was replaced by NM011, the siRNA conjugate RZ003017 without NM011, the siRNA conjugates RZ003047 to RZ003049 after NM011 substitution, the siRNA conjugate RZ003020 without NM011, and the siRNA conjugates RZ003058 to RZ003061 after NM011 substitution and the siRNA conjugate RZ003021 without NM011 for their inhibitory activity on the target gene AGT in the Huh7 cell line.

[0285] Table 10 Primer sequence information in Example 2

[0286]

[0287] The results of Example 2 showed that at concentrations of 1 nM and 0.1 nM, compared with the control RZ003017, the conjugate RZ003033 modified at the 4th position of the antisense strand, the conjugate RZ003035 modified at the 7th position of the antisense strand, and the conjugate RZ003036 modified at the 9th position of the antisense strand had comparable or better inhibitory activities; the conjugate RZ003037 modified at the 10th position of the antisense strand had comparable inhibitory activity compared with the control RZ003017 ( Figure 5 , Table 11).

[0288] Compared with the control RZ003020, the conjugate RZ003047 modified at the 8th position of the antisense strand, the conjugate RZ003048 modified at the 11th position of the antisense strand, and the conjugate RZ003049 modified at the 12th position of the antisense strand had comparable inhibitory activities ( Figure 6 , Table 12).

[0289] Compared with the control RZ003021, the conjugate RZ003058 modified at the 13th position of the antisense strand and the conjugate RZ003059 modified at the 15th position of the antisense strand had comparable inhibitory activities; the conjugate RZ003061 modified at the 19th position of the antisense strand was basically comparable to the control RZ003021 at a concentration of 1 nM and had slightly weaker inhibitory activity at a concentration of 0.1 nM ( Figure 7 , Table 13).

[0290] Based on the results of Comprehensive Example 2, it can be found that NM011 has good tolerance at most sites on the antisense strand of another target AGT sequence. Modification at some sites from positions 3 to 9 on the antisense strand can improve the activity to a certain extent. For example, the conjugate RZ003033 modified at position 4 of the antisense strand, the conjugate RZ003035 modified at position 7 of the antisense strand, and the conjugate modified at position 9 of the antisense strand have equivalent activity to the control at 1 nM, but show better inhibitory activity under the condition of 0.1 nM. Modification at positions 10, 11, 12, 13, 15, and 19 of the antisense strand has basically equivalent activity compared with the control.

[0291] Table 11 Relative expression levels of target genes on the Huh7 cell line after administration of siRNA conjugates

[0292]

[0293] Table 12 Inhibitory activities of target genes on the Huh7 cell line after administration of siRNA conjugates

[0294]

[0295] Table 13 Inhibitory activities of target genes on the Huh7 cell line after administration of siRNA conjugates

[0296]

[0297]

[0298] Evaluation method for the inhibitory activity of siRNA conjugates on target genes in primary mouse liver

[0299] Isolation of primary mouse hepatocytes:

[0300] Primary mouse hepatocytes were extracted from the fresh liver tissue of C56BL / 6J mice. The specific operation steps were as follows: The mice were anesthetized by intraperitoneal injection of 10% chloral hydrate solution, fixed, and their abdomen and chest were disinfected with 75% ethanol. The surgical instruments were sterilized, and the abdominal cavity was opened to expose the hepatic portal vein and the inferior vena cava. A heparin cap was installed on the indwelling needle, and the scalp needle connected to the infusion pump drip bottle (0.5 mM EDTA HBSS perfusion solution) was inserted. The needle was inserted through the inferior vena cava, and perfusion was carried out at a rate of 120 drops / min. The hepatic portal vein was cut open to allow the perfusion solution to flow out from the cut hepatic portal vein. Perfusion was carried out for 4 minutes, and then 0.8 mg / mL type IV collagenase HBSS solution (Sigma, C5138) (containing 0.08% DNAse I (sigma, DN25)) was used for continuous perfusion for 8 minutes. The perfused liver was removed from the animal and rinsed with HBSS (containing Ca 2+ 、Mg 2+, MACGENE, CC016) Wash the liver, place it in a sterile petri dish, add complete DMEM medium (DMEM medium + 10% serum), tear the liver into pieces, filter the cell suspension through a cell sieve to remove undigested tissues and connective tissues, centrifuge at 800 rpm for 3 min, discard the supernatant, add complete DMEM medium again to suspend and centrifuge to obtain primary mouse hepatocytes.

[0301] Cell culture and transfection:

[0302] Add complete DMEM medium to adjust the cell density to 2×10^5 cells / mL to obtain a suspension of primary mouse hepatocytes. Subsequently, inoculate the cells into a 12-well culture plate pre-coated with type I rat tail collagen (coating method according to the instructions of solarbio (C8062) at a concentration of 2 μg / cm 2 Coat), and the volume of the added cell suspension is 1000 μL / well, that is, the cell amount is 2×10^5 cells / well.

[0303] Dilute each group of conjugates with PBS to a working solution with a final test concentration 500 times that of siRNA. Add 2 μL / well of the siRNA conjugate working solution to the above 12-well culture plate, and set 2 culture wells for each siRNA conjugate. Additionally, add 2 μL / well of PBS to 2 - 3 culture wells as blank control wells. Shake the culture plate to mix evenly. Place the culture plate in a cell culture incubator at 37°C and 5% CO2 and continue to culture for 24 h.

[0304] mRNA expression level detection:

[0305] RNA extraction: Use the fully automatic nucleic acid extractor and nucleic acid extraction kit of Zhejiang Hanwei Technology Co., Ltd. to extract the total RNA of each group of primary hepatocyte samples according to the method described in the instructions. Reverse transcription reaction: Take 1000 ng of the total RNA of the extracted primary hepatocyte samples respectively, use the reverse transcription kit (Reverse Transcription System, A3500) of Promega Corporation and select Oligo(dT)15 reverse transcription primers, configure a 20 μL reverse transcription system according to the method described in the kit instructions and complete the reverse transcription reaction. After the reaction, add 80 μL of RNase-Free water to the reverse transcription system to obtain a cDNA solution for Real-time PCR detection. Real-time PCR detection: Use SYBR of ABI TMSelect Master Mix (4472908) reagent was used to prepare a 20 μL Real-time PCR reaction system for each PCR detection well according to the method described in the kit instructions. Each detection system contained 5 μL of the cDNA template obtained from the above reverse transcription reaction, 10 μL of SYBR TM Select Master Mix, 0.5 μL of 10 μM upstream primer, 0.5 μL of 10 μM downstream primer, and 4 μL of RNase-Free H2O. The prepared reaction system was placed on an ABI StepOnePlus PCR instrument, and Real-time PCR amplification was performed using the three-step method. The amplification program was pre-denaturation at 95 °C for 10 min, then denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s. The process of denaturation, annealing, and extension was repeated 40 cycles. After the program was completed, the gene expression difference was calculated by the ΔΔCt method.

[0306] Example 3. Inhibitory activity evaluation of Se nucleic acid multi-site modified siRNA conjugate on target gene superoxide dismutase 1 (Superoxide dismutase1, SOD1) on primary mouse hepatocytes

[0307] In this example, an evaluation method for the inhibitory activity of target genes on primary mouse hepatocytes was used to evaluate the inhibitory activity of siRNA conjugates RZ599063 to RZ599070 with Se nucleic acid-modified bases NM011, NM055, NM056, and NM057 substituted at multiple different consecutive sites of the sense or antisense strand and siRNA conjugate RZ599062 without Se nucleic acid-modified bases on the target gene SOD1.

[0308] Operations such as isolation and cell culture of primary mouse hepatocytes were as shown before. Each group of double-stranded siRNA conjugates was serially diluted with PBS to a working solution of 5 μM (calculated as siRNA). 2 μL / well of the working solution of each concentration of siRNA conjugate was added to the above 12-well culture plate, corresponding to a final transfection concentration of 10 nM of the siRNA conjugate (calculated as siRNA). Two culture wells were set for each concentration of siRNA. Additionally, 2 μL / well of PBS was added to another three culture wells as blank control wells. The culture plate was shaken to mix evenly. The cells were further cultured in a 37 °C, 5% CO2 cell culture incubator for 24 h.

[0309] Table 14 Primer sequence information in Example 3

[0310]

[0311] The results of Example 3 showed that under the concentration condition of 10 nM, the activities of the sense and antisense strands with different sites of Se nucleic acid modified base substitutions were basically equivalent to those of the control siRNA conjugate RZ599062, indicating that continuous Se nucleic acid modified base substitutions at most sites of the siRNA conjugate double strand were tolerated. Among them, after continuous Se nucleic acid modified base substitutions at positions 11 to 13 and 15 to 19 of the sense strand, the activity was slightly better than that of the control siRNA conjugate sequence (RZ599062)( Figure 8 , Table 15).

[0312] Table 15 Inhibitory activity of target genes on primary mouse liver after administration of siRNA conjugates

[0313] Group Average % inhibition rate ± STDEV value PBS 0.00 3.30 RZ599062 94.40 0.04 RZ599063 91.56 0.17 RZ599064 84.38 0.38 RZ599065 71.55 2.70 RZ599066 91.41 0.21 RZ599067 93.96 0.48 RZ599068 94.58 0.15 RZ599069 95.37 0.28 RZ599070 95.18 0.44

[0314] Evaluation method for the inhibitory activity of siRNA conjugates on target genes in mice

[0315] C57BL / 6J mice aged 6 - 8 weeks were randomly grouped by body weight (all female). The drug dose for each mouse in each group was calculated based on body weight, and a single dose was administered by subcutaneous injection in the abdomen. Each siRNA conjugate was prepared into a corresponding concentration (calculated based on siRNA) solution with PBS solution for administration, and the administration volume was 5 mL (calculated based on siRNA) / kg (calculated based on mice). The PBS control group was given 5 mL / kg (calculated based on mice) of PBS solution (without drug conjugate). The administration day was recorded as day 0 (recorded as D0). At the preset time after administration, 5 mice in each group were sacrificed. Each sacrificed mouse was dissected grossly and the liver tissue of each sacrificed mouse was collected. The liver tissue was cut into small pieces about 2 mm 3 and stored with RNA Later.

[0316] Liver tissue samples at different time points in different experimental groups were taken from the above RNA later. The liver tissue samples were disrupted in a Tissuelyser II type fully automatic tissue homogenizer for 60 s, and then total RNA was extracted using a fully automatic nucleic acid extractor (purchased from Zhejiang Hanwei Technology Co., Ltd.) and a nucleic acid extraction kit (purchased from Zhejiang Hanwei Technology Co., Ltd.) according to the standard operating procedures for total RNA extraction.

[0317] The detection of mRNA expression level was as shown in the evaluation method for the inhibitory activity of siRNA conjugates on target genes in primary mouse liver.

[0318] In the above real - time fluorescence quantitative PCR method, the ΔΔCt method was used to relatively quantitatively calculate the expression level and inhibition rate of the target gene mRNA in each test group. The calculation method was as follows:

[0319] ΔCt (test group) = Ct (target gene of test group) - Ct (housekeeping gene of test group)

[0320] ΔCt (control group) = Ct (target gene of control group) - Ct (housekeeping gene of control group)

[0321] ΔΔCt (test group) = ΔCt (test group) - ΔCt (average of control group)

[0322] ΔΔCt (control group) = ΔCt (control group) - ΔCt (average of control group)

[0323] Among them, ΔCt (average of control group) is the arithmetic mean of ΔCt (control group) of 5 mice sacrificed at the same time point in the control group. Therefore, each mouse in the test group and the control group corresponds to a ΔΔCt value.

[0324] Relative expression level of target gene mRNA in test group = 2 -ΔΔCt (test group) × 100%

[0325] Taking the control group as a reference, the mRNA expression level of the target gene in the test group was normalized, and the mRNA expression level of the target gene in the control group was defined as 100%.

[0326] Inhibitory rate of target gene mRNA expression in test group = (1 - relative expression level of target gene mRNA in test group) × 100%

[0327] Unless otherwise specified, the activity experimental data are all in shown, and the experimental data are all plotted and analyzed using GraphPad Prism 8.0 software.

[0328] Example 4. Inhibitory activity evaluation of siRNA conjugate on target gene CC3 in mice

[0329] In this example, a method for evaluating the inhibitory activity of siRNA conjugates on target genes in mice was used to evaluate the inhibitory activities of siRNA conjugate RZ502001 with the NM011 group substituted at the 4th position of the antisense strand, siRNA conjugate RZ502002 with the NM011 group substituted at the 7th position of the antisense strand, and control conjugate RZ002001 without this group on the target gene CC3 in mice.

[0330] 6 - 8 week - old C57BL / 6J mice were randomly divided into 4 groups according to body weight, with 20 mice in each group. Each group of mice was given the above - mentioned siRNA conjugate by subcutaneous administration in the abdomen. Among them, each mouse in the PBS control group was given a dose of 5 mL / kg of the administration volume, and each mouse in the siRNA conjugate experimental group was given a dose of 3 mg / kg (calculated based on siRNA), with an administration volume of 5 mL / kg. The day of administration was recorded as day 0 (D0). At 7 days (D7), 14 days (D14), 28 days (D28), and 42 days (D42) after administration, 5 mice were sacrificed from each group. The animals were grossly dissected, and liver tissues were collected for RNA extraction, reverse transcription reaction, and Real - time PCR detection. The relative quantification of the target gene mRNA in each test group was calculated according to the aforementioned ΔΔCt method.

[0331] Table 16 Primer sequence information in Example 4

[0332]

[0333] The results of Example 4 showed that the conjugate RZ502001 with the NM011 group at the 4th position of the antisense strand and the conjugate RZ502002 with the NM011 group at the 7th position were basically equivalent in activity to the control conjugate RZ002001 without the substitution group at D7 and D14, and were slightly superior in activity to the control sequence at D28 and D42 ( Figure 9 , Table 17).

[0334] Table 17 Inhibitory activity of the target gene in mice after administration of the siRNA conjugate

[0335]

[0336] Example 5. Inhibitory activity evaluation of Se nucleic acid modified siRNA conjugate on target gene angiopoietin-like protein 3 (angiopoietin-like protein3, ANGPTL3) in mice

[0337] In this example, the inhibitory activities of siRNA conjugates with the NM011 group at the 4th position (RZ597003) and 7th position (RZ597005) of the antisense strand and the siRNA control conjugate without NM011 (RZ597002) on the target gene ANGPTL3 in mice were evaluated by the method of evaluating the inhibitory activity of the target gene in mice in vivo.

[0338] As described above, C57BL / 6J mice at 6 - 8 weeks of age were randomly divided into 4 groups of 5 mice each according to body weight. Each group of mice was given the above siRNA conjugate by subcutaneous administration in the abdomen. Among them, each mouse in the PBS control group was given a dose of 5 mL / kg of the administration volume, and each mouse in the siRNA conjugate experimental group was given a dose of 3 mg / kg (calculated based on siRNA), with an administration volume of 5 mL / kg. The administration day was recorded as D0. On D14 after administration, 5 mice in each group were sacrificed, and the animals were grossly dissected to collect liver tissues, which were cut into several 2 - mm 3 small pieces and stored with RNAlater. RNA extraction, reverse transcription reaction, and Real - time PCR detection were carried out, and the relative quantification of the target gene mRNA in each test group was calculated according to the aforementioned ΔΔCt method.

[0339] Table 18 Primer sequence information in Example 5

[0340]

[0341] The results of Example 5 showed that the conjugate RZ597003 with the NM011 group at the 4th position of the antisense strand and the conjugate RZ597005 with the NM011 group at the 7th position had basically the same activity on D14 as the control conjugate RZ597002 without the substitution group ( Figure 10 , Table 19).

[0342] Table 19 Inhibitory activity of the target gene in mice after administration of the siRNA conjugate

[0343]

[0344] Toxicity evaluation method of siRNA conjugate in female mice

[0345] ICR female mice at 6 - 8 weeks of age were randomly divided into groups of 3 mice each according to body weight. Each test group was given a certain dose of the drug conjugate and a blank control group was added. All mice were calculated for the drug dose according to body weight and were administered by subcutaneous injection in the abdomen. Each drug conjugate was administered in the form of a PBS solution at 25 mg / mL (calculated based on siRNA), and the administration volume was 10 mL / kg of the mouse body weight, that is, the administration dose of each drug conjugate was 250 mg / kg of the mouse body weight (calculated based on siRNA). The drug was administered once a week for a total of two weeks. The blank control group was given a PBS solution without the siRNA conjugate at 10 mL / kg of the mouse body weight, and observed for 14 days.

[0346] The day of the first administration was recorded as day 0 (denoted as D0), and clinical observations were performed at least once a day after the first administration during the trial period. On the 14th day of the trial (denoted as D14), blood was collected from the veins of all animals (mice were fasted for at least 12 hours but not water-deprived before sampling). The collected blood samples (without anticoagulant) were placed at room temperature for about 30 minutes, and after blood coagulation, they were centrifuged at 2000g for 10 minutes at 4°C to obtain serum samples. An automatic biochemical analyzer (Mindray BS-430) was used to perform blood biochemical tests on the serum samples.

[0347] The calculation formula for the change rate of each serum biochemical index: Change rate = (experimental group - blank control group (PBS)) / blank control group

[0348] Example 6. Toxicity evaluation of siRNA conjugate in female mice

[0349] In this example, a toxicological evaluation method of siRNA conjugates in female mice was used to evaluate the nature and degree of the toxic reactions of siRNA conjugates RZ597003 and RZ597005, which contain the NM011 group at positions 4 and 7 of the antisense strand, and the reference conjugate RZ597002 without a substitution group, in ICR female mice. Figure 11 and Figure 12 are scatter plots of the concentrations of ALT and AST in the sera of mice after administration of 250 mg / kg of RZ597003, RZ597005, the reference conjugate RZ597002, and the blank control group PBS, respectively. As shown by Figure 11 , Figure 12 and Table 20, compared with the blank control group, after administration of the conjugate RZ597002 without a substitution group, the change rate of serum ALT in mice was 23-fold, increasing from 37.10 U / L in the PBS control group to 889.03 U / L; the change rate of AST was 4-fold, increasing from 199.57 U / L to 951.37 U / L, and the concentration also increased significantly. After administration of the siRNA conjugates of this example, the concentrations of serum ALT and AST both decreased to varying degrees. Among them, the serum ALT concentration of the RZ597003 conjugate containing the NM011 group at position 4 of the antisense strand decreased to 509.03 U / L, and the serum ALT concentration of the RZ597005 conjugate containing the NM011 group at position 7 of the antisense strand decreased to 549.23 U / L, reducing the change rate of ALT from 23-fold (RZ597002) to 12-fold and 13-fold, respectively. The AST concentration decreased to 546.10 U / L (RZ597003) and 587.93 (RZ597005), reducing the change rate of AST of this conjugate from 4-fold (RZ597002) to 2-fold, indicating that the siRNA conjugates of this example have a certain effect of reducing hepatotoxicity.

[0350] Table 20 Blood biochemical test results of female mice after administration of siRNA conjugates

[0351]

[0352]

[0353] Example 7. Efficacy evaluation of siRNA conjugate in hREN×hAGT hypertensive mice

[0354] In this example, a small animal non-invasive blood pressure measuring instrument was used to measure the systolic blood pressure of mice to evaluate the blood pressure regulation effects of RZ003063 and RZ003067 in mice. RZ003063 and RZ003067 are siRNA conjugates targeting angiotensinogen (AGT), which can produce a blood pressure-lowering effect in hREN×hAGT hypertensive mice.

[0355] Animal grouping, drug administration and tissue sample collection:

[0356] hREN×hAGT hypertensive mice aged 9 - 11 weeks (purchased from Cyagen Biosciences (Suzhou) Inc.) were grouped according to systolic blood pressure levels (all males), with 7 mice in each group. Each test group was given a predetermined drug dose of the drug conjugate and a PBS control group was added. The drug dose for all mice was calculated based on body weight, and a single administration was performed by abdominal subcutaneous injection. Each drug conjugate was administered in the form of a PBS solution at 0.3 mg / mL (calculated based on siRNA), and the administration volume was 10 mL / kg of mouse body weight, that is, the administration dose of each drug conjugate was 3 mg / kg of mouse body weight (calculated based on siRNA). The PBS control group was given the same volume of PBS solution (without the drug conjugate). The day of drug administration was recorded as day 0 (denoted as D0), and on the 3rd day (denoted as D3), 7th day (denoted as D7), 10th day (denoted as D10), 14th day (denoted as D14), and 17th day (denoted as D17) after drug administration, a small animal non-invasive blood pressure measuring instrument (Ruanlong, BP-2010A) was used to measure the systolic blood pressure (SBP) according to the instructions. The blood pressure detection data (blood pressure before the first drug administration (D0)) were calculated as follows:

[0357] Blood pressure reduction level (mmHg) = Dx - D0

[0358] The results of Example 7 showed that at a single administration dose of 3 mg / kg, RZ003063 and RZ003067 could effectively and continuously reduce the systolic blood pressure of hREN×hAGT hypertensive mice. The maximum blood pressure reduction of RZ003063 exceeded 10 mmHg (Table 21, Figure 13 ).

[0359] Table 21 Changes in systolic blood pressure levels (Δ mmHg) in hREN×hAGT hypertensive mice after administration of the siRNA conjugates described in this example

[0360]

[0361] Example 8. Efficacy evaluation of siRNA conjugate in normal cynomolgus monkeys

[0362] In this example, the inhibitory activities of RZ002104 and RZ002109 on the target gene C3 mRNA in the liver and C3 protein in the blood of cynomolgus monkeys were evaluated.

[0363] Animal grouping, administration, and tissue sample collection:

[0364] Healthy cynomolgus monkeys weighing 3 - 5 kg were grouped, with 4 monkeys in each group, half male and half female. Each test group was given a predetermined dose of the drug conjugate, and a PBS control group was added. The dosing dose for all animals was calculated based on body weight, and a single dose was administered by abdominal subcutaneous injection. Each drug conjugate was administered in the form of a PBS solution at 9 mg (calculated as siRNA) / mL, and the dosing volume was 1 mL / kg (cynomolgus monkey body weight), that is, the dosing dose of each drug conjugate was 9 mg (calculated as siRNA) / kg (cynomolgus monkey body weight). The PBS control group was given 1 mL / kg (cynomolgus monkey body weight) of PBS solution without the siRNA conjugate. The day of administration was recorded as day 0 (denoted as D0), and on the 14th day after administration (denoted as D14), liver biopsies were performed on each group of cynomolgus monkeys, and the liver biopsy tissues were immediately preserved with RNA later. For each cynomolgus monkey, the liver tissue sample was taken out from RNA later, and the liver tissue sample was disrupted in a Tissuelyser II type automatic tissue homogenizer for 60 s, and then total RNA was extracted using an automatic nucleic acid extractor (purchased from Zhejiang Hanwei Technology Co., Ltd.) and a nucleic acid extraction kit (purchased from Zhejiang Hanwei Technology Co., Ltd., GO-MNTR-100) according to the standard operating procedures for total RNA extraction. And RNA extraction, reverse transcription reaction, and Real-time PCR detection were performed according to the aforementioned methods, and relative quantification of the target gene mRNA in each test group was calculated by the ΔΔCt method (primer information is shown in Table 22).

[0365] Table 22 Sequences of the detection primers

[0366]

[0367] Simultaneously, cynomolgus monkey serum was collected before drug administration (recorded as pre-dose value), on the day of drug administration (recorded as Day 0, D0), on the 7th day after drug administration (recorded as D7), on the 14th day (recorded as D14), on the 21st day (recorded as D21), on the 28th day (recorded as D28), on the 35th day (recorded as D35), on the 42nd day (recorded as D42), on the 49th day (recorded as D49), and on the 56th day (recorded as D56). The C3 protein expression was measured using a Complement C3 ELISA kit (Hycult Biotech, HK366-01).

[0368] The results showed that at a single-dose of 9 mg / kg, RZ002104 and RZ002109 could significantly inhibit the mRNA expression of the C3 gene in the liver of cynomolgus monkeys at D14 (Table 23, Figure 14 ). At the same time, both RZ002104 and RZ002109 could significantly and persistently reduce the C3 protein level in the serum of cynomolgus monkeys (Table 24, Figure 15 ).

[0369] Table 23 Changes in the level of C3 mRNA in the liver of cynomolgus monkeys after administration of the siRNA conjugate described in this example

[0370]

[0371] Table 24 Changes in the level of C3 protein in the serum of cynomolgus monkeys after administration of the siRNA conjugate described in this example

[0372]

[0373] The above specific embodiments are only illustrative descriptions of the content of the present invention and do not represent limitations on the content of the present invention. For those of ordinary skill in the art, various modifications and improvements can be made without departing from the spirit and essence of the present disclosure, and these modifications and improvements are also regarded as the protection scope of the present disclosure.

Claims

1. A double-stranded oligonucleotide, said double-stranded oligonucleotide comprising a sense strand and an antisense strand, each of said sense strand and said antisense strand having 17 - 35 nucleotides, and said sense strand and said antisense strand being at least partially reverse complementary to form a duplex region; characterized in that, The sense strand and / or the antisense strand comprise at least one nucleotide modified with a 2'-Se(CH2) n CH3 group; wherein n is an integer selected from 0 to 3.

2. The double-stranded oligonucleotide according to claim 1, characterized in that, n is selected from 0 or 1.

3. The double-stranded oligonucleotide according to claim 1, characterized in that, The double-stranded region contains 17-23 pairs of nucleotides; optionally, it contains 19-21 pairs of nucleotides; Optionally, the antisense strand of the double-stranded region is reverse complementary or substantially reverse complementary to the nucleotide sequence of the sense strand; the substantially reverse complementary means that the number of nucleotide mismatches in the double-stranded region does not exceed three.

4. The double-stranded oligonucleotide according to any one of claims 1 - 3, characterized in that, The nucleotide modified with 2'-Se(CH2) n CH3 group is located on the antisense strand; optionally, in the direction from the 5'-end to the 3'-end, it is located at least at one of the remaining positions other than the 1st - 2nd positions on the antisense strand.

5. The double-stranded oligonucleotide according to any one of claims 1 - 3, characterized in that, The nucleotide modified by 2'-Se(CH2) n CH3 group is located on the sense strand.

6. The double-stranded oligonucleotide according to any one of claims 1 - 3, characterized in that, Each nucleotide in the duplex of the double-stranded oligonucleotide is modified; in the 5'-terminal to 3'-terminal direction, at least two nucleotides among the 7th to 10th positions in the sense strand are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl and / or 2'-Se(CH2) n CH3 group-modified nucleotides; and, the antisense strand contains 2'-O-methyl-modified nucleotides and at least one selected from 2'-Se(CH2) n CH3, 2'-O-MOE-modified nucleotides; Optionally, at least three nucleotides among the 7th to 10th positions in the sense strand are 2'-fluoro modified nucleotides; Optionally, there is a 2'-deoxy modified nucleotide among the 7th to 10th positions in the sense strand.

7. The double-stranded oligonucleotide according to any one of claims 1 - 3, characterized in that, Each nucleotide in the double-stranded region of the double-stranded oligonucleotide has any one of the following modification methods 1)-4): 1) In the direction from the 5'-end to the 3'-end, the 7th to 9th positions in the sense strand are nucleotides modified with 2'-fluoro, and the nucleotides at the remaining positions are nucleotides modified with 2'-O-methyl; the 2nd, 6th, 14th, and 16th positions in the antisense strand are nucleotides modified with 2'-fluoro, and there is one nucleotide modified with 2'-Se(CH2) n CH3 in the nucleotides at the 3rd to 19th positions, and the nucleotides at the remaining positions are nucleotides modified with 2'-O-methyl; 2) In the direction from the 5'-end to the 3'-end, the 7th and 9th positions in the sense strand are nucleotides with 2'-fluoro modification, the 8th position is a nucleotide with 2'-deoxy modification, and the nucleotides at the remaining positions are nucleotides with 2'-O-methyl modification; the 2nd, 6th, 14th, and 16th positions in the antisense strand are nucleotides with 2'-fluoro modification, and there is one nucleotide with 2'-Se(CH2) n CH3 modification, and the nucleotides at the remaining positions are nucleotides with 2'-O-methyl modification; 3) In the direction from the 5'-end to the 3'-end, nucleotides at positions 7-10 in the sense strand are 2'-fluoro-modified nucleotides, and nucleotides at the remaining positions are 2'-O-methyl-modified nucleotides; nucleotides at positions 2, 6, 9, 14, and 16 in the antisense strand are 2'-fluoro-modified nucleotides, and there is at least one nucleotide modified with 2'-Se(CH2) n CH3 in nucleotides at positions 3-8, and nucleotides at the remaining positions are 2'-O-methyl-modified nucleotides; Optionally, the 2'-Se(CH2) n CH3-modified nucleotide is present at the 3rd, or 4th, or 5th, or 7th position in the antisense strand; 4) In the direction from the 5'-end to the 3'-end, the sense strand contains at least two 2'-Se(CH2) n CH3-modified nucleotides, and the 7th - 10th positions are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl-modified nucleotides; at least four of the nucleotides at the 2nd, 6th, 9th, 14th, and 16th positions in the antisense strand are 2'-fluoro-modified nucleotides, the 15th position is a 2'-O-MOE-modified nucleotide, and the nucleotides at the remaining positions are 2'-O-methyl-modified nucleotides; Optionally, in the sense strand, in the direction from the 5'-end to the 3'-end, the positions of the 2'-Se(CH2) n CH3-modified nucleotides are selected from the following: positions 4-6, or positions 11-13, or positions 18-19, or positions 15-19.

8. The double-stranded oligonucleotide according to any one of claims 1 - 3, characterized in that, The 3'-end and / or 5'-end of the sense strand and the antisense strand contain one or more overhang regions. Optionally, the overhang region contains 1-3 nucleotides; Optionally, the sense strand and the antisense strand contain 1-3 phosphorothioate groups; Optionally, the phosphorothioate group is present in at least one of the following positions: 1) Between the 1st to 3rd nucleotides at the 5'-end of the sense strand; 2) Between the 0th to 3rd nucleotides at the 3'-end of the sense strand; 3) Between the 1st to 3rd nucleotides at the 5'-end of the antisense strand; 4) Between the 1st to 3rd nucleotides at the 3'-end of the antisense strand.

9. An oligonucleotide conjugate, characterized in that, The oligonucleotide conjugate comprises the double-stranded oligonucleotide according to any one of claims 1 to 8, and one or more ligands capable of binding to a cell surface receptor; Optionally, the ligand is selected from asialoglycoprotein receptor ligands containing galactosamine derivatives; Optionally, the galactosamine derivative is selected from galactosamine, N-formylgalactosamine, N-acetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine or N-isobutyrylgalactosamine; Optionally, the number of ligands is one, and it is conjugated to the 3'-end of the sense strand of the double-stranded oligonucleotide.

10. A composition, characterized in that, The composition comprises the double-stranded oligonucleotide according to any one of claims 1-8 or the double-stranded oligonucleotide conjugate according to any one of claims 9; Optionally, the composition further comprises one or more pharmaceutically acceptable carriers or excipients.

11. Use of the double-stranded oligonucleotide according to any one of claims 1-8, the double-stranded oligonucleotide conjugate according to claim 9, or the composition according to claim 10 in the preparation of a medicament for treating and / or preventing a disease or disorder associated with dysregulation of the mRNA level of a target gene expression.

12. A kit, the kit comprising the double-stranded oligonucleotide according to any one of claims 1-8, the double-stranded oligonucleotide conjugate according to claim 9, or the composition according to claim 10.

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