Modified double-stranded oligonucleotides and conjugates and compositions thereof

Abstract

The invention belongs to the field of biological medicines, and particularly relates to a modified double-stranded oligonucleotide, and a conjugate and a composition thereof. The double-stranded oligonucleotide provided by the invention effectively enhances the activity of the double-stranded oligonucleotide for inhibiting the expression of the target gene by performing specific modification on the nucleotide at a specific site, can efficiently inhibit the expression of the target gene in both in-vitro and in-vivo experiments, and surprisingly, the double-stranded oligonucleotide provided by the invention has a good application prospect. The modification on the double-stranded oligonucleotide can be generally used for siRNA designed for various different target genes, and the inhibition rate of the siRNA on the target genes can be effectively improved.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to Chinese patent application CN202411397097.4, filed on October 8, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure pertains to the field of biomedicine, and specifically relates to a modified double-stranded oligonucleotide, its conjugates, and compositions. Background Technology

[0004] RNA interference (RNAi) is a cellular physiological mechanism in which double-stranded RNA is cleaved by the protein complex Dicer, forming a complex with RISC. This complex recognizes and degrades the mRNA of a target gene, thereby inhibiting the expression of the corresponding protein. This mechanism has been successfully used in disease treatment, and this treatment approach is called RNAi therapy. RNAi therapy can target targets that were previously considered "undruggable." Developing RNAi drugs to treat various human diseases using this technology still requires solving many problems, one of which is addressing the inherent metabolic issues of these drugs. Although existing chemical modification and delivery methods have solved some of the metabolic problems of RNAi drugs, and six RNAi drugs have been developed, more RNAi agents are still needed to meet greater clinical needs.

[0005] The biggest obstacles to developing RNAi therapeutics, including siRNA, include poor compound stability, low in vivo delivery efficiency to target cells, and side effects such as off-target gene silencing and unexpected immune stimulation. To overcome these obstacles, researchers have explored various oligonucleotide chemical modifications. These modifications can be categorized into three types: ribose modifications, nucleotide inter-bond modifications, and nucleobase modifications. Different modifications can exhibit varying degrees of nuclease resistance and different hybridization characteristics with target mRNA, and may also introduce unpredictable side effects. Balancing the type, location, and amount of various modifications to double-stranded siRNA to maximize its nuclease resistance without affecting its activity, while simultaneously reducing toxic side effects, is an important direction in siRNA modification research and development. Summary of the Invention

[0006] In order to address the problems existing in the prior art, the purpose of this disclosure is to provide a double-stranded oligonucleotide whose activity in inhibiting the expression of target genes is enhanced.

[0007] In one aspect, this disclosure provides a double-stranded oligonucleotide comprising a sense strand and an antisense strand, each comprising 19-23 nucleotides, wherein the antisense strand is at least partially complementary to the mRNA expressed by a target gene, and the sense strand and antisense strand are at least partially anticomplementary to form a double-stranded region. The sense strand comprises a nucleotide sequence 1 of at least 11 nucleotides in length, and may further include additional nucleotide sequences at the 5' and / or 3' ends of the sense strand; the antisense strand comprises a nucleotide sequence 2 of at least 16 nucleotides in length, and may further include additional nucleotide sequences at the 5' and / or 3' ends of the antisense strand; wherein the nucleotides at positions 2, 12, 14, and 16 of the nucleotide sequence 2 along the 5'-3' direction are 2'-fluorinated nucleotides or the nucleotides at positions 2, 6, 9, 14, and 16 are 2'-fluorinated nucleotides.

[0008] In another aspect, this disclosure provides an oligonucleotide conjugate comprising the aforementioned double-stranded oligonucleotide or a pharmaceutically acceptable salt thereof and a conjugating group conjugated to the double-stranded oligonucleotide or a pharmaceutically acceptable salt thereof, the conjugating group comprising a pharmaceutically acceptable targeting group and / or a delivery assistant group, the conjugating group being covalently or non-covalently linked to the double-stranded oligonucleotide.

[0009] In another aspect, this disclosure provides a composition comprising the aforementioned double-stranded oligonucleotide or a pharmaceutically acceptable salt thereof, and / or the aforementioned oligonucleotide conjugate, and optionally a pharmaceutically acceptable carrier.

[0010] In another aspect, this disclosure provides a cell that contains or generates the aforementioned double-stranded oligonucleotide or a pharmaceutically acceptable salt thereof.

[0011] In another aspect, this disclosure provides a kit or cassette comprising the aforementioned double-stranded oligonucleotide or a pharmaceutically acceptable salt thereof, the aforementioned oligonucleotide conjugate, the aforementioned composition, and / or the aforementioned cells, wherein the double-stranded oligonucleotide or a pharmaceutically acceptable salt thereof, the oligonucleotide conjugate, the composition, and / or the cells are packaged in a cassette, container, packaging, dispenser, pre-filled syringe, or vial.

[0012] In another aspect, this disclosure provides the use of the aforementioned double-stranded oligonucleotides or pharmaceutically acceptable salts thereof, the aforementioned oligonucleotide conjugates, the aforementioned compositions, the aforementioned cells, and / or the aforementioned kits or pods in the preparation of medicaments for treating viral diseases, neuromuscular diseases, bacterial infections, inflammatory and immune diseases, metabolic diseases, liver diseases, kidney diseases, cardiovascular diseases, eye diseases, lung diseases, tumors, or rare diseases.

[0013] In another aspect, this disclosure provides the use of the aforementioned double-stranded oligonucleotides or pharmaceutically acceptable salts thereof, the aforementioned oligonucleotide conjugates, the aforementioned compositions, the aforementioned cells, and / or the aforementioned kits or pods in the preparation of reagents or medicaments for inhibiting the expression of target genes in cells.

[0014] In another aspect, this disclosure provides the aforementioned double-stranded oligonucleotides or pharmaceutically acceptable salts thereof, the aforementioned oligonucleotide conjugates, the aforementioned compositions, the aforementioned cells, and / or the aforementioned kits or pods for the treatment or prevention of an individual's disease or condition. In some embodiments, the disease or condition is selected from viral diseases, neuromuscular diseases, bacterial infections, inflammatory and immune diseases, metabolic diseases, liver diseases, kidney diseases, cardiovascular diseases, ophthalmic diseases, lung diseases, tumors, and rare diseases.

[0015] In another aspect, this disclosure provides the aforementioned double-stranded oligonucleotides or pharmaceutically acceptable salts thereof, the aforementioned oligonucleotide conjugates, the aforementioned compositions, the aforementioned cells and / or the aforementioned kits or pods for inhibiting the expression of target genes in cells.

[0016] In another aspect, this disclosure provides a method for inhibiting the expression of a target gene in a cell, comprising contacting the aforementioned double-stranded oligonucleotide or a pharmaceutically acceptable salt thereof, the aforementioned oligonucleotide conjugate, the aforementioned composition, and / or the aforementioned cell with the cell.

[0017] In another aspect, this disclosure provides a method for treating or preventing a disease or condition in an individual, wherein the method comprises administering to the individual an effective amount of the aforementioned double-stranded oligonucleotide or a pharmaceutically acceptable salt thereof, the aforementioned oligonucleotide conjugate, the aforementioned composition, and / or the aforementioned cells to inhibit the expression of genes causing the disease in the individual. In some embodiments, the disease or condition is selected from viral diseases, neuromuscular diseases, bacterial infections, inflammatory and immune diseases, metabolic diseases, liver diseases, kidney diseases, cardiovascular diseases, ophthalmic diseases, lung diseases, tumors, and rare diseases.

[0018] The double-stranded oligonucleotide disclosed herein effectively enhances its activity in inhibiting target gene expression by specifically modifying nucleotides at specific sites. It can efficiently inhibit target gene expression in both in vitro and in vivo experiments, and no toxic side effects have been found. Surprisingly, the modification of the double-stranded oligonucleotide disclosed herein can be applied to siRNAs designed for various different target genes, and can effectively improve the inhibition rate of siRNAs on target genes. Detailed Implementation

[0019] (I) Definitions or terms

[0020] In this disclosure, unless otherwise stated, scientific and technical terms used herein have meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. To better understand this disclosure, definitions and explanations of relevant terms are provided below. It should be understood that this disclosure is not limited to specific methods, reagents, compounds, compositions, or biological systems, and variations thereof are certainly possible. It should also be understood that the terminology used in this application is for describing specific embodiments only and is not intended to be limiting.

[0021] Unless otherwise expressly stated, the singular forms “a,” “an,” “the,” “the,” and similar designations used in this specification and the appended claims include plural designations.

[0022] As used in this article, the conjunction term “and / or” between multiple elements means to include both “and” and “or” meanings. For example, the phrase “A, B and / or C” is intended to cover each of the following: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0023] As used herein, the terms “comprises,” “comprising,” “having,” and “containing,” and any variations thereof, are intended to cover non-exclusive inclusion. The term is intended to be open-ended to specify the presence of any of the stated features, elements, integers, steps, or components, but does not exclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. Therefore, the term “comprising” includes the more restrictive terms “consisting of” and “substantially consisting of”.

[0024] The numerical ranges used in this article should be understood as including all numbers within that range. For example, the range 1 to 20 should be understood to include any number, combination of numbers, or subrange from the following group: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0025] As used herein, the term “about” or “approximately” when applied to one or more target values ​​refers to a value similar to a reference value. In some embodiments, unless otherwise stated or otherwise apparent from the context, the term “approximately” or “about” refers to a range of values ​​falling within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the reference value in any direction (unless such a number would exceed 100% of the possible value).

[0026] In the description herein, references to “some embodiments,” “some implementations,” or “some implementation schemes” describe a subset of all possible embodiments. However, it is understood that “some implementation schemes” may be the same subset or different subsets of all possible implementation schemes and may be combined with each other without conflict.

[0027] As used herein, the term "oligonucleotide" refers to a short nucleic acid, such as a short nucleic acid less than 100 nucleotides in length. Oligonucleotides may comprise ribonucleotides, deoxyribonucleotides, and / or modified nucleotides, including, for example, modified ribonucleotides. Oligonucleotides may be single-stranded or double-stranded. Oligonucleotides may or may not have a double-stranded region. As a set of non-limiting examples, oligonucleotides may be, but are not limited to, small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), Dicer substrate interfering RNA (dsiRNA), antisense oligonucleotides, short siRNA, or single-stranded siRNA. In some embodiments, the double-stranded oligonucleotide is an RNAi oligonucleotide.

[0028] As used herein, the term "chain" refers to a single, continuous sequence of nucleotides linked together by internucleotide bonds (e.g., phosphodiester bonds, thiophosphate bonds). In some embodiments, the chain has two free ends, such as a 5' end and a 3' end.

[0029] As used herein, the term "double-stranded oligonucleotide" refers to an oligonucleotide that is substantially in a double-stranded form. In some embodiments, complementary base pairings are formed between antiparallel sequences of nucleotides in covalently separated nucleic acid chains, forming one or more double-stranded regions of the double-stranded oligonucleotide. In some embodiments, complementary base pairings are formed between antiparallel sequences of nucleotides in covalently linked nucleic acid chains. In some embodiments, complementary base pairings of one or more double-stranded regions of the double-stranded oligonucleotide are formed from a single nucleic acid chain folded (e.g., via a hairpin) to provide complementary antiparallel sequences of nucleotides that are base-paired together. In some embodiments, the double-stranded oligonucleotide comprises two covalently separated nucleic acid chains that are fully double-stranded with each other. However, in some embodiments, the double-stranded oligonucleotide comprises two covalently separated nucleic acid chains that are partially double-stranded, for example, having overhangs at one or both ends. In some embodiments, the double-stranded oligonucleotide comprises antiparallel sequences of nucleotides that are partially complementary, and therefore may have one or more mismatches, which may include internal mismatches or terminal mismatches.

[0030] As used herein, the terms “complementary” or “reverse complementary” are used interchangeably and have the meaning known to those skilled in the art: in a double-stranded nucleic acid molecule, the bases of one strand are paired in a complementary manner with the bases of the other strand. In DNA, the purine base adenine (A) always pairs with the pyrimidine base thymine (T) (or uracil (U) in RNA); the purine base guanine (C) always pairs with the pyrimidine base cytosine (G). Each base pair consists of one purine and one pyrimidine. When adenine on one strand always pairs with thymine (or uracil) on the other strand, and guanine always pairs with cytosine, the two strands are considered complementary, and the sequence of the complementary strand can be inferred from its sequence. Correspondingly, “mismatch” in the art means, in the case of a double-stranded nucleic acid, that the bases at corresponding positions are not paired in a complementary manner.

[0031] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide protruding from the double-stranded structure of a double-stranded oligonucleotide. For example, a nucleotide overhang exists when the 3' end of one strand of a double-stranded RNA (dsRNA) extends beyond the 5' end of the other strand, or vice versa. dsRNA may include an overhang of at least one nucleotide; alternatively, an overhang may include at least two, three, four, five, or more nucleotides. A nucleotide overhang may include or consist of nucleotide / nucleoside analogs comprising deoxynucleotides / nucleosides. The overhang may be on the sense strand, antisense strand, or any combination thereof. Furthermore, the overhanging nucleotide may be present at the 5', 3', or both ends of the antisense or sense strand of the dsRNA. The term "blunt end" or "flat-ended" means that there is no unpaired nucleotide at that end of the double-stranded siRNA, i.e., no nucleotide overhang. A "blunt" siRNA is a double-stranded siRNA throughout its entire length, i.e., without a nucleotide overhang at either end of the molecule.

[0032] As used herein, the term "double-stranded RNA" or "dsRNA" refers to a complex of ribonucleic acid molecules having a double-stranded structure comprising two antiparallel and substantially complementary nucleic acid strands with "sense" and "antisense" orientations relative to the target RNA. In some embodiments of this disclosure, the double-stranded RNA (dsRNA) triggers the degradation of the target RNA (e.g., mRNA) through a post-transcriptional gene silencing mechanism referred to herein as RNA interference or RNAi. Generally, most nucleotides in each strand of the dsRNA molecule are ribonucleotides, but each or both strands may also contain one or more non-ribonucleotides, such as deoxyribonucleotides or modified nucleotides, as described in detail herein. Additionally, as used herein, "iRNA" may contain chemically modified ribonucleotides; iRNA may contain substantial modifications at multiple nucleotide sites.

[0033] As used herein, the terms “siRNA,” “iRNA,” “RNAi agent,” “iRNA agent,” “RNAi drug,” and “RNA interference agent” are used interchangeably. siRNA mediates the targeted cleavage of RNA transcripts via the RNA-inducible silencing complex (RISC) pathway, thereby mediating the silencing of complementary target RNAs (e.g., mRNA, such as transcripts of protein-coding genes). siRNAs are typically double-stranded, consisting of an antisense strand (AS) complementary to the target RNA and a sense strand (SS) complementary to that antisense strand. For convenience, such mRNAs are also referred to herein as mRNAs to be silenced. Such genes are also referred to as target genes. Typically, the RNA to be silenced is an endogenous gene or a pathogen gene. Additionally, RNAs other than mRNA (e.g., tRNA) and viral RNAs can also be targeted. RNA interference (RNAi) is a process that directs the specific degradation of mRNA sequences.

[0034] As used herein, the term "antisense strand" refers to a strand of siRNA that contains a region that is fully or substantially complementary to the target sequence. The term "sense strand" refers to a strand of siRNA that includes a region substantially complementary to the region that is defined herein as the antisense strand.

[0035] As used herein, the term "modified nucleotide" refers to a nucleotide that independently has a modified sugar moiety, a modified internucleotide bond, or a modified nucleobase, or any combination thereof. Therefore, the term "modified nucleotide" encompasses substitutions, additions, or removals of, for example, functional groups or atoms, of internucleotide bonds, sugar moieties, or nucleobases. Modifications of nucleotides applicable to this disclosure include all types of modifications disclosed herein or known in the art.

[0036] As used herein, "fluorinated nucleotides," "2′-fluorinated nucleotides," or "fluorinated nucleotides" refer to nucleotides formed by replacing the hydroxyl group at the 2' position of the ribosome with fluorine. "Non-fluorinated nucleotides" refer to nucleotides or nucleotide analogs formed by replacing the hydroxyl group at the 2' position of the ribosome with a non-fluorinated group, such as 2′-O-methyl-modified nucleotides, 2′-O-methoxyethyl-modified nucleotides, 2′-O-alkyl-modified nucleotides, 2′-O-allyl-modified nucleotides, and 2′-amino-modified nucleotides. "Nucleotide analogs" refer to groups that can replace nucleotides in nucleic acids but have a structure different from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, or thymine deoxyribonucleotides. Examples include isonucleotides, bridged nucleic acids (BNAs), or acyclic nucleotides. "Methoxy-modified nucleotides" refer to nucleotides formed by replacing the hydroxyl group at the 2' position of the ribosome with a methoxy group.

[0037] As used herein, the term "deoxyribonucleotide" refers to a nucleotide that has a hydrogen atom at the 2' position of its pentose sugar compared to a ribonucleotide. A modified deoxyribonucleotide is a deoxyribonucleotide that has one or more modifications or substitutions (including modifications or substitutions in the sugar, phosphate group, or base) other than at the 2' position.

[0038] As used in this article, the term "naked sequence" refers to an unmodified nucleotide sequence.

[0039] The double-stranded oligonucleotides provided in this disclosure can be obtained using conventional double-stranded oligonucleotide preparation methods in the art (e.g., solid-phase synthesis and liquid-phase synthesis methods), the only difference being that the phosphoramidite monomer corresponding to the SN substitution group is used instead of the nucleoside phosphoramidite monomer corresponding to the substituted nucleotide. Solid-phase synthesis is already available as a commercially available custom service. Modified nucleotide groups can be introduced into the double-stranded oligonucleotides described in this disclosure using appropriately modified nucleoside monomers. Methods for preparing appropriately modified nucleoside monomers and methods for introducing modified nucleotide groups into double-stranded oligonucleotides are also well known to those skilled in the art.

[0040] In this document, particularly in describing methods for preparing double-stranded oligonucleotides, pharmaceutical compositions, or oligonucleotide conjugates of this disclosure, unless otherwise specified, the nucleoside monomer refers to the unmodified or modified RNA phosphoramidites (sometimes also called nucleoside-phosphoramidites) used in phosphoramidite solid-phase synthesis, depending on the type and sequence of nucleotides in the desired double-stranded oligonucleotide or oligonucleotide conjugate. Phosphoramidite solid-phase synthesis is a method known to those skilled in the art for RNA synthesis. All nucleoside monomers used in this disclosure are commercially available.

[0041] As used herein, “conjugation” refers to the covalent connection between two or more chemical moieties, each with a specific function; correspondingly, “conjugated compound” refers to a compound formed by the covalent connection of these chemical moieties. Further, “siRNA conjugated compound” refers to a compound formed by the covalent attachment of one or more chemical moieties with specific functions to siRNA. In the following text, the siRNA conjugated compounds of this disclosure will sometimes be simply referred to as “conjugated compounds.” The term “siRNA conjugated compound” should be understood, depending on the context, as a general term for siRNA conjugated compounds, including first-type or second-type siRNA conjugated compounds, or siRNA sense strand conjugated compounds or siRNA antisense strand conjugated compounds.

[0042] As used herein, the term “inhibition” is used interchangeably with “knockdown,” “reduction,” “silence,” “downregulation,” “suppression,” and other similar terms, and includes any degree of inhibition.

[0043] As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents compatible with drug administration. The use of such media and reagents for pharmaceutically active substances is well known in the art. Its use in these compositions is covered unless any conventional media or reagent is incompatible with the active compound. Pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials known in the art. Exemplary pharmaceutically acceptable carriers are described in U.S. Patent No. 5,211,657, while other carriers are known to those skilled in the art. Such formulations may typically contain salts, buffers, preservatives, compatible carriers, and optional other therapeutic agents. When used in medicine, the salt should be pharmaceutically acceptable, but non-pharmaceuticalally acceptable salts may be readily used to prepare their pharmaceutically acceptable form, without exclusion from the scope of this invention. Such pharmacologically and pharmaceutically acceptable salts include, but are not limited to, salts prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, formic acid, malonic acid, succinic acid, etc. Furthermore, pharmaceutically acceptable salts can be prepared as alkali metal salts or alkaline earth metal salts, such as sodium salts, potassium salts, or calcium salts.

[0044] The double-stranded oligonucleotides disclosed herein can be various double-stranded oligonucleotides that regulate gene expression. In some embodiments, they can be double-stranded oligonucleotides that inhibit or downregulate gene expression, such as siRNA; in some embodiments, they can be double-stranded oligonucleotides that activate or upregulate gene expression, such as saRNA.

[0045] As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents compatible with drug administration. The use of such media and reagents for pharmaceutically active substances is well known in the art. Its use in these compositions is covered unless any conventional media or reagent is incompatible with the active compound. Pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers, protectants, and osmotic regulators, among other materials well known in the art. Exemplary pharmaceutically acceptable carriers are described in U.S. Patent No. 5,211,657, while other carriers are known to those skilled in the art. Such formulations may typically contain salts, buffers, preservatives, compatible carriers, and optionally other therapeutic agents. When used in medicine, the salt should be pharmaceutically acceptable, but non-pharmaceuticalally acceptable salts may be readily used to prepare their pharmaceutically acceptable form, without exclusion from the scope of this invention. Such pharmacologically and pharmaceutically acceptable salts include, but are not limited to, salts prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, formic acid, malonic acid, succinic acid, etc. Furthermore, pharmaceutically acceptable salts can be prepared as alkali metal salts or alkaline earth metal salts, such as sodium salts, potassium salts, or calcium salts.

[0046] As used herein, the term “treatment” is intended to include prevention and treatment, methods by which beneficial or desired outcomes, including clinical outcomes, can be obtained. For the purposes of this application, beneficial or desired clinical outcomes include, but are not limited to, one or more of the following: relief of one or more symptoms caused by the disease, reduction of the severity of the disease, stabilization of the disease (e.g., prevention or delay of disease progression), prevention or delay of disease spread (e.g., metastasis), prevention or delay of disease recurrence, delay or slowing of disease progression, improvement of disease status, provision of disease remission (partial or complete), reduction of the dosage of one or more other medications required to treat the disease, delay of disease progression, improvement or enhancement of quality of life, improvement of weight gain and / or prolongation of survival. “Treatment” also includes a reduction in the pathological outcome of cancer (e.g., tumor volume). In the context of cancer, “treatment” includes any or all of the following: inhibition of cancer cell growth, inhibition of cancer cell replication, reduction of overall tumor burden, and improvement of one or more symptoms associated with the disease.

[0047] As used herein, the term "effective dose" or "therapeutic effective dose" includes an amount sufficient to improve or prevent the symptoms or condition of a medical condition. An effective dose also means an amount sufficient to allow or facilitate diagnosis. The effective dose for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the patient's overall health, the route and dosage of administration, and the severity of side effects. An effective dose can be the maximum dose or administration regimen that avoids significant side effects or toxicity.

[0048] As used herein, the terms “subject,” “individual,” or “patient” are used interchangeably and refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, mice, apes, humans, farm animals, racing animals, and pets. The amount of drug administered may depend on the subject being treated, the subject’s age, health status, sex, and weight, the type of concurrent treatment (if any), the severity of the condition, the nature of the desired effect, the manner and frequency of treatment, and the prescribing physician’s judgment. The frequency of administration may also depend on the pharmacodynamic effect on arterial oxygen partial pressure. However, the optimal dose may be adjusted for individual subjects, as understood by those skilled in the art and determined without tolerance experiments. This typically involves adjusting the standard dose (e.g., reducing the dose if the patient is underweight).

[0049] (II) Detailed Technical Solution

[0050] On one hand, this disclosure provides a double-stranded oligonucleotide comprising a sense strand and an antisense strand, each comprising 19-23 nucleotides, wherein the antisense strand is at least partially complementary to the mRNA expressed by a target gene, and the sense strand and antisense strand are at least partially anticomplementary to form a double-stranded region. The sense strand comprises a nucleotide sequence 1 of at least 11 nucleotides in length, and may further include additional nucleotide sequences at the 5' and / or 3' ends of the sense strand; the antisense strand comprises a nucleotide sequence 2 of at least 16 nucleotides in length, and may further include additional nucleotide sequences at the 5' and / or 3' ends of the antisense strand; wherein the nucleotides at positions 2, 12, 14, and 16 of the nucleotide sequence 2 along the 5'-3' direction are 2'-fluorinated nucleotides or the nucleotides at positions 2, 6, 9, 14, and 16 are 2'-fluorinated nucleotides.

[0051] In some embodiments, the nucleotide sequence 1 is 11 nucleotides in length, and the positive strand further includes an additional nucleotide sequence of 8, 9, 10, 11 or 12 nucleotides in length at the 3' end of the nucleotide sequence 1.

[0052] In some embodiments, the nucleotide sequence 1 is 11 nucleotides in length, and the positive strand further includes an additional nucleotide sequence of 5, 6, 7, 8, 9, 10 or 11 nucleotides in length at the 3' end of the nucleotide sequence 1, and the positive strand further includes another nucleotide sequence of 1, 2, 3 or 4 nucleotides in length at the 5' end of the nucleotide sequence 1.

[0053] In some embodiments, the nucleotide sequence 1 is 11 nucleotides in length, the positive strand further includes an additional nucleotide sequence of 7 nucleotides in length at the 3' end of the nucleotide sequence 1, and the positive strand further includes another nucleotide sequence of 1 nucleotide in length at the 5' end of the nucleotide sequence 1.

[0054] In some embodiments, the nucleotide sequence 1 is 11 nucleotides in length, the positive strand further includes an additional nucleotide sequence of 6 nucleotides in length at the 3' end of the nucleotide sequence 1, and the positive strand further includes another nucleotide sequence of 2 nucleotides in length at the 5' end of the nucleotide sequence 1.

[0055] In some embodiments, the nucleotide sequence 1 is 11 nucleotides in length, and the positive strand further includes an additional nucleotide sequence of 5 nucleotides in length at the 3' end of the nucleotide sequence 1, and the positive strand further includes another nucleotide sequence of 3 nucleotides in length at the 5' end of the nucleotide sequence 1.

[0056] In some embodiments, the nucleotide sequence 1 is 11 nucleotides in length, the positive strand further includes an additional nucleotide sequence of 5 nucleotides in length at the 3' end of the nucleotide sequence 1, and the positive strand further includes another nucleotide sequence of 4 nucleotides in length at the 5' end of the nucleotide sequence 1.

[0057] In some embodiments, the nucleotide sequence 1 is 11 nucleotides in length, the positive strand further includes an additional nucleotide sequence of 8 nucleotides in length at the 3' end of the nucleotide sequence 1, and the positive strand further includes another nucleotide sequence of 1 nucleotide in length at the 5' end of the nucleotide sequence 1.

[0058] In some embodiments, the nucleotide sequence 1 is 11 nucleotides in length, the positive strand further includes an additional nucleotide sequence of 9 nucleotides in length at the 3' end of the nucleotide sequence 1, and the positive strand further includes another nucleotide sequence of 2 nucleotides in length at the 5' end of the nucleotide sequence 1.

[0059] In some embodiments, the nucleotide sequence 1 is 11 nucleotides in length, the positive strand further includes an additional nucleotide sequence of 9 nucleotides in length at the 3' end of the nucleotide sequence 1, and the positive strand further includes another nucleotide sequence of 3 nucleotides in length at the 5' end of the nucleotide sequence 1.

[0060] In some embodiments, the nucleotide sequence 1 is 12 nucleotides in length, and the positive strand further includes an additional nucleotide sequence of 7, 8, 9, 10 or 11 nucleotides in length at the 3' end of the nucleotide sequence 1.

[0061] In some embodiments, the nucleotide sequence 1 is 15 nucleotides in length, and the positive strand further includes an additional nucleotide sequence of 4, 5, 6, 7 or 8 nucleotides in length at the 3' end of the nucleotide sequence 1.

[0062] In some embodiments, the length of nucleotide sequence 1 is 15 nucleotides, and the positive strand further includes an additional nucleotide sequence of 1, 2, 3, 4, 5, 6 or 7 nucleotides at the 3' end of nucleotide sequence 1, and the positive strand further includes another nucleotide sequence of 1, 2, 3 or 4 nucleotides at the 5' end of nucleotide sequence 1.

[0063] In some embodiments, the length of nucleotide sequence 1 is 19, 20, 21, 22, or 23 nucleotides, and the positive strand does not include additional nucleotide sequences at the 5' and 3' ends of nucleotide sequence 1.

[0064] In some embodiments, the nucleotide sequence 2 is 16 nucleotides in length, and the antisense strand further includes an additional nucleotide sequence of 3, 4, 5, 6 or 7 nucleotides in length at the 3' end of the nucleotide sequence 2.

[0065] In some embodiments, the nucleotide sequence 2 is 16 nucleotides in length, and the antisense strand further includes an additional nucleotide sequence of 1, 2, 3, 4, 5 or 6 nucleotides in length at the 3' end of the nucleotide sequence 2, and the antisense strand further includes another nucleotide sequence of 1, 2, 3 or 4 nucleotides in length at the 5' end of the nucleotide sequence 2.

[0066] In some embodiments, the nucleotide sequence 2 is 16 nucleotides in length, the antisense strand further includes an additional nucleotide sequence of 4 nucleotides in length at the 3' end of the nucleotide sequence 2, and the antisense strand further includes another nucleotide sequence of 1 nucleotide in length at the 5' end of the nucleotide sequence 2.

[0067] In some embodiments, the nucleotide sequence 2 is 16 nucleotides in length, the antisense strand further includes an additional nucleotide sequence of 3 nucleotides in length at the 3' end of the nucleotide sequence 2, and the antisense strand further includes another nucleotide sequence of 2 nucleotides in length at the 5' end of the nucleotide sequence 2.

[0068] In some embodiments, the nucleotide sequence 2 is 16 nucleotides in length, the antisense strand further includes an additional nucleotide sequence of 2 nucleotides in length at the 3' end of the nucleotide sequence 2, and the antisense strand further includes another nucleotide sequence of 3 nucleotides in length at the 5' end of the nucleotide sequence 2.

[0069] In some embodiments, the nucleotide sequence 2 is 16 nucleotides in length, the antisense strand further includes an additional nucleotide sequence of 1 nucleotide in length at the 3' end of the nucleotide sequence 2, and the antisense strand further includes another nucleotide sequence of 4 nucleotides in length at the 5' end of the nucleotide sequence 2.

[0070] In some embodiments, the nucleotide sequence 2 is 16 nucleotides in length, the antisense strand further includes an additional nucleotide sequence of 3 nucleotides in length at the 3' end of the nucleotide sequence 2, and the antisense strand further includes another nucleotide sequence of 3 nucleotides in length at the 5' end of the nucleotide sequence 2.

[0071] In some embodiments, the nucleotide sequence 2 is 16 nucleotides in length, and the antisense strand further includes an additional nucleotide sequence of 5 nucleotides in length at the 3' end of the nucleotide sequence 2, and the antisense strand further includes another nucleotide sequence of 2 nucleotides in length at the 5' end of the nucleotide sequence 2.

[0072] In some embodiments, the nucleotide sequence 2 is 16 nucleotides in length, the antisense strand further includes an additional nucleotide sequence of 3 nucleotides in length at the 3' end of the nucleotide sequence 2, and the antisense strand further includes another nucleotide sequence of 1 nucleotide in length at the 5' end of the nucleotide sequence 2.

[0073] In some embodiments, the nucleotide sequence 2 is 16 nucleotides in length, the antisense strand further includes an additional nucleotide sequence of 2 nucleotides in length at the 3' end of the nucleotide sequence 2, and the antisense strand further includes another nucleotide sequence of 1 nucleotide in length at the 5' end of the nucleotide sequence 2.

[0074] In some embodiments, the nucleotide sequence 2 is 17 nucleotides in length, and the antisense strand further includes an additional nucleotide sequence of 2, 3, 4, 5 or 6 nucleotides in length at the 3' end of the nucleotide sequence 2.

[0075] In some embodiments, the nucleotide sequence 2 is 18 nucleotides in length, and the antisense strand further includes an additional nucleotide sequence of 1, 2, 3 or 4 nucleotides in length at the 3' end of the nucleotide sequence 2, and the antisense strand further includes another nucleotide sequence of 1, 2, 3 or 4 nucleotides in length at the 5' end of the nucleotide sequence 2.

[0076] In some embodiments, the length of the nucleotide sequence 2 is 19, 20, 21, 22, or 23 nucleotides, and the antisense strand does not include additional nucleotide sequences at the 5' and 3' ends of the nucleotide sequence 2.

[0077] In some implementations, the sense and antisense strands are substantially anticomplementary, substantially anticomplementary, or completely anticomplementary. Unless otherwise specified above and below, "substantially anticomplementary" means that there are no more than three base mismatches between the two nucleotide sequences involved; "substantially anticomplementary" means that there are no more than one base mismatch between the two nucleotide sequences; and "completely anticomplementary" means that there are no base mismatches between the two nucleotide sequences.

[0078] In some embodiments, the sense strand comprises 19, 20, or 21 nucleotides, and the antisense strand comprises 19, 20, 21, 22, or 23 nucleotides. The double-stranded nucleotides may include a protruding end of at least one nucleotide; optionally, the protruding end may include 1, 2, 3, or 4 nucleotides, and the protruding end may be located at the 5' end, 3' end, or both ends of the sense and / or antisense strands. In some embodiments, the double-stranded nucleotides have blunt ends. In some preferred embodiments, the sense strand comprises 19 nucleotides, the antisense strand comprises 21 nucleotides, and the antisense strand has a 2-nucleotide protruding end at the 3' end.

[0079] In some embodiments, the nucleotides in the sense strand and / or antisense strand are each independently modified nucleotides, wherein the modified nucleotides are selected from at least one of 2′-fluoro-modified nucleotides, 2′-O-methyl-modified nucleotides, 2′-O-methoxyethyl-modified nucleotides, 2′-O-alkyl-modified nucleotides, 2′-O-allyl-modified nucleotides, bicyclic nucleic acids (BNA), deoxyribonucleotides, 2′-amino-modified nucleotides (such as the amino-modified nucleotides disclosed in CN11806353A), baseless nucleotides, reverse baseless nucleotides, and reverse deoxyribonucleotides. In some preferred embodiments, the nucleotides in the sense strand and / or antisense strand are each independently modified nucleotides.

[0080] Table 1 shows the abbreviations for nucleotide monomers or modified nucleotide monomers used in nucleic acid sequence representation (according to the st.26 sequence listing standard, where the letter U in the abbreviations of uridine and modified uridine is T in the st.26 sequence listing file). It will be understood that these monomers, when present in oligonucleotides, are interconnected by 5'-3' phosphodiester bonds unless otherwise stated. And it should be understood that when a nucleotide contains a 2'-fluorine modification, the fluorine replaces the hydroxyl group at that position in the parent nucleotide (i.e., it is a 2'-fluorine nucleotide).

[0081] Table 1. Abbreviations for nucleotide monomers used in nucleic acid sequence representation

[0082]

[0083] VPUm is a 2'-methoxy-modified uridine, and its structure is as follows:

[0084] M is 2'-O-methyl-6-(3-(2-carboxyethyl)phenyl)-adenosine, and its structure is as follows:

[0085] Some oligonucleotides may contain GNA (glucan-diol nucleic acid) modifications. In some oligonucleotides, individual ribonucleic acid units are replaced with (Tgn), which is the S-isomer of thymidine-diol nucleic acid (GNA) shown in formula (I). In some oligonucleotides, individual ribonucleic acid units are replaced with (Cgn), which is the S-isomer of cytidine-diol nucleic acid (GNA) shown in formula (II). In some oligonucleotides, individual ribonucleic acid units are replaced with (Agn), which is the S-isomer of adenosine-diol nucleic acid (GNA) shown in formula (III). In some oligonucleotides, individual ribonucleic acid units are replaced with (Ggn), which is the S-isomer of guanosine-diol nucleic acid (GNA) shown in formula (IV). The structural formulas of Tgn, Cgn, Agn, and Ggn are as follows:

[0086]

[0087] In some embodiments, the 5' and / or 3' ends of the sense and / or antisense strands comprise at least one of a baseless nucleotide, a reverse a baseless nucleotide, and a reverse deoxyribonucleotide.

[0088] In some embodiments, the 5' and / or 3' ends of the sense and / or antisense strands each independently contain at least one phosphate thioester bond. In some embodiments, the 5' and / or 3' ends of the sense and / or antisense strands each independently contain at least two phosphate thioester bonds. In some embodiments, the phosphate thioester bonds are present between the first and second nucleotides, and between the second and third nucleotides starting from the 5' end; and / or between the first and second nucleotides, and between the second and third nucleotides starting from the 3' end.

[0089] In some embodiments, the positive and / or negative strands do not contain a thiophosphate bond at the 5' end and contain one, two, or three thiophosphate bonds at the 3' end. In some embodiments, the positive and / or negative strands contain one, two, or three thiophosphate bonds at the 5' end and do not contain a thiophosphate bond at the 3' end. In some embodiments, the positive and / or negative strands contain one thiophosphate bond at the 5' end and one, two, or three thiophosphate bonds at the 3' end. In some embodiments, the positive and / or negative strands contain two thiophosphate bonds at the 5' end and one, two, or three thiophosphate bonds at the 3' end. In some embodiments, the positive and / or negative strands contain three thiophosphate bonds at the 5' end and one, two, or three thiophosphate bonds at the 3' end. In some embodiments, the one, two, or three phosphate thioester bonds are located between one, two, or three consecutive nucleotides starting from the 5' end or the 3' end. In some embodiments, the number of phosphate thioester bonds is related to the presence of the carrier; depending on the type of carrier introduced, one end of the introduced carrier may contain 0, 1, 2, or 3 phosphate thioester bonds. For example, in the case of introducing the L96 carrier at the 3' end, the 3' end does not contain phosphate thioester bonds.

[0090] In some embodiments, the antisense strand comprises at least five 2'-fluorinated nucleotides, wherein at least five nucleotides of the antisense strand at positions 2, 4, 6, 7, 9, 12, 14, and 16 of the nucleotide sequence 2 in the 5'-3' direction are 2'-fluorinated nucleotides.

[0091] In some embodiments, the antisense strand contains nucleotides at positions 2, 4, 12, 14, and 16 of the nucleotide sequence 2 in the 5'-3' direction, which are 2'-fluorinated nucleotides.

[0092] In some embodiments, the antisense strand contains nucleotides at positions 2, 7, 12, 14, and 16 of the nucleotide sequence 2 in the 5'-3' direction, which are 2'-fluorinated nucleotides.

[0093] In some embodiments, the antisense strand contains nucleotides at positions 2, 6, 9, 14, and 16 of the nucleotide sequence 2 in the 5'-3' direction, which are 2'-fluorinated nucleotides.

[0094] In some embodiments, the nucleotides in the antisense strand, except for those that are 2'-fluorinated, are all non-fluorinated nucleotides, such as one or more of the following: 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, and 2'-amino modified nucleotides.

[0095] In some preferred embodiments, the nucleotides in the antisense strand, except for those modified with 2'-fluorine, are all modified with 2'-O-methyl.

[0096] In some embodiments, the 5' end and 3' end of the antisense chain each independently contain at least one thiophosphate bond.

[0097] In some embodiments, the 5' end and 3' end of the antisense chain each contain two thiophosphate bonds.

[0098] In some embodiments, the thiophosphate bond of the antisense chain is located between the first and second nucleotides, and between the second and third nucleotides, starting from the 5' end of the antisense chain; and between the first and second nucleotides, and between the second and third nucleotides, starting from the 3' end of the antisense chain.

[0099] In some embodiments, the positive strand contains at least three 2'-fluorinated nucleotides. Specifically, at least three nucleotides in the positive strand at positions 5, 7, 8, 9, and 11 of the nucleotide sequence 1 along the 5'-3' direction are 2'-fluorinated nucleotides.

[0100] In some embodiments, the nucleotides at positions 5, 7, and 9 of the positive strand of the nucleotide sequence 1 in the 5'-3' direction are 2'-fluorinated nucleotides.

[0101] In some embodiments, the nucleotides at positions 7, 8, and 9 of the positive strand of the nucleotide sequence 1 in the 5'-3' direction are 2'-fluorinated nucleotides.

[0102] In some embodiments, the nucleotides at positions 7, 9, and 11 of the positive strand of the nucleotide sequence 1 in the 5'-3' direction are 2'-fluorinated nucleotides.

[0103] In some embodiments, the nucleotides at positions 5, 8, and 9 of the positive strand of the nucleotide sequence 1 in the 5'-3' direction are 2'-fluorinated nucleotides.

[0104] In some embodiments, the nucleotides at positions 5, 9, and 11 of the positive strand of the nucleotide sequence 1 in the 5'-3' direction are 2'-fluorinated nucleotides.

[0105] In some embodiments, the nucleotides at positions 8, 9, and 11 of the positive strand of the nucleotide sequence 1 in the 5'-3' direction are 2'-fluorinated nucleotides.

[0106] In some embodiments, the positive strand comprises at least four 2'-fluorinated nucleotides, wherein at least four nucleotides of the positive strand at positions 5, 7, 8, 9, and 11 of the nucleotide sequence 1 in the 5'-3' direction are 2'-fluorinated nucleotides.

[0107] In some embodiments, the nucleotides at positions 5, 7, 8, and 9 of the positive strand of the nucleotide sequence 1 in the 5'-3' direction are 2'-fluorinated nucleotides.

[0108] In some embodiments, the nucleotides at positions 7, 8, 9, and 11 of the positive strand of the nucleotide sequence 1 in the 5'-3' direction are 2'-fluorinated nucleotides.

[0109] In some embodiments, the nucleotides at positions 5, 8, 9, and 11 of the positive strand of the nucleotide sequence 1 in the 5'-3' direction are 2'-fluorinated nucleotides.

[0110] In some embodiments, the nucleotides at positions 5, 7, 9, and 11 of the positive strand of the nucleotide sequence 1 in the 5'-3' direction are 2'-fluorinated nucleotides.

[0111] In some embodiments, the nucleotides in the positive strand, except for those that are 2'-fluorinated, are all non-fluorinated nucleotides.

[0112] In some preferred embodiments, the nucleotides in the positive strand, except for those that are 2'-fluorine modified, are all 2'-O-methyl modified nucleotides.

[0113] In some implementations, the 5' end and / or 3' end of the positive chain each independently contain at least one thiophosphate bond.

[0114] In some embodiments, the 5' end of the positive chain contains two thiophosphate bonds, and the 3' end of the positive chain contains 0-3 thiophosphate bonds.

[0115] In some implementations, the chain of justice is selected from any combination of the following:

[0116] (1) There are two phosphate thioester bonds between the first and second nucleotides and between the second and third nucleotides starting from the 5' end of the positive strand, and there are no phosphate thioester bonds at the 3' end of the positive strand;

[0117] (2) There are two phosphate thioester bonds between the first and second nucleotides starting from the 5' end of the positive strand, and between the second and third nucleotides; and there is one phosphate thioester bond between the first and second nucleotides starting from the 3' end of the positive strand.

[0118] (3) There are two thiophosphate bonds between the first and second nucleotides and between the second and third nucleotides starting from the 5' end of the positive strand, and there are two thiophosphate bonds between the first and second nucleotides and between the second and third nucleotides starting from the 3' end of the positive strand;

[0119] (4) There are two thiophosphate bonds between the first and second nucleotides and between the second and third nucleotides starting from the 5' end of the positive strand, and there are three thiophosphate bonds between the first and second nucleotides, between the second and third nucleotides and between the third and fourth nucleotides starting from the 3' end of the positive strand.

[0120] In some embodiments, the double-stranded oligonucleotides of this disclosure are modified nucleotides at the sites shown in Table 2, and these modification patterns are generally applicable to RNAi agents targeting different sites.

[0121] Table 2. Nucleotide modification patterns

[0122]

[0123] In some embodiments, the sense strand has nucleotides at positions 7, 8, and 9 of the nucleotide sequence 1 in the 5'-3' direction that are 2'-fluorinated nucleotides, and the antisense strand has nucleotides at positions 2, 4, 12, 14, and 16 of the nucleotide sequence 2 in the 5'-3' direction that are 2'-fluorinated nucleotides.

[0124] In some embodiments, the sense strand has nucleotides at positions 5, 7, and 9 of the nucleotide sequence 1 in the 5'-3' direction that are 2'-fluorinated nucleotides, and the antisense strand has nucleotides at positions 2, 4, 12, 14, and 16 of the nucleotide sequence 2 in the 5'-3' direction that are 2'-fluorinated nucleotides.

[0125] In some embodiments, the sense strand has nucleotides at positions 7, 9, and 11 of the nucleotide sequence 1 in the 5'-3' direction that are 2'-fluorinated nucleotides, and the antisense strand has nucleotides at positions 2, 7, 12, 14, and 16 of the nucleotide sequence 2 in the 5'-3' direction that are 2'-fluorinated nucleotides.

[0126] In some embodiments, the sense strand has nucleotides at positions 7, 8, and 9 of the nucleotide sequence 1 in the 5'-3' direction that are 2'-fluorinated nucleotides, and the antisense strand has nucleotides at positions 2, 12, 14, and 16 of the nucleotide sequence 2 in the 5'-3' direction that are 2'-fluorinated nucleotides.

[0127] In some embodiments, the sense strand has nucleotides at positions 7, 8, and 9 of the nucleotide sequence 1 in the 5'-3' direction that are 2'-fluorinated nucleotides, and the antisense strand has nucleotides at positions 2, 6, 9, 14, and 16 of the nucleotide sequence 2 in the 5'-3' direction that are 2'-fluorinated nucleotides.

[0128] In some embodiments, the sense strand has nucleotides at positions 5, 7, 8, and 9 of the nucleotide sequence 1 in the 5'-3' direction that are 2'-fluorinated nucleotides, and the antisense strand has nucleotides at positions 2, 6, 14, and 16 of the nucleotide sequence 2 in the 5'-3' direction that are 2'-fluorinated nucleotides.

[0129] In some embodiments, the sense strand has nucleotides at positions 5, 7, and 9 of the nucleotide sequence 1 in the 5'-3' direction that are 2'-fluorinated nucleotides, and the antisense strand has nucleotides at positions 2, 12, 14, and 16 of the nucleotide sequence 2 in the 5'-3' direction that are 2'-fluorinated nucleotides.

[0130] In some embodiments, the sense strand has nucleotides at positions 7, 9, and 11 of the nucleotide sequence 1 in the 5'-3' direction that are 2'-fluorinated nucleotides, and the antisense strand has nucleotides at positions 2, 4, 12, 14, and 16 of the nucleotide sequence 2 in the 5'-3' direction that are 2'-fluorinated nucleotides.

[0131] In some embodiments, the sense strand has nucleotides at positions 7, 8, and 9 of the nucleotide sequence 1 in the 5'-3' direction that are 2'-fluorinated nucleotides, and the antisense strand has nucleotides at positions 2, 7, 12, 14, and 16 of the nucleotide sequence 2 in the 5'-3' direction that are 2'-fluorinated nucleotides.

[0132] In some embodiments, the sense strand has nucleotides at positions 7, 9, and 11 of the nucleotide sequence 1 in the 5'-3' direction that are 2'-fluorinated nucleotides, and the antisense strand has nucleotides at positions 2, 12, 14, and 16 of the nucleotide sequence 2 in the 5'-3' direction that are 2'-fluorinated nucleotides.

[0133] In some embodiments, the sense strand has nucleotides at positions 5, 7, and 9 of the nucleotide sequence 1 in the 5'-3' direction that are 2'-fluorinated nucleotides, and the antisense strand has nucleotides at positions 2, 7, 12, 14, and 16 of the nucleotide sequence 2 in the 5'-3' direction that are 2'-fluorinated nucleotides.

[0134] In some preferred embodiments, the sense strand comprises 19 nucleotides, wherein the 7th, 8th, and 9th nucleotides of the sense strand in the nucleotide sequence 1 along the 5'-3' direction are 2'-fluorinated nucleotides, and the remaining nucleotides are non-fluorinated nucleotides; the antisense strand comprises 21 nucleotides, wherein the 2nd, 4th, 12th, 14th, and 16th nucleotides of the antisense strand in the nucleotide sequence 2 along the 5'-3' direction are 2'-fluorinated nucleotides, and the remaining nucleotides are non-fluorinated nucleotides.

[0135] In some preferred embodiments, the sense strand comprises 19 nucleotides, wherein the 5th, 7th, and 9th nucleotides of the sense strand in the nucleotide sequence 1 along the 5'-3' direction are 2'-fluorinated nucleotides, and the remaining nucleotides are non-fluorinated nucleotides; the antisense strand comprises 21 nucleotides, wherein the 2nd, 4th, 12th, 14th, and 16th nucleotides of the antisense strand in the nucleotide sequence 2 along the 5'-3' direction are 2'-fluorinated nucleotides, and the remaining nucleotides are non-fluorinated nucleotides.

[0136] In some embodiments, the 5' end of the sense chain contains two thiophosphate bonds, and the 3' end of the sense chain contains 0-3 thiophosphate bonds; the 5' end and the 3' end of the antisense chain each contain two thiophosphate bonds.

[0137] In some embodiments, the antisense strand has two phosphate thioester bonds between the first and second nucleotides, and between the second and third nucleotides, starting from the 5' end; the antisense strand has two phosphate thioester bonds between the first and second nucleotides, and between the second and third nucleotides, starting from the 3' end; the sense strand has two phosphate thioester bonds between the first and second nucleotides, and between the second and third nucleotides, starting from the 5' end; and the sense strand has two phosphate thioester bonds between the first and second nucleotides, and between the second and third nucleotides, starting from the 3' end. There are 0-3 phosphate thioester bonds between the first, second, and third nucleotides of the positive chain. For example, there is no phosphate thioester bond at the 3' end of the positive chain. There is one phosphate thioester bond between the first and second nucleotides starting from the 3' end of the positive chain. There are two phosphate thioester bonds between the first and second nucleotides and between the second and third nucleotides starting from the 3' end of the positive chain. Alternatively, there are three phosphate thioester bonds between the first and second nucleotides, between the second and third nucleotides, and between the third and fourth nucleotides starting from the 3' end of the positive chain.

[0138] In some embodiments, the double-stranded oligonucleotide of this disclosure is any of the following siRNAs:

[0139]

[0140]

[0141] In another aspect, this disclosure provides an oligonucleotide conjugate comprising the aforementioned double-stranded oligonucleotide or a pharmaceutically acceptable salt thereof and a conjugating group conjugated to the double-stranded oligonucleotide or a pharmaceutically acceptable salt thereof, the conjugating group comprising a pharmaceutically acceptable targeting group and / or a delivery assistant group, the conjugating group being covalently or non-covalently linked to the double-stranded oligonucleotide.

[0142] In some embodiments, the targeting group and / or delivery auxiliary group includes proteins, carbohydrates, or lipids, wherein the proteins include natural proteins, synthetic polyamino acids, and / or antibodies; the carbohydrates include dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, amino sugars, and / or hyaluronic acid; and the lipids include fatty acids, sterols, and / or phospholipids.

[0143] In some embodiments, the targeting group may be a ligand conventionally used in the field of double-stranded oligonucleotide drug delivery, such as the various ligands described in WO2009082607A2, the entire disclosure of which is incorporated herein by reference. In some embodiments, the targeting group is selected from ligands capable of binding to cell surface receptors expressing the target gene, such as ligands capable of binding to receptors on the surface of mammalian liver parenchymal cells or ligands capable of binding to receptors on the surface of lung epithelial cells. In some embodiments, the delivery helper group is selected from groups capable of increasing the biocompatibility of the oligonucleotide conjugate in mammals.

[0144] In some embodiments, the conjugating group is L96. L96 is a well-known GalNAc delivery carrier in the art. As a conjugating group, it achieves targeted delivery of nucleic acid drugs by binding to ASGPR (desialyl glycoprotein receptor) in the liver, thereby improving drug concentration and efficacy in the liver. The structure of L96 is as follows:

[0145]

[0146] In some embodiments, the conjugating group is C16. C16 is a long-chain alkyl group having 16 carbon atoms. C16 conjugations enhance the targeting and delivery efficiency of oligonucleotides to specific cells or tissues (such as the central nervous system, eyes, and lungs) by coupling the oligonucleotide to a long-chain alkyl group (typically hexadecyl). The structure of a C16 conjugation is as follows: Where Base is a nucleotide base or a nucleotide base analogue, for example, 2'-O-hexadecyluridine has the following structure:

[0147] In some embodiments, the conjugation group is covalently or non-covalently linked to the double-stranded oligonucleotide or its pharmaceutically acceptable salt via a linker. The conjugation group can be linked to the double-stranded oligonucleotide molecule via a suitable linker, and those skilled in the art can select an appropriate linker based on the specific type of conjugation group. Such linkers, types of conjugation groups, and methods of linking to double-stranded oligonucleotides can be found in the disclosure of WO2015006740A2, the entire contents of which are incorporated herein by reference.

[0148] Generally, the conjugation group comprises at least one pharmaceutically acceptable target group and an optional linker, and the double-stranded oligonucleotide, the linker, and the target group are sequentially linked. In some embodiments, there are 1-6 target groups. In one embodiment, there are 2-4 target groups. The double-stranded oligonucleotide molecule can be non-covalently or covalently conjugated to the conjugation group, for example, it can be covalently conjugated to the conjugation group. The conjugation site of the double-stranded oligonucleotide to the conjugation group can be at the 3' or 5' end of the sense strand of the double-stranded oligonucleotide, at the 5' end of the antisense strand, or within the internal sequence of the double-stranded oligonucleotide. In some preferred embodiments, the conjugation site of the double-stranded oligonucleotide to the conjugation group is at the 3' end of the sense strand of the double-stranded oligonucleotide.

[0149] In some embodiments, the double-stranded oligonucleotide and the conjugation group are linked by acid-labile or reducible chemical bonds that can degrade in the acidic environment of the endosome, thus freeing the double-stranded oligonucleotide. For non-degradable conjugations, the conjugation group can be attached to the positive and negative strands of the double-stranded oligonucleotide to minimize the impact of the conjugation on the activity of the double-stranded oligonucleotide.

[0150] In some embodiments, the conjugation group may be attached to a phosphate group, a 2'-hydroxyl group, or a base of the nucleotide. In some embodiments, the conjugation group may be attached to a 3'-hydroxyl group, in which case the nucleotides are linked by a 2'-5' phosphodiester bond. When the conjugation group is attached to the end of a double-stranded oligonucleotide chain, it is typically attached to a phosphate group of the nucleotide; when the conjugation group is attached to the inner sequence of a double-stranded oligonucleotide, it is typically attached to a ribose ring or a base. Various connection methods can be found in: Muthiah Manoharan et al., ACS Chemical Biology, 2015, 10(5): 1181-7, the contents of which are incorporated herein by reference.

[0151] In another aspect, this disclosure provides a composition comprising the aforementioned double-stranded oligonucleotide or a pharmaceutically acceptable salt thereof, and / or the aforementioned oligonucleotide conjugate, and optionally a pharmaceutically acceptable carrier.

[0152] In some implementations, "pharmaceutically acceptable carrier" means a pharmaceutically acceptable substance, composition, or carrier involved in carrying or transporting the target compound from one organ or site of the body to another, such as liquid or solid fillers, diluents, excipients, manufacturing aids (e.g., lubricants, magnesium talc, calcium stearate, zinc stearate, or stearic acid), or solvent encapsulation materials. Each carrier must be "acceptable" in the sense of compatibility with other components of the formulation and harmlessness to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) powdered astragalus; (5) malt; (6) gelatin; (7) lubricants, such as magnesium stearate, sodium lauryl sulfate, and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) polyols. (12) Alcohols, such as glycerol, sorbitol, mannitol, and polyethylene glycol; (13) Esters, such as ethyl oleate and ethyl laurate; (14) Agar; (15) Buffers, such as magnesium hydroxide and aluminum hydroxide; (16) Alginate; (17) Atherless water; (18) Isotonic saline; (19) Ringer's solution; (20) Ethanol; (21) pH buffer solutions; (22) Polyesters, polycarbonates, and / or polyanhydrides; (23) Additives, such as peptides and amino acids; (24) Serum components, such as serum albumin, HDL, and LDL; and (25) Other non-toxic and compatible substances used in pharmaceutical formulations.

[0153] In some embodiments, the pharmaceutically acceptable carrier may be a carrier conventionally used in the field of double-stranded oligonucleotide delivery, such as, but not limited to, magnetic nanoparticles (e.g., Fe3O4 or Fe2O3-based nanoparticles), carbon nanotubes, mesoporous silica, calcium phosphate nanoparticles, polyethyleneimine, polylysine, chitosan, 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), poly(D- or L-type lactic acid / hydroxyacetic acid copolymer) (PLGA), poly(2-aminoethylethylene phosphate) (PPEEA), and poly(2-dimethylaminoethylmethacrylate) (PDMAEMA), and one or more of their derivatives.

[0154] In some embodiments, the composition further comprises one or more additional therapeutic components.

[0155] In some embodiments, the composition is formulated for administration via ocular, vaginal, rectal, nasal, transdermal, subcutaneous, intravenous, intra-arterial, intralymphatic, intrabronchial, intrapleural, intraperitoneal, cerebrospinal, or intramuscular injection, or for administration to the lungs, intrathecal, or intracardiac.

[0156] These formulations can be conveniently presented in unit doses and can be prepared by any method well known in the field of pharmaceutical science. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the host being treated and the specific route of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be the amount of the compound that produces the therapeutic effect. Typically, this amount will be in the range of about 0.1% to about 99%, preferably from about 5% to about 70%, and most preferably from about 10% to about 30% of the active ingredient. In some embodiments, the formulations of the present invention comprise excipients selected from the group consisting of cyclodextrins, cellulose, liposomes, micelle forming agents (e.g., bile acids) and polymer carriers (e.g., polyesters and polyanhydrides); and the compounds of the present invention. In some embodiments, the aforementioned formulations make the compounds of the present invention orally bioavailable.

[0157] In another aspect, this disclosure provides a cell comprising the aforementioned double-stranded oligonucleotide or a pharmaceutically acceptable salt thereof.

[0158] In another aspect, this disclosure provides a kit or cassette comprising the aforementioned double-stranded oligonucleotide or a pharmaceutically acceptable salt thereof, the aforementioned oligonucleotide conjugate, the aforementioned composition, and / or the aforementioned cells, wherein the double-stranded oligonucleotide or a pharmaceutically acceptable salt thereof, the oligonucleotide conjugate, the composition, and / or the cells are packaged in a cassette, container, packaging, dispenser, pre-filled syringe, or vial.

[0159] In another aspect, this disclosure provides the use of the aforementioned double-stranded oligonucleotides or pharmaceutically acceptable salts thereof, the aforementioned oligonucleotide conjugates, the aforementioned compositions, the aforementioned cells, and / or the aforementioned kits or pods in the preparation of medicaments for treating viral diseases, neuromuscular diseases, bacterial infections, inflammatory and immune diseases, metabolic diseases, liver diseases, kidney diseases, cardiovascular diseases, eye diseases, lung diseases, tumors, or rare diseases.

[0160] Depending on whether local or systemic treatment is desired and depending on the area to be treated, the compositions of this disclosure can be administered in a variety of ways. In some embodiments, the compositions are formulated for administration via ocular, vaginal, rectal, intranasal, transdermal, subcutaneous, intravenous infusion, intra-arterial, intra-lymphatic, intrabronchial, intrapleural, intraperitoneal, cerebrospinal, or intramuscular injection, or for administration to the lungs, intrathecal, or intracardiac.

[0161] In some embodiments, the unit dose administered is less than 10 mg / kg body weight, or less than 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001 mg / kg body weight, and less than 200 nanomoles of RNA reagent (e.g., about 4.4 x 10⁻⁶). 16 RNA reagents at a concentration of 1500, 750, 300, 150, 75, 15, 7.5, 1.5, 0.75, 0.15, 0.075, 0.015, 0.0075, 0.0015, 0.00075, or 0.00015 nanomoles per kg of body weight.

[0162] The determined amount can be an amount that is effective in treating or preventing a disease or disorder (e.g., a disease or disorder related to the target RNA). This unit dose can be administered, for example, by injection (e.g., intravenous, subcutaneous, or intramuscular), inhalation, or topical application. In some embodiments, the dose may be less than 10, 5, 2, 1, or 0.1 mg / kg of body weight.

[0163] In some implementations, the unit dose is given less frequently than once a day, for example less than once every 2, 4, 8, or 30 days.

[0164] In another implementation, the unit dose is not administered at a frequency (e.g., not at a regular frequency). For example, the unit dose may be administered in a single dose.

[0165] In another aspect, this disclosure provides the use of the aforementioned double-stranded oligonucleotides or pharmaceutically acceptable salts thereof, the aforementioned oligonucleotide conjugates, the aforementioned compositions, the aforementioned cells, and / or the aforementioned kits or pods in the preparation of reagents or medicaments for inhibiting the expression of target genes in cells.

[0166] In another aspect, this disclosure provides a method for inhibiting the expression of a target gene in a cell, comprising delivering the aforementioned double-stranded oligonucleotide or a pharmaceutically acceptable salt thereof, the aforementioned oligonucleotide conjugate, the aforementioned composition, and / or the aforementioned cell to the cell to inhibit the expression of the target gene in the cell.

[0167] In some embodiments, the target gene can be various endogenous genes in the human or animal body, or pathogen genes that multiply in the human or animal body. Double-stranded oligonucleotides with specific nucleotide sequences and specific modification schemes can be designed and prepared based on the mRNA expressed by the target gene.

[0168] In some implementations, the target genes are selected from APP, DPP4, MAPT, INHBE, ACE2, ANGPTL3, ApoA, ApoB, ApoC, AR, ASK1, C5, Col1A1, CTGF, Ebola, FOXO1, FTO, FVII, FXI, FXII, GCGR, HBV, HCV, HSD17B13, p53, PCSK9, PNP, PLG, PKK, KNG, SARS-CoV-2, SCD1, SCNN1A, SOD1, STAT3, TIMP-1, TMPRSS6, XO, INSR, SREBF1, HDV, RPTOR, TLK2, LPA, C3, AGT, PNPLA3, ASGR1, F7, F12, F XI, APOCIII, APOB, APOL1, TTR, PCSK9, SCAP, KRAS, CD274, PDCD1, CFB, MASP2, ALAS1, MSTN, HAO1, LDHA, ANGPTL3, SERPINA1, HSD17B13, HAMP, LECT2, EGFR, VEGF, KIF11, AT3, CTNNB1, HMGB1, HIF1A, ATXN2, C9orf72, TARDBP, HTT, SNCA, FUS, ATXN3, SCN9A, SCN10A, CACNA1B, ATXN1, SCA1, SCA7, SCA8, MeCP2, PRNP, SOD1, CIDEB, DMPK, and STAT3. Among these, pathogen genes (e.g., HBV genes) include any gene from the pathogen.

[0169] In some embodiments, the target gene is selected from the amyloid precursor protein (APP), dipeptidyl peptidase-4 (DPP4), microtubule-associated protein tau (MAPT), and inhibin subunit beta E (INHBE).

[0170] In another aspect, this disclosure provides a method for treating or preventing a disease or condition in a subject, wherein the method comprises administering to the subject an amount sufficient to inhibit the expression of a gene causing the subject's disease, including the aforementioned double-stranded oligonucleotide or a pharmaceutically acceptable salt thereof, the aforementioned oligonucleotide conjugate, the aforementioned composition, and / or the aforementioned cells. In some embodiments, the disease or condition is selected from viral diseases, neuromuscular diseases, bacterial infections, inflammatory and immune diseases, metabolic diseases, liver diseases, kidney diseases, cardiovascular diseases, ophthalmic diseases, lung diseases, tumors, or rare diseases.

[0171] For the purpose of clarity and concise description, the features are described herein as part of some identical or separate embodiments; however, it will be understood that the scope of this disclosure may include some embodiments having a combination of all or some of the features described.

[0172] Example

[0173] To make the objectives, technical solutions, and advantages of the embodiments of this disclosure clearer, the technical solutions in the embodiments of this disclosure will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this disclosure, not all embodiments. Unless otherwise specified, the following embodiments and features can be combined with each other. The following embodiments are used to further illustrate this disclosure, but should not be construed as limiting this disclosure. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of this disclosure should be considered equivalent substitutions and are included within the protection scope of this disclosure.

[0174] Unless otherwise specified in this article, such reagents can be obtained from any molecular biology reagent supplier, and their quality / purity standards are applicable to molecular biology.

[0175] Example 1. Preparation of siRNA

[0176] The siRNA sequence was synthesized separately on a solid support via the sense strand (SS) and antisense strand (AS), and was obtained after deprotection, cleavage, purification, annealing, purification and lyophilization.

[0177] Solid-phase synthesis: Sensitive and antisense oligonucleotides were synthesized separately on a solid support using an automated oligonucleotide synthesizer, employing phosphoramide technology. The synthesizer, such as the AKTA Oligopilot (Cytiva) or Dr. Oligo 192XLc (Kunshan Berlik Precision Instruments Co., Ltd.), was used. Solid-phase synthesis began at the 3' end of the sequence, with monomers sequentially coupled into the sequence. Each coupling of a phosphoramide monomer involved four chemical steps: 1) unblocking or deprotection (de-hydroxyl protecting group); 2) coupling; 3) oxidation; and 4) end-capping. All phosphoramidite monomers, reagents, and purification consumables used were commercially available. For example, various phosphoramidite monomers (such as 5'-O-(4,4'-Dimethoxytrityl)-2'-O-methyl-Uridine-3'-CE-Phosphoramidite) were purchased from Shanghai Zhaowei Technology Development Co., Ltd., and reaction reagents (such as 40wt% methylamine aqueous solution, 28wt% ammonium hydroxide aqueous solution, etc.) were purchased from Sigma-Aldrich LLC. The siRNA synthesis and purification methods used in this paper are described in US20130178612A1 and US2015100197A1, the contents of which are incorporated herein by reference. The synthesis methods containing VPUm and APU structural sequences are described in J.Med.Chem. 2018, 61, 734-744, the contents of which are incorporated herein by reference.

[0178] (1) The synthesis of the justice chain

[0179] Solid-phase phosphoramide synthesis is a mature method for synthesizing oligonucleotides. A computer-controlled synthesizer is used, and the reaction takes place in a stainless steel column. The positive chain synthesis begins with a solid support loaded with a targeting ligand (e.g., L96), or directly with the solid support. Different starting materials, reagents, and solvents are injected sequentially from sequence 3' to 5' using the solid-phase synthesizer, linking phosphoramide nucleoside monomers one by one. The reaction process involves four cyclic steps: DMT protection removal, condensation, oxidation or thiolation, and end-capping. One nucleotide unit is linked in each cycle, yielding an oligonucleotide sequence of 19 or 21 nucleotides. After synthesis, the protecting group (2-cyanoethyl) is removed on the solid-phase column, and the synthesized sequence is cleaved from the solid support via ammonolysis. The sequence is filtered, the filter cake is washed with ethanol, and the filtrate and washings are collected and concentrated to obtain the crude positive chain. The crude product is purified by chromatography (SOURCE 15Q) and lyophilized to obtain the target product, the positive chain.

[0180] (2) Synthesis of antisense chains

[0181] Similar to the sense strand synthesis, the antisense strand is synthesized using a solid-phase synthesizer. Different starting materials, reagents, and solvents are injected sequentially from the 3' to 5' ends of the sequence through different tubing, linking phosphoramidine nucleoside monomers one by one. The reaction process involves four cyclic steps: DMT protection removal, condensation, oxidation or thiolation, and end-capping. One nucleotide unit is linked in each cycle, yielding an oligonucleotide sequence of 21 or 23 nucleotides. After synthesis, the protecting group (2-cyanoethyl) is removed on a solid-phase column, and the synthesized sequence is cleaved from the solid support via ammonolysis. The sequence is filtered, the filter cake is washed with ethanol, and the filtrate and washings are collected and concentrated to obtain the crude antisense strand. The crude product is purified by chromatography (SOURCE 15Q), ultrafiltered, and dried or lyophilized to obtain the target product, antisense siRNA.

[0182] (3) Preparation of double-stranded siRNA

[0183] The sense and antisense strands were dissolved separately in injection water and mixed in a defined ratio (1.01:1.0–1.2:1.0). The mixture was incubated at 30–50°C for 30–90 minutes and then cooled to room temperature. The double-stranded siRNA product was obtained by freeze-drying.

[0184] Example 2. In vitro activity assay of APP-siRNA

[0185] 1. Preparation of APP-siRNA

[0186] Candidate siRNA sequences were designed based on the mRNA sequence of the APP (Amyloid Precursor Protein) gene. The candidate siRNA sequences (specifically shown in Table 3) were complementary to human APP mRNA (Genbank accession number: NM_201414.3) or had one mismatch at the 3' end of the positive strand, or were also complementary to cynomolgus monkey APP mRNA (Genbank accession number: XM_005548883.3) or had no more than two mismatches. The siRNA naked sequences shown in Table 3 were modified according to the nucleotide modification pattern of this disclosure, and APP-siRNA modified sequences were prepared according to the method in Example 1. The obtained modified sequences are shown in Table 4.

[0187] Table 3. Exemplary APP-siRNA naked sequences

[0188]

[0189] Table 4. Exemplary APP-siRNA Modification Sequences

[0190]

[0191]

[0192] 2. In vitro activity assay

[0193] (1) Cell culture and transfection:

[0194] Human neuroblastoma cells (also known as Be2C cells) (Tongpai (Shanghai) Biotechnology Co., Ltd., catalog number BE(2)-C cell identification) were collected and placed in a 37℃, 5% CO2 incubator. They were cultured using DMEM, high glucose (Thermo, catalog number 11965-092), with the addition of 10% FBS (GIBCO, 12483020) and 1% penicillin-streptomycin (GIBCO, 15140-122). When the cell confluence reached 90%, the cells were digested with trypsin-EDTA (Thermo, 25200-072), counted using a Countstar (IC1000), and seeded with 190 μl of cell suspension per well in 96-well plates. The seeding number of Be2C cells was 1*102. 4 Cells / wells will adhere to the culture vessel the following day for transfection.

[0195] Transfection was performed using Lipofectamine™ RNAiMAX (thermofisher, 13778150). A transfection complex was prepared by mixing 2.2 μl (2 μM) diluted siRNA (as shown in Table 4), 19.1 μl Opti-MEM (thermofisher, 1105821), and 0.7 μl RNAiMAX. After incubation for 5 minutes, the transfection complex was added to the cells (two technical replicates per complex), 10 μl per well, resulting in a final siRNA concentration of 10 nM. The cells were incubated at 37°C in a 5% CO2 incubator for 24 hours.

[0196] (2) RNA extraction and detection

[0197] (i) One day after transfection, remove the culture medium; wash with PBS; add 200 μl of lysis buffer to each well, incubate at room temperature for 15 minutes, and then rehydrate. RNA was extracted using a 96-throughput automated nucleic acid extractor (HW-96 series). RNA concentration was measured for subsequent experiments. The extracted RNA can be used immediately for subsequent experiments or stored at -80℃ for later use.

[0198] (ii) Use IIQ RT SuperMix for qPCR (+gDNA wiper) reverse transcription kit (Novazia, R223-01) for cDNA synthesis:

[0199] Prepare a mixture in an RNase-free centrifuge tube: 4 μl 4×g DNA wiper Mix, 1 μg template RNA, and RNase-free ddH2O to a final volume of 16 μl to remove genomic DNA. Gently pipette to mix and incubate at 42°C for 2 min. Then, directly add 4 μl 5×HiScript II qRT SuperMix II to the reaction tube and gently pipette to mix. Incubate in a PCR instrument (Bio-Rayer C1000 Touch PCR instrument) at 50°C for 15 min, then at 85°C for 5 sec, and finally at 4°C. The product can be used immediately for qPCR reactions or stored at -20°C and used within six months. For long-term storage, aliquot and store at -80°C. Avoid repeated freeze-thaw cycles for cDNA.

[0200] (iii) Quantitative analysis using ChamQ SYBR qPCR Master Mix (Novazia, Q311-02):

[0201] Prepare a 20 μl mixture by adding 10 μl 2×ChamQ SYBR qPCR Master Mix, 0.5 μl Forward primer (Ruiboxingke), 0.5 μl Reverse primer (Ruiboxingke), 1 μl Template cDNA, and 8 μl ddH2O. Each sample was tested in triplicate. The 96-well plate was placed in a qPCR instrument (Quantstudio 5), and the following program was executed: pre-denaturation, 95℃, 30 sec; amplification, 95℃, 10 sec, 60℃, 30 sec, 40 cycles; melting curve, 95℃, 15 sec, 60℃, 60 sec, 95℃, 15 sec.

[0202] (3) Data statistical analysis:

[0203] Export the data to Excel format and normalize the control group using CTAPP-CTGAPDH. To calculate the fold change in relative silencing efficiency, the data were analyzed using the ΔΔCT method. The mean and standard deviation of the three parallel replicates were calculated.

[0204] The results of the APP siRNA activity assay in Be2C cells are shown in Table 5.

[0205] As shown in Table 5, when the dosage is 10 nM, the inhibition rate of APP mRNA modified by some siRNAs can reach or approach 50%, such as M1, M5, M6, M8, M9, and M10; the inhibition rate of APP mRNA modified by some siRNAs can reach or approach 70%, such as M8 and M9.

[0206] Table 5. APP siRNA knockdown levels in Be2C cells

[0207]

[0208]

[0209] Example 3. In vitro activity experiment of DPP4-siRNA in Hep3B cells

[0210] 1. Preparation of DPP4-siRNA

[0211] Candidate siRNA sequences were designed based on the mRNA sequence of the DPP4 (Dipeptidyl peptidase-4) gene. The candidate siRNA sequences (specifically shown in Table 6) were complementary to human DPP4 mRNA (Genbank accession number: NM_001935.4) or had one mismatch at the 3' end of the positive strand, or were also complementary to cynomolgus monkey DPP4 mRNA (Genbank accession number: XM_005573318.4) or had no more than two mismatches. The siRNA naked sequences shown in Table 6 were modified according to the nucleotide modification pattern of this disclosure, and APP-siRNA modified sequences were prepared according to the method in Example 1. The obtained modified sequences are shown in Table 7.

[0212] Table 6. Exemplary naked DPP4-siRNA sequences

[0213]

[0214] Table 7. Exemplary DPP4-siRNA Modification Sequences

[0215]

[0216]

[0217]

[0218] 2. In vitro activity assay

[0219] (1) Cell culture and transfection:

[0220] Hep3B cells (Wuhan Pronoss Biotechnology Co., Ltd., catalog number CL-0102) were placed in a 37℃, 5% CO2 incubator using DMEM medium (Shanghai Biotechnology Co., Ltd., catalog number iCell-0001) with 10% FBS (GIBCO, 12483020) and 1% penicillin-streptomycin (GIBCO, 15140-122) added. When the cell confluence reached 90%, the cells were digested with trypsin-EDTA (Thermo, 25200-072), counted using a counter (Countstar, IC1000), and seeded with 190 μl of cell suspension per well into 96-well plates at a seeding density of 2*10⁴ cells / well. The cells were then allowed to adhere the following day for transfection.

[0221] Transfection was performed using Lipofectamine™ RNAiMAX (thermofisher, 13778150). A transfection complex was prepared by mixing 2.2 μl (100 nM, 20 nM, or 10 nM) diluted siRNA (as shown in Table 7), 19.1 μl Opti-MEM (thermofisher, 1105821), and 0.7 μl RNAiMAX. After incubation for 5 minutes, the transfection complex was added to the cells (two technical replicates per complex), 10 μl per well, resulting in a final siRNA concentration of 0.5 nM. The cells were incubated at 37°C in a 5% CO2 incubator for 24 hours.

[0222] (2) RNA extraction and detection

[0223] (i) Total RNA extraction using the RNA-Quick Purification Kit (RN001, Yishan Biotechnology): Remove the 12-well plate from the incubator, aspirate the culture medium, wash once with an appropriate amount of PBS, add 500 μl of lysis buffer to each well, and transfer the supernatant to a new 1.5 ml centrifuge tube. Add 500 μl of anhydrous ethanol to the lysed cells and mix thoroughly (if precipitation occurs, this is normal; continue the operation). Invert the centrifuge tube several times, or use a pipette to forcefully aspirate 10 times to disperse the precipitate, then add the liquid to the centrifuge column. Place the centrifuge tube symmetrically in a centrifuge (Eppendorf, 5430) and centrifuge at 4000×g for 1 min. Remove the centrifuge tube, add 500 μl of wash buffer to the column, centrifuge at 12000×g for 1 min, remove the column after centrifugation, discard the waste liquid, reassemble the RNA column into the collection tube, centrifuge the empty tube once to remove any remaining wash buffer. Place the column on a clean, RNase-free 1.5 ml centrifuge tube and allow it to air dry for 2 minutes. Add 30 μl of elution buffer to the center of the RNA column membrane, incubate at room temperature for 2 minutes, centrifuge at 2000 × g for 1 min, and after the RNA is eluted, place it on ice. Measure the concentration of the eluted RNA for subsequent experiments. The extracted RNA can be used immediately for subsequent experiments or stored at -80℃ for later use.

[0224] (ii) Use IIQ RT SuperMix for qPCR (+gDNA wiper) reverse transcription kit (Novazia, R223-01) for cDNA synthesis:

[0225] Prepare a mixture in an RNase-free centrifuge tube: 4 μl 4×g DNA wiper Mix, 1 μg template RNA, and RNase-free ddH2O to a final volume of 16 μl to remove genomic DNA. Gently pipette to mix and incubate at 42°C for 2 min. Then, directly add 4 μl 5×HiScript II qRT SuperMix II to the reaction tube and gently pipette to mix. Incubate in a PCR instrument (Applied Biosystems, 9700) at 50°C for 15 min, then at 85°C for 5 sec, and finally at 4°C. The product can be used immediately for qPCR or stored at -20°C and used within six months. For long-term storage, aliquot and store at -80°C. Avoid repeated freeze-thaw cycles for cDNA.

[0226] (iii) Quantitative analysis using ChamQ SYBR qPCR Master Mix (Novazia, Q311-02):

[0227] Prepare a 20 μl mixture by adding 10 μl of 2×ChamQ SYBR qPCR Master Mix, 0.5 μl of Forward primer (Ruiboxingke), 0.5 μl of Reverse primer (Ruiboxingke), 1 μl of Template cDNA, and 8 μl of ddH2O. Each sample was tested in triplicate. The 96-well plate was placed in a qPCR instrument (ROCGENE, Archimed). The following program was executed: pre-denaturation, 95℃, 30 sec; amplification, 95℃, 10 sec, 60℃, 30 sec, 40 cycles; melting curve, 95℃, 15 sec, 60℃, 60 sec, 95℃, 15 sec.

[0228] (3) Data statistical analysis:

[0229] Export the data to Excel format using CT. DPP4 -CT GAPDH The control group was normalized. To calculate the fold change in relative silencing efficiency, the data were analyzed using the ΔΔCT method. The mean and standard deviation of the three parallel replicates were calculated.

[0230] The results of the DPP4 siRNA activity assay in Hep3B cells are shown in Table 8.

[0231] As shown in Table 8, when the dosage is 0.5 nM, the inhibition rate of DPP4 mRNA modified by some siRNAs can reach or approach 50%, such as M1, M4, M5, M8, and M9; the inhibition rate of DPP4 mRNA modified by some siRNAs can reach or approach 70-80%, such as M8 and M9.

[0232] Table 8. DPP4 siRNA knockdown levels in Hep3B

[0233]

[0234] Example 4. In vitro activity assay of MAPT-siRNA

[0235] 1. Preparation of MAPT-siRNA

[0236] Candidate siRNA sequences were designed based on the mRNA sequence of the MAPT (microtubule-associated protein tau) gene. The candidate siRNA sequences (specifically shown in Table 9) were complementary to human MAPT mRNA (Genbank accession number: NM_016841.4) or had one mismatch at the 3' end of the positive strand, or were also complementary to cynomolgus monkey MAPT mRNA (Genbank accession number: XM_005584540.2) or had no more than two mismatches. The siRNA naked sequences shown in Table 9 were modified according to the nucleotide modification pattern of this disclosure, and APP-siRNA modified sequences were prepared according to the method in Example 1. The obtained modified sequences are shown in Table 10.

[0237] Table 9. Exemplary MAPT-siRNA naked sequences

[0238]

[0239] Table 10. Exemplary MAPT-siRNA Modification Sequences

[0240]

[0241]

[0242] 2. In vitro activity assay

[0243] (1) Cell culture and transfection:

[0244] Human neuroblastoma cells (also known as Be2C cells) (Tongpai (Shanghai) Biotechnology Co., Ltd., catalog number BE(2)-C cell identification) were collected and placed in a 37℃, 5% CO2 incubator. They were cultured using DMEM, high glucose (Thermo, catalog number 11965-092), with the addition of 10% FBS (GIBCO, 12483020) and 1% penicillin-streptomycin (GIBCO, 15140-122). When the cell confluence reached 90%, the cells were digested with trypsin-EDTA (Thermo, 25200-072), counted using a Countstar (IC1000), and seeded with 190 μl of cell suspension per well in 96-well plates. The seeding number of Be2C cells was 1*102. 4 Cells / wells will adhere to the culture vessel the following day for transfection.

[0245] Transfection was performed using Lipofectamine™ RNAiMAX (thermofisher, 13778150). A transfection complex was prepared by mixing 2.2 μl (2 μM) diluted siRNA (as shown in Table 10), 19.1 μl Opti-MEM (thermofisher, 1105821), and 0.7 μl RNAiMAX. After incubation for 5 minutes, the transfection complex was added to the cells (two technical replicates per complex), 10 μl per well, resulting in a final siRNA concentration of 10 nM. The cells were incubated at 37°C in a 5% CO2 incubator for 24 hours.

[0246] (2) RNA extraction and detection

[0247] (i) One day after transfection, remove the culture medium; wash with PBS; add 200 μl of lysis buffer to each well, incubate at room temperature for 15 minutes, and then rehydrate. RNA was extracted using a 96-throughput automated nucleic acid extractor (HW-96 series). RNA concentration was measured for subsequent experiments. The extracted RNA can be used immediately for subsequent experiments or stored at -80℃ for later use.

[0248] (ii) Use IIQ RT SuperMix for qPCR (+gDNAwiper) Reverse Transcription Kit (Novazia, R223-01) for cDNA synthesis:

[0249] Prepare a mixture in an RNase-free centrifuge tube: 4 μl 4×g DNA wiper Mix, 1 μg template RNA, and RNase-free ddH2O to a final volume of 16 μl to remove genomic DNA. Gently pipette to mix and incubate at 42°C for 2 min. Then, directly add 4 μl 5×HiScript II qRT SuperMix II to the reaction tube and gently pipette to mix. Incubate in a PCR instrument (Bio-Rayer C1000 Touch PCR instrument) at 50°C for 15 min, then at 85°C for 5 sec, and finally at 4°C. The product can be used immediately for qPCR reactions or stored at -20°C and used within six months. For long-term storage, aliquot and store at -80°C. Avoid repeated freeze-thaw cycles for cDNA.

[0250] (iii) Quantitative analysis using ChamQ SYBR qPCR Master Mix (Novazia, Q311-02):

[0251] Prepare a 20 μl mixture by adding 10 μl 2×ChamQ SYBR qPCR Master Mix, 0.5 μl Forward primer (Ruiboxingke), 0.5 μl Reverse primer (Ruiboxingke), 1 μl Template cDNA, and 8 μl ddH2O. Each sample was tested in triplicate. The 96-well plate was placed in a qPCR instrument (Quantstudio 5), and the following program was executed: pre-denaturation, 95℃, 30 sec; amplification, 95℃, 10 sec, 60℃, 30 sec, 40 cycles; melting curve, 95℃, 15 sec, 60℃, 60 sec, 95℃, 15 sec.

[0252] (3) Data statistical analysis:

[0253] Export the data to Excel format using CT. MAPT -CT GAPDH The control group was normalized. To calculate the fold change in relative silencing efficiency, the data were analyzed using the ΔΔCT method. The mean and standard deviation of the three parallel replicates were calculated.

[0254] The results of the MAPT-siRNA activity experiment in Be2C cells are shown in Table 11.

[0255] As shown in Table 11, when the dosage is 10 nM, the inhibition rate of certain modified siRNAs of MAPT mRNA can reach or approach 60%, such as M1, M5, M6, M8, and M9; the inhibition rate of certain modified siRNAs of MAPT mRNA can reach or approach 70-80%, such as M8 and M9.

[0256] Table 11. MAPT siRNA knockdown levels in Be2C cells

[0257]

[0258]

[0259] Example 5. In vitro activity experiment of INHBE-siRNA in Hep3B cells

[0260] 1. Preparation of INHBE-siRNA

[0261] Candidate siRNA sequences were designed based on the mRNA sequence of the INHBE (Inhibin Subunit Beta E) gene. The candidate siRNA sequences (as shown in Table 3) were complementary to human INHBE mRNA (Genbank accession number: NM_031479.5) or had a mismatch at the 3' end of the positive strand, or were also compatible with cynomolgus monkey INHBE mRNA (Genbank accession number:

[0262] XM_005571319.4) complementary or no more than 2 mismatches. The siRNA naked sequences shown in Table 12 were modified according to the nucleotide modification pattern of this disclosure, and APP-siRNA modified sequences were prepared according to the method of Example 1. The specific modified sequences obtained are shown in Table 13.

[0263] Table 12. Naked sequences of INHBE-siRNA

[0264]

[0265] Table 13. INHBE-siRNA Modification Sequences

[0266]

[0267]

[0268] 2. In vitro activity assay

[0269] (1) Cell culture and transfection:

[0270] Hep3B cells (Wuhan Pronoss Biotechnology Co., Ltd., catalog number CL-0102) were placed in a 37℃, 5% CO2 incubator using DMEM medium (Shanghai Biotechnology Co., Ltd., catalog number iCell-0001) with 10% FBS (GIBCO, 12483020) and 1% penicillin-streptomycin (GIBCO, 15140-122) added. When the cell confluence reached 90%, the cells were digested with trypsin-EDTA (Thermo, 25200-072), counted using a counter (Countstar, IC1000), and seeded with 190 μl of cell suspension per well into 96-well plates at a seeding density of 2*10⁴ cells / well. The cells were then allowed to adhere the following day for transfection.

[0271] Transfection was performed using Lipofectamine™ RNAiMAX (thermofisher, 13778150). A transfection complex was prepared by mixing 2.2 μl (2 μM) diluted siRNA (as shown in Table 13), 19.1 μl Opti-MEM (thermofisher, 1105821), and 0.7 μl RNAiMAX. After incubation for 5 minutes, the transfection complex was added to the cells (two technical replicates per complex), 10 μl per well, resulting in a final siRNA concentration of 10 nM. The cells were incubated at 37°C in a 5% CO2 incubator for 24 hours.

[0272] (2) RNA extraction and detection

[0273] (i) Total RNA was extracted using the RNA-Quick Purification Kit (RN001, Yishan Biotechnology):

[0274] Remove the 12-well plate from the incubator, aspirate the culture medium, wash once with an appropriate amount of PBS, add 500 μl of lysis buffer to each well, and transfer the supernatant to a new 1.5 ml centrifuge tube. Add 500 μl of anhydrous ethanol to the lysed cells and mix thoroughly (if precipitation occurs, this is normal; continue the procedure). Invert the centrifuge tube several times, or use a pipette to forcefully aspirate 10 times to disperse the precipitate, then add the liquid to the centrifuge column. Place the centrifuge tube symmetrically in a centrifuge (Eppendorf, 5430) and centrifuge at 4000 × g for 1 min. Remove the centrifuge tube, add 500 μl of wash buffer to the column, and centrifuge at 12000 × g for 1 min. After centrifugation, remove the column, discard the waste liquid, and reassemble the RNA column into the collection tube. Centrifuge the empty tube once to remove any remaining wash buffer. Place the column on a clean, RNase-free 1.5 ml centrifuge tube and allow it to air dry for 2 minutes. Add 30 μl of elution buffer to the center of the RNA column membrane, incubate at room temperature for 2 minutes, centrifuge at 2000×g for 1 min to elute the RNA, and then place on ice. Measure the concentration of the eluted RNA for subsequent experiments. The extracted RNA can be used immediately for subsequent experiments or stored at -80℃ for later use.

[0275] (ii) Use IIQ RT SuperMix for qPCR (+gDNA wiper) reverse transcription kit (Novazia, R223-01) for cDNA synthesis:

[0276] Prepare a mixture in an RNase-free centrifuge tube: 4 μl 4×g DNA wiper Mix, 1 μg template RNA, and RNase-free ddH2O to a final volume of 16 μl to remove genomic DNA. Gently pipette to mix and incubate at 42°C for 2 min. Then, directly add 4 μl 5×HiScript II qRT SuperMix II to the reaction tube and gently pipette to mix. Incubate in a PCR instrument (Applied Biosystems, 9700) at 50°C for 15 min, then at 85°C for 5 sec, and finally at 4°C. The product can be used immediately for qPCR or stored at -20°C and used within six months. For long-term storage, aliquot and store at -80°C. Avoid repeated freeze-thaw cycles for cDNA.

[0277] (iii) Quantitative analysis using ChamQ SYBR qPCR Master Mix (Novazia, Q311-02):

[0278] Prepare a 20 μl mixture by adding 10 μl of 2×ChamQ SYBR qPCR Master Mix, 0.5 μl of Forward primer (Ruibo Kexing), 0.5 μl of Reverse primer (Ruibo Kexing), 1 μl of Template cDNA, and 8 μl of ddH2O. Each sample was tested in triplicate. The 96-well plate was placed in a qPCR instrument (ROCGENE, Archimed). The following program was executed: pre-denaturation, 95℃, 30 sec; amplification, 95℃, 10 sec, 60℃, 30 sec, 40 cycles; melting curve, 95℃, 15 sec, 60℃, 60 sec, 95℃, 15 sec.

[0279] (3) Data statistical analysis:

[0280] Export the data to Excel format using CT. INHBE -CT GAPDH The control group was normalized. To calculate the fold change in relative silencing efficiency, the data were analyzed using the ΔΔCT method. The mean and standard deviation of the three parallel replicates were calculated.

[0281] The results of the activity experiment of INHBE-siRNA in Hep3B cells are shown in Table 14.

[0282] As shown in Table 14, when the dosage is 10 nM, the inhibition rate of some modified siRNAs of INHBE mRNA can reach or approach 60-70%, such as M1, M2, M6, M8, and M9; the inhibition rate of some modified siRNAs of INHBE mRNA can reach or approach 80%, such as M8 and M9.

[0283] Table 14. INHBE siRNA knockdown levels in Hep3B cells

[0284]

[0285]

[0286] Example 6. Activity evaluation experiment of DPP4-siRNA in mice

[0287] SPF-grade male C57BL / 6J mice, aged 6–8 weeks (Speford (Beijing) Biotechnology Co., Ltd.), were randomly divided into groups of 6 mice each. C57BL / 6 mice were subcutaneously administered 3 mg / kg of the DPP4-siRNA conjugated vector L96 (shown in Table 15) and physiological saline (NC, negative control). On day 14 post-administration, mice were euthanized, liver samples were collected, and liver mRNA was extracted and analyzed by RT-PCR. Specifically, reverse transcription, qPCR quantification, and statistical analysis were performed according to the method described in Example 3. During the experiment, no animals showed signs of death or impending death. Clinical observation revealed no significant abnormalities in any animal. Experimental data are shown in Table 16. As shown in Table 16, at a dosage of 3 mg / kg, the in vivo inhibition rate of DPP4 mRNA modified with the siRNA reagent reached or approached 65%, for example, M4, M5, M8, and M9.

[0288] Table 15. DPP4-siRNA reagents for conjugated vector L96

[0289]

[0290] Table 16. In vivo inhibition rate of DPP4 siRNA modified sequence of conjugated vector L96

[0291]

[0292]

Claims

1. A double-stranded oligonucleotide comprising a sense strand and an antisense strand, each of the sense strand and the antisense strand comprising 19-23 nucleotides, wherein the antisense strand is at least partially complementary to the mRNA expressed by a target gene, and the sense strand and the antisense strand are at least partially anticomplementary to form a double-stranded region, characterized in that, The positive strand includes nucleotide sequence 1, which is at least 11 nucleotides in length, and may also include additional nucleotide sequences at the 5' end and / or 3' end of nucleotide sequence 1; the antisense strand includes nucleotide sequence 2, which is at least 16 nucleotides in length, and may also include additional nucleotide sequences at the 5' end and / or 3' end of nucleotide sequence 2, wherein the nucleotides at positions 2, 12, 14, and 16 in the 5'-3' direction are 2'-fluorinated nucleotides or the nucleotides at positions 2, 6, 9, 14, and 16 are 2'-fluorinated nucleotides.

2. The double-stranded oligonucleotide according to claim 1, wherein, The justice chain and antisense chain are basically oppositely complementary, substantially oppositely complementary, or completely oppositely complementary. Preferably, the sense strand contains 19, 20, or 21 nucleotides, and the antisense strand contains 19, 20, 21, 22, or 23 nucleotides; more preferably, the sense strand contains 19 nucleotides, and the antisense strand contains 21 nucleotides. Preferably, the nucleotides in the sense strand and / or antisense strand are each independently modified nucleotides, and the modified nucleotides are selected from at least one of 2′-fluoro-modified nucleotides, 2′-O-methyl-modified nucleotides, 2′-O-methoxyethyl-modified nucleotides, 2′-O-alkyl-modified nucleotides, 2′-O-allyl-modified nucleotides, bicyclic nucleic acids (BNA), deoxyribonucleotides, 2′-amino-modified nucleotides, baseless nucleotides, reverse baseless nucleotides, and reverse deoxyribonucleotides; Preferably, the 5' end and / or 3' end of the sense strand and / or antisense strand comprises at least one of a baseless nucleotide, a reverse a baseless nucleotide, and a reverse deoxyribonucleotide; Preferably, the 5' end and / or 3' end of the sense chain and / or antisense chain each independently contain at least one phosphate thioester bond; More preferably, the 5' end and / or 3' end of the sense chain and / or antisense chain each independently contain at least two phosphate thioester bonds; More preferably, the thiophosphate bond is present between the first and second nucleotides starting from the 5' end, between the second and third nucleotides; and / or between the first and second nucleotides starting from the 3' end, between the second and third nucleotides.

3. The double-stranded oligonucleotide according to claim 1 or 2, wherein, The antisense strand contains at least five 2'-fluorine modified nucleotides; Preferably, at least five nucleotides at positions 2, 4, 6, 7, 9, 12, 14 and 16 of the antisense strand in the nucleotide sequence 2 along the 5'-3' direction are 2'-fluorinated nucleotides; More preferably, the antisense chain is selected from any one of the following combinations: (1) The antisense strand consists of nucleotides at positions 2, 4, 12, 14 and 16 of the nucleotide sequence 2 in the 5'-3' direction, which are 2'-fluorine modified nucleotides; (2) The antisense strand consists of nucleotides at positions 2, 7, 12, 14 and 16 of the nucleotide sequence 2 in the 5'-3' direction, which are 2'-fluorine modified nucleotides; (3) The antisense strand consists of nucleotides at positions 2, 6, 9, 14 and 16 of the nucleotide sequence 2 in the 5'-3' direction, which are 2'-fluorine modified nucleotides; Preferably, the nucleotides in the antisense strand, except for those that are 2'-fluorinated, are all non-fluorinated nucleotides. Preferably, the 5' end and 3' end of the antisense chain each independently contain at least one phosphate thioester bond; More preferably, the 5' end and 3' end of the antisense chain each contain two thiophosphate bonds; More preferably, the thiophosphate bond of the antisense chain is present between the first and second nucleotides starting from the 5' end, between the second and third nucleotides; and between the first and second nucleotides starting from the 3' end, and between the second and third nucleotides; Preferably, the positive strand contains at least three 2'-fluorine modified nucleotides; Preferably, at least three nucleotides at positions 5, 7, 8, 9, and 11 of the positive strand of the nucleotide sequence 1 in the 5'-3' direction are 2'-fluorinated nucleotides; More preferably, the justice chain is selected from any one of the following combinations: (1) The nucleotides at positions 5, 7 and 9 of the positive strand of the nucleotide sequence 1 in the 5'-3' direction are 2'-fluorine modified nucleotides; (2) The nucleotides at positions 7, 8 and 9 of the positive strand of the nucleotide sequence 1 in the 5'-3' direction are 2'-fluorine modified nucleotides; (3) The nucleotides at positions 7, 9 and 11 of the positive strand of the nucleotide sequence 1 in the 5'-3' direction are 2'-fluorine modified nucleotides; (4) The nucleotides at positions 5, 8 and 9 of the positive strand of the nucleotide sequence 1 in the 5'-3' direction are 2'-fluorine modified nucleotides; (5) The nucleotides at positions 5, 9 and 11 of the positive strand of the nucleotide sequence 1 in the 5'-3' direction are 2'-fluorine modified nucleotides; (6) The nucleotides at positions 8, 9 and 11 of the positive strand of the nucleotide sequence 1 in the 5'-3' direction are 2'-fluorine modified nucleotides; Preferably, the positive strand contains at least four 2'-fluorine modified nucleotides; Preferably, at least four nucleotides at positions 5, 7, 8, 9, and 11 of the positive strand of the nucleotide sequence 1 in the 5'-3' direction are 2'-fluorinated nucleotides; More preferably, the justice chain is selected from any one of the following combinations: (1) The nucleotides at positions 5, 7, 8 and 9 of the positive strand of the nucleotide sequence 1 in the 5'-3' direction are 2'-fluorine modified nucleotides; (2) The nucleotides at positions 7, 8, 9 and 11 of the positive strand of the nucleotide sequence 1 in the 5'-3' direction are 2'-fluorine modified nucleotides; (3) The nucleotides at positions 5, 8, 9, and 11 of the positive strand of nucleotide sequence 1 in the 5'-3' direction are 2'-fluorinated nucleotides; and (4) The nucleotides at positions 5, 7, 9 and 11 of the positive strand of the nucleotide sequence 1 in the 5'-3' direction are 2'-fluorine modified nucleotides; Preferably, the nucleotides in the positive strand, except for those modified by 2'-fluorine, are all non-fluorinated nucleotides; Preferably, the 5' end and / or 3' end of the positive chain each independently contain at least one phosphate thioester bond; More preferably, the 5' end of the positive chain contains two thiophosphate bonds, and the 3' end of the positive chain contains 0-3 thiophosphate bonds; More preferably, the justice chain is selected from any one of the following combinations: (1) There are two phosphate thioester bonds between the first and second nucleotides and between the second and third nucleotides starting from the 5' end of the positive strand, and there are no phosphate thioester bonds at the 3' end of the positive strand; (2) There are two phosphate thioester bonds between the first and second nucleotides starting from the 5' end of the positive strand, and between the second and third nucleotides; and there is one phosphate thioester bond between the first and second nucleotides starting from the 3' end of the positive strand. (3) There are two thiophosphate bonds between the first and second nucleotides and between the second and third nucleotides starting from the 5' end of the positive strand, and there are two thiophosphate bonds between the first and second nucleotides and between the second and third nucleotides starting from the 3' end of the positive strand; (4) There are two thiophosphate bonds between the first and second nucleotides and between the second and third nucleotides starting from the 5' end of the positive strand, and there are three thiophosphate bonds between the first and second nucleotides, between the second and third nucleotides and between the third and fourth nucleotides starting from the 3' end of the positive strand.

4. The double-stranded oligonucleotide according to any one of claims 1-3, wherein, The sense strand contains 19 nucleotides, the antisense strand contains 21 nucleotides, and the double-stranded oligonucleotide is selected from any of the following combinations: (1) The sense strand has nucleotides at positions 7, 8 and 9 of the nucleotide sequence 1 in the 5'-3' direction that are 2'-fluorinated nucleotides, and the antisense strand has nucleotides at positions 2, 4, 12, 14 and 16 of the nucleotide sequence 2 in the 5'-3' direction that are 2'-fluorinated nucleotides. (2) The sense strand has 2'-fluorinated nucleotides at positions 5, 7 and 9 of the nucleotide sequence 1 in the 5'-3' direction, and the antisense strand has 2'-fluorinated nucleotides at positions 2, 4, 12, 14 and 16 of the nucleotide sequence 2 in the 5'-3' direction. (3) The sense strand has nucleotides at positions 7, 9 and 11 of the nucleotide sequence 1 in the 5'-3' direction that are 2'-fluorinated nucleotides, and the antisense strand has nucleotides at positions 2, 7, 12, 14 and 16 of the nucleotide sequence 2 in the 5'-3' direction that are 2'-fluorinated nucleotides. (4) The sense strand has nucleotides at positions 7, 8 and 9 of the nucleotide sequence 1 in the 5'-3' direction that are 2'-fluorinated nucleotides, and the antisense strand has nucleotides at positions 2, 12, 14 and 16 of the nucleotide sequence 2 in the 5'-3' direction that are 2'-fluorinated nucleotides. (5) The sense strand has nucleotides at positions 7, 8 and 9 of the nucleotide sequence 1 in the 5'-3' direction that are 2'-fluorinated nucleotides, and the antisense strand has nucleotides at positions 2, 6, 9, 14 and 16 of the nucleotide sequence 2 in the 5'-3' direction that are 2'-fluorinated nucleotides. (6) The sense strand is composed of 2'-fluorinated nucleotides at positions 5, 7, 8 and 9 of the nucleotide sequence 1 in the 5'-3' direction, and the antisense strand is composed of 2'-fluorinated nucleotides at positions 2, 6, 14 and 16 of the nucleotide sequence 2 in the 5'-3' direction. (7) The sense strand has 2'-fluorinated nucleotides at positions 5, 7 and 9 of the nucleotide sequence 1 in the 5'-3' direction, and the antisense strand has 2'-fluorinated nucleotides at positions 2, 12, 14 and 16 of the nucleotide sequence 2 in the 5'-3' direction. (8) The sense strand has nucleotides at positions 7, 9 and 11 of the nucleotide sequence 1 in the 5'-3' direction that are 2'-fluorinated nucleotides, and the antisense strand has nucleotides at positions 2, 4, 12, 14 and 16 of the nucleotide sequence 2 in the 5'-3' direction that are 2'-fluorinated nucleotides. (9) The sense strand has nucleotides at positions 7, 8 and 9 of the nucleotide sequence 1 in the 5'-3' direction that are 2'-fluorinated nucleotides, and the antisense strand has nucleotides at positions 2, 7, 12, 14 and 16 of the nucleotide sequence 2 in the 5'-3' direction that are 2'-fluorinated nucleotides. (10) The sense strand comprises nucleotides at positions 7, 9, and 11 of the nucleotide sequence 1 in the 5'-3' direction, which are 2'-fluorinated nucleotides; and the antisense strand comprises nucleotides at positions 2, 12, 14, and 16 of the nucleotide sequence 2 in the 5'-3' direction, which are 2'-fluorinated nucleotides; and (11) The sense strand has 2'-fluorinated nucleotides at positions 5, 7 and 9 of the nucleotide sequence 1 in the 5'-3' direction, and the antisense strand has 2'-fluorinated nucleotides at positions 2, 7, 12, 14 and 16 of the nucleotide sequence 2 in the 5'-3' direction. Preferably, the nucleotides in the sense and antisense strands, except for those modified with 2'-fluorine, are all non-fluorinated nucleotides. Preferably, the 5' end of the sense chain contains two thiophosphate bonds, and the 3' end of the sense chain contains 0-3 thiophosphate bonds; the 5' end and the 3' end of the antisense chain each contain two thiophosphate bonds. More preferably, there are two phosphate thioester bonds between the first and second nucleotides, and between the second and third nucleotides, starting from the 5' end of the positive strand; There are 0-3 phosphate thioester bonds between the first and second nucleotides, between the second and third nucleotides, and between the third and fourth nucleotides starting from the 3' end of the positive chain; There are two phosphate thioester bonds between the first and second nucleotides, and between the second and third nucleotides, starting from the 5' end of the antisense strand; There are two phosphate thioester bonds between the first and second nucleotides, and between the second and third nucleotides, starting from the 3' end of the antisense strand.

5. An oligonucleotide conjugate comprising the double-stranded oligonucleotide of any one of claims 1-4 or a pharmaceutically acceptable salt thereof and a conjugating group conjugated to the double-stranded oligonucleotide or a pharmaceutically acceptable salt thereof, the conjugating group comprising a pharmaceutically acceptable targeting group and / or a delivery assistant group, the conjugating group being covalently or non-covalently linked to the double-stranded oligonucleotide; Preferably, the conjugating group is covalently or non-covalently linked to the double-stranded oligonucleotide or its pharmaceutically acceptable salt via a linker; Preferably, the targeting group and / or delivery auxiliary group comprises a protein, carbohydrate, or lipid, wherein the protein comprises a natural protein, a synthetic polyamino acid, and / or an antibody; the carbohydrate comprises dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, amino sugar, and / or hyaluronic acid; and the lipid comprises fatty acids, sterols, and / or phospholipids.

6. A composition comprising the double-stranded oligonucleotide of any one of claims 1-4 or a pharmaceutically acceptable salt thereof, and / or the oligonucleotide conjugate of claim 5, and optionally a pharmaceutically acceptable carrier; Preferably, the composition further comprises one or more additional therapeutic components; Preferably, the composition is formulated for administration via ocular, vaginal, rectal, nasal, transdermal, subcutaneous, intravenous, intra-arterial, intralymphatic, intrabronchial, intrapleural, intraperitoneal, cerebrospinal, or intramuscular injection, or for administration via the lungs, intrathecal, or intracardiac.

7. A cell comprising or generating the double-stranded oligonucleotide of any one of claims 1-4 or a pharmaceutically acceptable salt thereof.

8. A kit or cassette comprising a double-stranded oligonucleotide of any one of claims 1-4 or a pharmaceutically acceptable salt thereof, an oligonucleotide conjugate of any one of claims 5, a composition of any one of claims 6, and / or cells of claim 7, wherein the double-stranded oligonucleotide or a pharmaceutically acceptable salt thereof, the oligonucleotide conjugate, the composition and / or the cells are packaged in a cassette, container, packaging, dispenser, pre-filled syringe or vial.

9. The use of the double-stranded oligonucleotide of any one of claims 1-4 or a pharmaceutically acceptable salt thereof, the oligonucleotide conjugate of any one of claims 5, the composition of any one of claims 6, the cell of claim 7, and / or the kit or pod of claim 8 in the preparation of medicaments for treating viral diseases, neuromuscular diseases, bacterial infections, inflammatory and immune diseases, metabolic diseases, liver diseases, kidney diseases, cardiovascular diseases, ophthalmic diseases, lung diseases, tumors, or rare diseases, or in the preparation of reagents or medicaments for inhibiting the expression of target genes in cells.

10. The double-stranded oligonucleotide of any one of claims 1-4 or a pharmaceutically acceptable salt thereof, the oligonucleotide conjugate of any one of claims 5, the composition of any one of claims 6, the cell of claim 7, and / or the kit or cassette of claim 8, for use in inhibiting the expression of a target gene in a cell, or for use in treating or preventing a disease or condition in an individual; Preferably, the disease or condition is selected from viral diseases, neuromuscular diseases, bacterial infections, inflammatory and immune diseases, metabolic diseases, liver diseases, kidney diseases, cardiovascular diseases, ophthalmic diseases, lung diseases, tumors, and rare diseases.

11. A method for inhibiting the expression of a target gene in a cell, comprising contacting the cell with the double-stranded oligonucleotide of any one of claims 1-4 or a pharmaceutically acceptable salt thereof, the oligonucleotide conjugate of any one of claims 5, the composition of any one of claims 6, and / or the cell of claim 7.

12. A method of treating or preventing a disease or condition in an individual, comprising administering to the individual an effective amount of the double-stranded oligonucleotide of any one of claims 1-4 or a pharmaceutically acceptable salt thereof, the oligonucleotide conjugate of any one of claims 5, the composition of any one of claims 6, and / or the cell of claim 7, to inhibit the expression of a gene causing the disease in the individual; Preferably, the disease or condition is selected from viral diseases, neuromuscular diseases, bacterial infections, inflammatory and immune diseases, metabolic diseases, liver diseases, kidney diseases, cardiovascular diseases, ophthalmic diseases, lung diseases, tumors, and rare diseases.

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