Nucleotide-like-containing double-stranded oligonucleotides and uses thereof

Abstract

The invention provides a nucleotide-like-containing double-stranded oligonucleotide and application thereof, and belongs to the technical field of nucleic acid drugs. According to the present invention, the nucleotide-like is introduced into the double-strand oligonucleotide, such that the modified oligonucleotide molecule can improve the targeting delivery effect of the oligonucleotide drug on the myocardial tissue or the adipose tissue, and can be used for treating and / or preventing the pathological condition or the disease caused by the abnormal expression of the specific gene in the myocardial cell or the adipose cell.

Description

Technical Field

[0001] This disclosure relates to the field of nucleic acid drug technology, specifically to a double-stranded oligonucleotide containing nucleotide-like molecules and its uses. Background Technology

[0002] Worldwide, diseases associated with abnormal expression of specific genes in adipose tissue, including lipid metabolism disorders, hypertension, cardiovascular disease, and obesity, are on the rise. Currently, oligonucleotides are difficult to target effectively to adipose tissue for disease treatment. Therefore, there is a need to develop oligonucleotide molecules and methods that can be targeted to adipocytes in vivo or have relatively high selectivity for adipose tissue cells. Summary of the Invention

[0003] This disclosure provides a nucleotide analogue (or nucleotide analogue), a double-stranded oligonucleotide containing the nucleotide analogue, and uses thereof. By introducing the nucleotide analogue into a double-stranded oligonucleotide, this disclosure enhances the targeted delivery of oligonucleotide drugs to myocardial or adipose tissue, and can be used to treat and / or prevent pathological conditions or diseases caused by the abnormal expression of specific genes in cardiomyocytes or adipocytes.

[0004] This disclosure includes the following technical solutions: In a first aspect, this disclosure provides a double-stranded oligonucleotide, characterized in that the double-stranded oligonucleotide comprises a sense strand and an antisense strand, each strand having 17-23 nucleotides, and the sense strand and the antisense strand are complementary or substantially complementary to form a double-stranded region, wherein substantially complementary means that the mismatch between the sense strand and the antisense strand in the double-stranded region does not exceed 3 nucleotides; wherein the double-stranded oligonucleotide comprises at least two (preferably two or three) nucleotides (Ns) represented by Formula I, or their tautomers, stereoisomers, or pharmaceutically acceptable salts thereof: (I) in," " " represents the connection site between the nucleotide and the adjacent nucleotide; B is selected from substituted or unsubstituted nucleoside bases A, U, G, C or T. If B contains a substituent group, the substituent group is independently selected from halogen, C1-C3 alkyl, C1-C3 alkoxy or iminoyl. L is selected from saturated or unsaturated C. 14 -C 22 hydrocarbon group; p is selected from 1 or 2; q is selected from 1 or 2; n is selected from 1 or 2; Z is selected from hydroxyl or thiol groups.

[0005] In some embodiments of this disclosure, L is selected from saturated C. 16 -C 20 Hydrocarbon group.

[0006] In some embodiments of this disclosure, L is selected from saturated C. 16 C 17 C 18 C 19 Or C 20 Hydrocarbon group.

[0007] In some embodiments of this disclosure, n is selected from 1.

[0008] In some embodiments of this disclosure, p is selected from 1, and q is selected from 1.

[0009] In some embodiments of this disclosure, the nucleotide-like structure is selected from the structure shown in Formula II, or its tautomer, stereoisomer, or pharmaceutically acceptable salt thereof: (II) Z is selected from hydroxyl or mercapto groups; B is selected from unsubstituted nucleoside bases A, U, G, C, or T; L is selected from saturated C. 16 C 18 Or C 20 Straight-chain alkyl groups.

[0010] In some embodiments of this disclosure, the nucleotide-like structures are each independently selected from the structures shown in NM1 or NM2, or their tautomers, stereoisomers, or pharmaceutically acceptable salts: (NM1) (NM2).

[0011] In some embodiments of this disclosure, the double-stranded oligonucleotide comprises two identical or different nucleotide-like molecules, and the nucleotide-like molecules are located in the double-stranded region.

[0012] In some embodiments of this disclosure, the nucleotide-like structure is located on the positive strand.

[0013] In some embodiments of this disclosure, at least one nucleotide-like substance is located at the 5' or 3' end of the positive strand.

[0014] In some embodiments of this disclosure, one nucleotide-like substance is located at the 5' end of the positive strand and the other nucleotide-like substance is located at the 3' end of the positive strand.

[0015] In some embodiments of this disclosure, one of the nucleotide-like molecules is located at any position in the positive strand of the double-stranded region, and the other nucleotide-like molecule is located at the 5' end of the positive strand.

[0016] In some embodiments of this disclosure, one of the nucleotide-like molecules is located at any position in the positive strand of the double-stranded region, and the other nucleotide-like molecule is located at the 3' end of the positive strand.

[0017] In some embodiments of this disclosure, the positive strand of the double-stranded oligonucleotide comprises two nucleotide-like structures, one of which is located at any position from the 4th to the 8th position of the positive strand starting from the 5' end; the other is located at the 3' end of the positive strand.

[0018] In some embodiments of this disclosure, the positive strand of the double-stranded oligonucleotide comprises two nucleotide-like structures, one of which is located at position 4, counting from the 5' end of the positive strand; the other is located at the 3' end of the positive strand.

[0019] In some embodiments of this disclosure, the positive strand of the double-stranded oligonucleotide contains two identical nucleotide-like NM1s, one NM1 located at the 4th position counting from the 5' end of the positive strand; the other NM1 located at the 3' end of the positive strand.

[0020] In some embodiments of this disclosure, the positive strand of the double-stranded oligonucleotide comprises two identical nucleotide-like NM2s, one of which is located at the 4th position of the positive strand starting from the 5' end; the other NM2 is located at the 3' end of the positive strand.

[0021] In some embodiments of this disclosure, the positive strand of the double-stranded oligonucleotide comprises two distinct nucleotide-like molecules, NM1 and NM2, wherein NM1 is located at the 4th position of the positive strand starting from the 5' end; and the other NM2 is located at the 3' end of the positive strand.

[0022] In some embodiments of this disclosure, the positive strand of the double-stranded oligonucleotide comprises two distinct nucleotide-like molecules, NM1 and NM2, wherein NM2 is located at the 4th position counting from the 5' end of the positive strand; and the other NM1 is located at the 3' end of the positive strand.

[0023] In some alternative embodiments of this disclosure, all nucleotides in the double-stranded oligonucleotide, except for the nucleotides replaced by the nucleotide-like group, are modified; each is independently selected from the following modified nucleotides: 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-O-methoxyethyl (2'-O-MOE) modified nucleotides, or 2',2'-disubstituted modified nucleotides.

[0024] In some embodiments of this disclosure, in the double-stranded region of the double-stranded oligonucleotide, apart from the nucleotides replaced by the nucleotide-like compounds, at least three positions from the 7th to the 10th positions of the sense strand, starting from the 5' end, are selected from 2'-fluoro-modified nucleotides, and the remaining nucleotides are selected from 2'-O-methyl-modified nucleotides; and / or, at least four positions from the 2nd, 6th, 9th, 12th, 14th, and 16th positions of the antisense strand, starting from the 5' end, are selected from 2'-fluoro-modified nucleotides, and the nucleotide at position 15 is selected from 2'-O-methoxyethyl or 2'-O-methyl-modified nucleotides, and the remaining nucleotides are selected from 2'-O-methyl-modified nucleotides.

[0025] In some embodiments of this disclosure, in the double-stranded region of the double-stranded oligonucleotide, apart from the nucleotides replaced by the nucleotide-like compounds, at least three positions from the 7th to the 10th positions of the sense strand, starting from the 5' end, are selected from 2'-fluoro-modified nucleotides, and the remaining nucleotides are selected from 2'-O-methyl-modified nucleotides; and / or, at least five positions from the 2nd, 6th, 9th, 12th, 14th, and 16th positions of the antisense strand, starting from the 5' end, are selected from 2'-fluoro-modified nucleotides, and the nucleotide at position 15 is selected from a 2'-O-methoxyethyl-modified nucleotide, and the remaining nucleotides are selected from 2'-O-methyl-modified nucleotides.

[0026] In some embodiments of this disclosure, in the double-stranded region of the double-stranded oligonucleotide, except for the nucleotides replaced by the nucleotide-like compounds, the nucleotides at positions 7-10 of the sense strand, starting from the 5' end, are selected from 2'-fluoro-modified nucleotides, and the remaining nucleotides are selected from 2'-O-methyl-modified nucleotides; and the nucleotides at positions 2, 6, 12, 14, and 16 of the antisense strand, starting from the 5' end, are selected from 2'-fluoro-modified nucleotides, and the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the remaining nucleotides are selected from 2'-O-methyl-modified nucleotides.

[0027] In some embodiments of this disclosure, the double-stranded oligonucleotide further comprises at least one dangling end, and the dangling end is not located at the 5' end of the antisense strand; the dangling end is composed of 1-4 nucleotides; each nucleotide comprising the dangling end is independently selected from the structure shown in formula (III), or its tautomer, or its stereoisomer, or its pharmaceutically acceptable salt: Where Base represents nucleoside bases A, U, G, C, or T; Z represents a hydroxyl or thiol group; R1 and R2 are each independently selected from H, halogen, optionally substituted C1-C6 alkyl or optionally substituted C1-C6 alkoxy; In some embodiments of this disclosure, both R1 and R2 are selected from H; In some embodiments of this disclosure, R1 is selected from methoxy groups, and R2 is selected from H; In some embodiments of this disclosure, both R1 and R2 are selected from F; In some embodiments of this disclosure, R1 is selected from F, and R2 is selected from methyl.

[0028] In some embodiments of this disclosure, R1 is selected from methoxy and R2 is selected from methyl; In some embodiments of this disclosure, the overhanging end is composed of two nucleotides; In some embodiments of this disclosure, the double-stranded oligonucleotide has a dangling end, and the dangling end is located at the 3' end of the antisense strand.

[0029] In some embodiments of this disclosure, the 5' end of the antisense chain comprises a phosphate ester or a phosphate ester analogue. In one embodiment, the phosphate ester analogue is selected from 5'-vinylphosphonates, such as 5'-(E)-vinylphosphonate (5'-(E)-VP).

[0030] In some embodiments of this disclosure, the antisense strand does not contain the nucleotide-like structure, and the antisense strand is substantially anticomplementary, substantially anticomplementary, or completely anticomplementary to a nucleotide sequence in the mRNA expressed by the target gene.

[0031] In some embodiments of this disclosure, the sense chain and / or the antisense chain each independently contain one or more thiophosphate bonds.

[0032] Specifically, the internucleotide bonds between the following two adjacent nucleotides in the positive strand are phosphate thioester bonds: The nucleotide bond between the first and second nucleotides of the positive strand, starting at the 5' end; and, The positive chain consists of the nucleotide bond between the second and third nucleotides starting at the 5' end. The nucleotide internucleotides between two adjacent nucleotides in the antisense strand are phosphate thioester bonds: The nucleotide bond between the first and second nucleotides of the antisense strand, starting at the 5' end; and, The nucleotide bond between the second and third nucleotides of the antisense strand, starting from the 5' end; and, The antisense strand consists of the nucleotide bond between the first and second nucleotides, starting at the 3' end; and, The antisense strand consists of the nucleotide bond between the second and third nucleotides starting at the 3' end.

[0033] The antisense chain contains at least three thiophosphate bonds in the bistrand region, and the sense chain contains at least two thiophosphate bonds in the bistrand region.

[0034] In some embodiments of this disclosure, the antisense strand contains at least three phosphate thioester bonds in the double-stranded region, with the direction from the 5' end to the 3' end, and the three phosphate thioester bonds are respectively located between the first and second nucleotides, between the second and third nucleotides, and between the tenth and eleventh nucleotides of the antisense strand; In some embodiments of this disclosure, the positive strand contains at least two thiophosphate bonds in the double-stranded region, with the direction from the 5' end to the 3' end, and wherein the two thiophosphate bonds are located between the first and second nucleotides and between the second and third nucleotides in the double-stranded region of the positive strand; In some embodiments of this disclosure, the positive strand contains at least three phosphate thioester bonds in the double-stranded region, wherein the three phosphate thioester bonds are respectively located between the first and second nucleotides in the direction from the 5' end to the 3' end in the double-stranded region of the positive strand, between the second and third nucleotides, and between the first and second nucleotides in the direction from the 3' end to the 5' end. In some embodiments of this disclosure, the nucleotides in the sense strand that are base-complementary to the 10th nucleotide of the antisense strand and the nucleotides that are base-complementary to the 11th nucleotide of the antisense strand are connected by a phosphate diester bond, with the nucleotides in the sense strand paired with each other in the direction from the 5' end to the 3' end.

[0035] At least one thiophosphate bond exists in the internucleotide bonds between the dangling end and the double-stranded region, and in the internucleotide bonds between nucleotides in the dangling end of the double-stranded oligonucleotide; optionally, the internucleotide bonds between the dangling end and the double-stranded region, and in the internucleotide bonds between nucleotides in the dangling end of the double-stranded oligonucleotide, are all selected from thiophosphate bonds. The antisense strand has a nucleotide-phosphate bond between the 10th and 11th nucleotides starting from the 5' end; And / or, the nucleotide pairing between the nucleotide at position 10 (starting from the 5' end) of the sense strand and the nucleotide at position 11 (starting from the 5' end) of the antisense strand via a base complementarity is a phosphate diester bond.

[0036] In some embodiments of this disclosure, a double-stranded oligonucleotide is provided, comprising a sense strand and an antisense strand as shown below, oriented from the 5' end to the 3' end: SS: UmsUmsUm Ns1 AmAmUfCfCfUfCmAmCmUmCmUmAmAmAm Ns2 ; AS: VPUmsUfsUmAmGmAfGmUmGmAmGmGfAmUfU(moe)AfAmAmAmsUmsGm.

[0037] Ns1 and Ns2 may be the same or different, and Ns1 and Ns2 are each independently selected from the nucleotide class represented by NM1 or NM2.

[0038] In some embodiments of this disclosure, the double-stranded oligonucleotide is selected from any one of groups A) to C): Group A) Following the direction from the 5' end to the 3' end, the justice chain is as follows: UmsUmsUm(NM1)AmAmUfCfCfUfCmAmCmUmCmUmAmAmAm(NM1); Following the direction from the 5' end to the 3' end, the antisense chain is as follows: VPUmsUfsUmAmGmAfGmUmGmAmGmGfAmUfU(moe)AfAmAmAmsUmsGm.

[0039] Group B) Following the direction from the 5' end to the 3' end, the justice chain is as follows: UmsUmsUm(NM2)AmAmUfCfCfUfCmAmCmUmCmUmAmAmAm(NM1); Following the direction from the 5' end to the 3' end, the antisense chain is as follows: VPUmsUfsUmAmGmAfGmUmGmAmGmGfAmUfU(moe)AfAmAmAmsUmsGm.

[0040] Group C) Following the direction from the 5' end to the 3' end, the justice chain is as follows: UmsUmsUm(NM2)AmAmUfCfCfUfCmAmCmUmCmUmAmAmAm(NM2); Following the direction from the 5' end to the 3' end, the antisense chain is as follows: VPUmsUfsUmAmGmAfGmUmGmAmGmGfAmUfU(moe)AfAmAmAmsUmsGm.

[0041] It should be noted that the nucleotide sequence of the disclosed double-stranded oligonucleotide can be adjusted and designed according to the target gene, as long as it has a complementary region with at least partial base pairing with the target gene mRNA to ensure that at least one strand of the double-stranded oligonucleotide can be complementary to the target gene mRNA, and its specific sequence is not restricted.

[0042] In a second aspect of this disclosure, a pharmaceutical composition is provided comprising the double-stranded oligonucleotide described in the first aspect of this disclosure, and pharmaceutically acceptable excipients.

[0043] The pharmaceutical compositions disclosed herein include formulations suitable for parenteral administration. The formulations can be conveniently available in unit dosage forms and can be prepared by any method known in the pharmaceutical field. The amount of active ingredient in a single-dose form, typically the amount of siRNA producing the therapeutic effect, can be prepared in combination with excipients.

[0044] In a third aspect of this disclosure, the following are provided for use in the preparation of medicaments for the prevention and / or treatment of diseases associated with abnormal expression of specific genes in target tissues: (I) The double-stranded oligonucleotides described in the first aspect of this disclosure; and / or (II) The pharmaceutical composition described in the second aspect of this disclosure.

[0045] In some embodiments of this disclosure, the target tissue is selected from adipose tissue.

[0046] In some embodiments of this disclosure, the diseases associated with abnormal expression of specific genes in adipose tissue are selected from lipid metabolism disorders, hypertension, cardiovascular diseases, or overweight-related conditions.

[0047] In some embodiments of this disclosure, the target tissue is selected from myocardial tissue.

[0048] In some embodiments of this disclosure, the diseases associated with abnormal expression of specific genes in myocardial tissue are selected from obstructive hypertrophic cardiomyopathy (HOCM), familial hypertrophic cardiomyopathy (FHC), heart failure with preserved ejection fraction (HFPEF), atrial fibrillation (AFIB), ventricular fibrillation (VFIB), angina pectoris, myocardial infarction (MI), heart failure with reduced ejection fraction (HFREF), supraventricular tachycardia (SVT), hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), arrhythmia, or congestive heart failure (CHF).

[0049] The effective amount of the double-stranded oligonucleotides or pharmaceutical compositions described in this disclosure may vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, such as four times a day, three times a day, twice a day, once a day, or every other day, or the number of daily doses may be proportionally reduced.

[0050] The drug may be administered to the subject via any suitable route known in the art, including but not limited to: oral or parenteral routes, including intravenous administration, intramuscular administration, subcutaneous administration, transdermal administration, airway administration (aerosol), pulmonary administration, nasal administration, rectal administration, and local administration (including oral administration and sublingual administration), with preferred routes of administration selected from intravenous injection.

[0051] This disclosure introduces nucleotide-like molecules into double-stranded oligonucleotides, and the modified oligonucleotide molecules can enhance the targeted delivery of oligonucleotide drugs to myocardial or adipose tissue, which can be used to treat and / or prevent pathological conditions or diseases caused by abnormal expression of specific genes in cardiomyocytes or adipose cells. Attached Figure Description

[0052] Figure 1 The relative expression level of the target gene in mice after administration of the siRNA conjugate in Example 1. Detailed Implementation

[0053] The technical solutions of this disclosure will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of this disclosure, not all embodiments. Based on the embodiments of this disclosure, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this disclosure.

[0054] Terminology Explanation In this article, "phosphate diester nucleoside internucleotide bond" and "phosphate diester bond" can be used interchangeably, and their structural formula is as follows: .

[0055] In this article, "thiophosphate diester nucleoside internucleotide bond" and "thiophosphate ester bond" can be used interchangeably, and their structural formula is as follows: .

[0056] In the context of this disclosure, unless otherwise stated, the “nucleic acid analogues” of this disclosure may exist independently in the form of salts, mixed salts, or non-salts (e.g., free acids or free bases). When present in the form of salts or mixed salts, they may be pharmaceutically acceptable salts.

[0057] In this disclosure, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this disclosure but do not exclude other contents.

[0058] In this disclosure, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0059] In this disclosure, the term "optionally substituted" is used to define a variable that may be unsubstituted or substituted.

[0060] In this disclosure, the term "unsubstituted" means that the specified group does not contain substituents.

[0061] In this disclosure, the terms “substituted,” “replaced,” and “substituted” are used interchangeably to indicate that any one or more hydrogen atoms in the given structure are specifically substituented (e.g., C). 1-3 Alkyl, C 1-3 The substituted group may be replaced by an alkoxy or halogen group, provided that the normal valence of the specified atom does not exceed the valence of the substituted atom and the substitution produces a stable compound. Unless otherwise indicated, a substituted group may have one substituent at each substituted position of the group. When more than one position in the given structural formula can be substituted by one or more substituents selected from a particular group, then the substituents may be substituted at each substituted position in the same or different manner.

[0062] In this disclosure, the terms “each…independently selected”, “…independently selected”, and “…independently selected” are interchangeable and should be interpreted broadly. They can mean that the specific options expressed by the same symbols in different groups do not affect each other, or that the specific options expressed by the same symbols in the same group do not affect each other.

[0063] In this disclosure, the term "stereoisomer" refers to compounds having the same chemical structure but with different spatial arrangements of atoms or groups. Stereoisomers include enantiomers, diastereomers, conformational isomers (rotational isomers), geometrical isomers (cis / trans) isomers, blocked isomers, and so on.

[0064] In this disclosure, the term "chirality" refers to a molecule that has the property of not being superimposed on its mirror image; while "chirality" refers to a molecule that is superimposed on its mirror image.

[0065] In this disclosure, the term "enantiomer" refers to two non-overlapping but mirror-image isomers of a compound.

[0066] In this disclosure, the term "diastereomer" refers to a stereoisomer that has two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers have different physical properties, such as melting point, boiling point, spectral properties, and reactivity. Mixtures of diastereomers can be separated by high-resolution analytical operations such as electrophoresis and chromatography, for example, HPLC.

[0067] In this disclosure, the term “~~~~” indicates a site where a group is covalently linked.

[0068] In this disclosure, in the chemical structure, the bond "—" indicates that the configuration is not specified. If chiral isomerism exists in the chemical structure, the bond "—" can be " "", ", or both contain " "and" "Two configurations. Although all the above structural formulas are shown in some isomer forms for simplicity, this disclosure can include all isomers, such as: tautomers, rotational isomers, geometric isomers, diastereomers, racemates and enantiomers."

[0069] In this disclosure, the term "small interfering RNA (siRNA)" is a double-stranded RNA of 17 to 25 nucleotides in length, comprising a sense strand and an antisense strand. siRNA mediates targeted cleavage of RNA transcripts via the RISC pathway by forming an RNA-induced silencing complex (RISC). Specifically, siRNA directs the specific degradation of mRNA sequences through a known RNA interference (RNAi) process, inhibiting the translation of mRNA into amino acids and its conversion into proteins.

[0070] In this disclosure, the term "antisense strand (or guide strand)" includes a region substantially complementary to a target sequence. The term "sense strand (or follower strand)" refers to an iRNA strand containing an iRNA strand substantially complementary to the antisense strand. The term "substantially complementary" means completely complementary or at least partially complementary, for example, the antisense strand being completely complementary or at least partially complementary to the target sequence. In the case of partial complementarity, mismatches may be present in the internal or terminal regions of the molecule, wherein the most tolerant mismatches are present in the terminal regions, for example, within 5, 4, 3, or 2 nucleotides at the 5'- and / or 3' ends of the iRNA. It should be noted that "at least partially substantially complementary" of the antisense strand to the mRNA means that the antisense strand has a polynucleotide substantially complementary to a continuous portion of the mRNA of interest.

[0071] In this disclosure, "substantially reverse complementary" means that there are no more than three base mismatches between the two nucleotide sequences involved.

[0072] In this disclosure, "substantially reverse complementary" means that there is no more than one base mismatch between the two nucleotide sequences involved.

[0073] In this disclosure, "perfectly reverse complementary" means that there is no base mismatch between the two nucleotide sequences involved.

[0074] In this disclosure, the terms “treatment,” “relief,” or “improvement” are used interchangeably. These terms refer to methods of achieving beneficial or desired outcomes, including, but not limited to, treatment benefits. A “treatment benefit” means the eradication or improvement of the underlying disorder being treated. Here, a treatment benefit is achieved by eradicating or improving one or more physical symptoms associated with the underlying disorder, thereby observing improvement in the subject, although the subject may still be suffering from the underlying disorder.

[0075] In this disclosure, the terms “prevention” and “avoidance” are used interchangeably to refer to methods for obtaining beneficial or desired results, including but not limited to preventive benefits. To obtain a “preventive benefit,” the conjugate, RNAi reagent, or composition may be given to a subject at risk of developing a specific disease, or to a subject who reports one or more physiological symptoms of a disease, even if a diagnosis of the disease may not have been made.

[0076] In this disclosure, the term "ligand" generally refers to any compound or molecule capable of covalently or otherwise chemically binding to a biologically active substance, such as an oligonucleotide. In some embodiments, the ligand is capable of interacting directly or indirectly with another compound, such as a receptor. The receptor interacting with the ligand may be present on the cell surface, or alternatively may be an intracellular and / or intercellular receptor. The interaction between the ligand and the receptor may result in a biochemical reaction, or may simply be a physical interaction or binding.

[0077] In this disclosure, the term "administration" generally refers to the introduction of a pharmaceutical preparation of this disclosure into the body of a subject by any route of introduction or delivery. Any method known to those skilled in the art for contacting cells, organs, or tissues with the drug may be employed. Administration may include, but is not limited to, intravenous, intra-arterial, intranasal, intraperitoneal, intramuscular, subcutaneous, or oral administration. A daily dose may be divided into one, two, or more doses in suitable forms to be administered at one, two, or more times during a period of time.

[0078] In this disclosure, "pharmaceutical composition" can refer to a drug for the treatment of a disease or for use in in vitro cell culture experiments. When used for the treatment of a disease, the term "pharmaceutical composition" generally refers to a unit dose form and can be prepared by any method well known in the pharmaceutical industry. All methods involve the step of combining the active ingredient with excipients constituting one or more adjunct components. Typically, the composition is prepared by uniformly and adequately combining active siRNA with liquid excipients, finely pulverized solid excipients, or both.

[0079] In this disclosure, the term "pharmaceutical acceptable" means that a substance or composition must be chemically and / or toxicologically compatible with other components of the formulation and / or the mammals to which it is treated. Preferably, "pharmaceutical acceptable" as used in this disclosure means approved by a federal regulatory agency or national government, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in animals, particularly in humans.

[0080] In this disclosure, the term "pharmaceuticalally acceptable excipient" may include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for a specific target dosage form. The use of any conventional excipients that are incompatible with the siRNA of this disclosure, such as those that produce any adverse biological effects or interactions with any other component of the pharmaceutically acceptable composition in a harmful manner, is also within the scope of this disclosure.

[0081] As in this disclosure, the term "regulation of gene expression" means that the expression of a gene, or the level of an RNA molecule or equivalent RNA molecule encoding one or more proteins or protein subunits, is upregulated or downregulated such that the expression, level, or activity is greater or less than that observed in the absence of a regulator. For example, the term "regulation" may mean "inhibition," but the use of the word "regulation" is not limited to this definition.

[0082] In addition to any conventional excipients, the use of any range of siRNAs incompatible with the present disclosure, such as any adverse biological effects produced or interactions with any other component of a pharmaceutically acceptable composition in a harmful manner, is also within the scope of this disclosure.

[0083] Preparation of compounds Unless otherwise stated, all siRNA sequences used in this disclosure were synthesized by Suzhou Beixin Biotechnology Co., Ltd.; all PCR primers used in this disclosure were synthesized by Beijing Qingke Biotechnology Co., Ltd.; and all C57BL / 6J mice used in this disclosure were purchased from Spiford (Beijing) Biotechnology Co., Ltd.

[0084] Unless otherwise stated, all reagents, reagent consumables, and instruments used in this disclosure are commercially available products. The main reagents and consumables are shown in Table 1, and the main instruments and equipment are shown in Table 2.

[0085] Table 1 Main Reagents and Consumables Table 2 Main Instruments and Equipment Preparation Example 1: Preparation of Compound NM1 In this preparation example, the synthetic route of compound NM1 is as follows: (1-1) Synthesis of compound NM1-2: Compound NM1-1 (20 g, 36.6 mmol, 1 eq, CAS No. 81246-79-9) was dissolved in a mixed solution of 1,4-dioxane (160 ml) and water (40 ml). Sodium periodate (7.82 g, 36.6 mmol, 1 eq) was added, and the mixture was purged with argon three times. The reaction solution was stirred at 25 °C for 2 hours under an argon atmosphere until the reaction was complete. The reaction solution was concentrated to remove the solvent, diluted with water (300 ml), and extracted five times with dichloromethane (200 ml each time). The organic phases were combined. The organic phase was washed twice with saturated sodium bicarbonate aqueous solution (100 ml each time) and twice with saturated sodium chloride aqueous solution (100 ml each time). The solution was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a white solid (16.2 g).

[0086] At 25°C, the white solid (16.2 g) obtained in this step was dissolved in 1,4-dioxane (160 ml), and sodium borohydride (2.08 g, 54.9 mmol, 1.5 eq) was added in portions. The mixture was purged three times with argon gas, and the reaction solution was stirred at 25°C for 2 hours under an argon atmosphere. The reaction was then quenched with saturated ammonium chloride aqueous solution (120 ml), and the reaction was complete. The reaction solution was extracted four times with dichloromethane (150 ml each time), and the organic phases were combined. The organic phase was washed twice with saturated sodium chloride aqueous solution (100 ml each time), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound NM1-2 (13.5 g, yield 67.5%) as a white solid. MS ESI (m / z) = 547.15 [M - H] - .

[0087] 1 H NMR (400 MHz, DMSO-d6) δ 11.34 (br s, 1H), 7.64 (d, J = 8.0 Hz,1H), 7.40 – 7.24 (m, 5H), 7.23 – 7.11 (m, 5H), 6.89 – 6.82 (m, 4H), 5.81 (t,J = 5.6 Hz, 1H), 5.52 (dd, J = 8.0, 2.0 Hz, 1H), 5.13 (t, J = 6.4 Hz, 1H), 4.74 (t, J = 5.2 Hz, 1H), 3.73 (s, 6H), 3.71 – 3.60 (m, 2H), 3.40 (t, J = 5.2Hz, 2H), 3.07 – 2.88 (m, 2H). (1-2) Synthesis of compound NM1-3: At 25°C, compound NM1-2 (13g, 23.7mmol, 1eq) was dissolved in toluene (120ml), and Novozymes immobilized lipase TL IM (0.5g, with an enzyme activity of 360 IUN / g) and benzoyl chloride (3.33g, 23.7mmol, 1eq, CAS No. 98-88-4) were added. The mixture was purged with nitrogen three times, and the reaction solution was stirred at 25°C for 6 hours under a nitrogen atmosphere. The reaction was then quenched by adding saturated ammonium chloride aqueous solution (150ml), and the reaction was completed. The reaction mixture was extracted three times with dichloromethane (120 ml each time), and the organic phases were combined. The organic phase was washed twice with saturated sodium bicarbonate aqueous solution (200 ml each time) and twice with saturated sodium chloride aqueous solution (100 ml each time). The mixture was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reversed-phase chromatography (column: C18 silica gel, elution gradient: acetonitrile / water = 5 / 95–95 / 5, v / v) to give compound NM1-3 as a yellow solid (10.2 g, yield 65.9%). MS ESI (m / z) = 651.20 [M - H] - . 1 H NMR (400 MHz, Chloroform-d) δ 8.00 – 7.91 (m, 2H), 7.63 – 7.53 (m,1H), 7.50 – 7.37 (m, 3H), 7.30 – 7.09 (m, 12H), 6.88 – 6.74 (m, 4H), 6.30 –6.07 (m, 1H), 5.61 (dd, J = 8.0, 2.0 Hz, 1H), 4.62 – 4.43 (m, 2H), 3.84 –3.61 (m, 8H), 3.22 – 3.20 (m, 2H). (1-3) Synthesis of compound NM1-4: At 25°C, compound NM1-3 (10.1 g, 15.5 mmol, 1 eq) was dissolved in pyridine (100 ml), and tert-butyldimethylchlorosilane (2.80 g, 18.6 mmol, 1.2 eq, CAS No. 18162-48-6) was added. The mixture was purged with nitrogen three times, and the reaction solution was stirred at 25°C under a nitrogen atmosphere for 16 hours until the reaction was complete. The reaction mixture was evaporated to dryness to remove the solvent, diluted with water (400 ml), and extracted four times with dichloromethane (150 ml each time). The organic phases were combined, washed twice with saturated sodium bicarbonate solution (80 ml each time) and once with saturated sodium chloride solution (100 ml each time), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reversed-phase chromatography (column: C18 silica gel, elution gradient: acetonitrile / water = 5 / 95–95 / 5, v / v) to give compound NM1-4 as a yellow solid (8.5 g, yield 71.6%). MS ESI (m / z) = 765.30 [M - H] - .

[0088] 1 H NMR (400 MHz, Chloroform-d) δ 7.96 (d, J = 8.4 Hz, 2H), 7.60 –7.51 (m, 2H), 7.45 – 7.33 (m, 4H), 7.29 – 7.19 (m, 8H), 6.84 – 6.73 (m, 4H), 6.36 (t, J = 5.2 Hz, 1H), 5.58 (dd, J = 8.0, 2.0 Hz, 1H), 4.59 – 4.33 (m, 2H), 3.77 (s, 6H), 3.72 – 3.58 (m, 3H), 3.17 (d, J = 4.0 Hz, 2H), 0.84 (s,9H), -0.01 ~ -0.03 (m, 6H). (1-4) Synthesis of compound NM1-5: At 25°C, compound NM1-4 (17.1 g, 22.3 mmol, 1 eq) was dissolved in a mixed solution of pyrimidine (102 mL) and methanol (41 mL). The reaction system was cooled to 0°C using an ice bath. At 0°C, 1M sodium hydroxide aqueous solution (16.7 mL, 1.2 eq) was added. The mixture was purged with nitrogen three times. The reaction solution was stirred at 0°C under a nitrogen atmosphere for 1 hour until the reaction was complete. The solvent was removed by rotary evaporation, and the solution was diluted with water (150 mL). The mixture was extracted three times with dichloromethane (200 mL each time), and the organic phases were combined. The organic phase was washed twice with saturated ammonium chloride aqueous solution (100 mL each time) and twice with saturated sodium chloride aqueous solution (100 mL each time). The solution was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by normal phase chromatography (eluent: petroleum ether / ethyl acetate = 1 / 1, v / v) to give compound NM1-5 (13.1 g, yield 88.6%) as a white solid. MS ESI (m / z) = 663.3 [M+ H] + .

[0089] (1-5) Synthesis of compound NM1-6: Compound NM1-5 (3.84 g) was dissolved in N,N-dimethylformamide (50 ml). The temperature of the reaction system was lowered to 0 °C using an ice bath. A 60 wt% sodium hydride solution (0.70 g, 29.3 mmol, 3 eq, CAS No. 7646-69-7) in N,N-dimethylformamide was added at 0 °C. The mixture was purged with nitrogen three times. The reaction solution was stirred at 0 °C under a nitrogen atmosphere for 20 minutes. Hexadecane bromo (4.48 g, 14.7 mmol, 1.5 eq, CAS No. 112-82-3) was added. The temperature of the reaction solution was slowly raised to 25 °C and stirred at 25 °C for 16 hours. The reaction was quenched by adding saturated ammonium chloride aqueous solution (150 ml). The reaction was then complete. The reaction mixture was extracted three times with dichloromethane (150 ml each time), and the organic phases were combined. The organic phase was washed twice with saturated sodium bicarbonate aqueous solution (80 ml each time) and once with saturated sodium chloride aqueous solution (100 ml each time). The mixture was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by normal-phase chromatography (eluent: petroleum ether / ethyl acetate = 1 / 1, v / v) to give compound NM1-6 as a yellow solid (1.82 g, yield 21.6%). MSESI (m / z) = 885.50 [M - H] - .

[0090] (1-6) Synthesis of compound NM1-7: At 25°C, compound NM1-6 (1.8 g, 2.03 mmol, 1 eq) was dissolved in tetrahydrofuran (20 ml). The temperature of the reaction system was lowered to 0°C using an ice bath. At 0°C, a tetrahydrofuran solution of 1 M tetrabutylammonium fluoride (0.79 g, 3.04 mmol, 1.5 eq, CAS No. 429-41-4) was added dropwise. The reaction was purged with nitrogen three times. The reaction solution was stirred at 0°C under a nitrogen atmosphere for 4 hours until the reaction was complete. The reaction mixture was diluted with 50 ml of water, extracted three times with dichloromethane (50 ml each time), and the organic phases were combined. The organic phase was washed twice with saturated sodium bicarbonate solution (30 ml each time) and once with saturated sodium chloride solution (40 ml each time). The mixture was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by normal-phase chromatography (elution: petroleum ether / ethyl acetate = 3 / 7, v / v) to give compound NM1-7 (1.05 g, yield 67.3%) as a yellow solid. MS ESI (m / z) = 771.45 [M - H] - .

[0091] 1H NMR (300 MHz, DMSO-d6) δ 11.38 (d, J = 2.1 Hz, 1H), 7.67 (d, J =8.1 Hz, 1H), 7.38 – 7.10 (m, 9H), 6.86 (d, J = 9.0, 2.1 Hz, 4H), 5.98 (t, J =6.0 Hz, 1H), 5.53 (dd, J = 8.1, 2.1 Hz, 1H), 4.87 – 4.67 (m, 1H), 3.73 (s,6H), 3.66 (d, J = 6.0 Hz, 2H), 3.43 (d, J = 5.7 Hz, 2H), 3.10 – 2.82 (m, 2H),1.43 (s, 2H), 1.23 – 1.20 (m, 30H), 0.87 – 0.83 (m, 3H). (1-7) Synthesis of compound NM1: Compound NM1-7 (1 g, 1.29 mmol, 1 eq), which had been dehydrated three times by acetonitrile (20 ml each time), was dissolved in dichloromethane (15 ml). A solution of bis(diisopropylamino)(2-cyanoethoxy)phosphine (584.88 mg, 1.94 mmol, 1.5 eq, CAS No. 102691-36-1) in dichloromethane (15 ml) was added, followed by 1H-imidazolium-4,5-dianitronidazole (106.94 mg, 0.91 mmol, 0.7 eq, CAS No. 1122-28-7, abbreviated as DCI). The mixture was purged with argon three times, and the reaction solution was stirred at 25 °C for 1 hour under an argon atmosphere until the reaction was complete. The reaction mixture was diluted with 100 ml of saturated sodium bicarbonate solution, extracted three times with 100 ml of dichloromethane each time, and the organic phases were combined. The organic phase was washed twice with 50 ml of saturated sodium chloride solution each time, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reversed-phase chromatography (column: C18 silicagel, elution gradient: acetonitrile / water = 30 / 70–100 / 0, v / v) to give a yellow oily compound NM1 (605 mg, yield 47.6%). MS ESI (m / z) = 971.50 [M - H] - .

[0092] 1 H NMR (400 MHz, Acetonitrile-d3) δ 9.00 (s, 1H), 7.38 (dd, J = 8.0,3.2 Hz, 1H), 7.32 - 7.24 (m, 2H), 7.22 - 7.09 (m, 7H), 6.79 - 6.69 (m, 4H),5.96 - 5.83 (m, 1H), 5.39 (dd, J = 8.0, 1.2 Hz, 1H), 3.70 - 3.58 (m, 10H),3.58 - 3.41 (m, 5H), 3.33 (dd, J = 6.4, 4.0 Hz, 2H), 3.12 - 2.89 (m, 2H),2.50 (d, J = 7.6 Hz, 2H), 1.37 (q, J = 6.4 Hz, 2H), 1.16 (d, J = 10.4 Hz, 26H), 1.08 - 0.94 (m, 12H), 0.78 (t, J = 6.8 Hz, 3H). Preparation Example 2: Preparation of Compound NM2 In this preparation example, the synthetic route of compound NM2 is as follows: (2-1) Synthesis of compound NM2-1: Compound NM1-5 (4 g, 6.04 mmol, 1 eq) was dissolved in N,N-dimethylformamide (40 ml). The temperature of the reaction system was lowered to 0 °C using an ice bath. At 0 °C, a 60 wt% sodium hydride solution (0.72 g, 18.11 mmol, 3 eq) in N,N-dimethylformamide was added. The mixture was purged with nitrogen three times. The reaction solution was stirred at 0 °C under a nitrogen atmosphere for 30 minutes. Then, eicosane (3.27 g, 9.06 mmol, 1.5 eq, CAS No. 4276-49-7) was added. The reaction solution was slowly heated to 25 °C and stirred at 25 °C for 16 hours. The reaction was quenched by adding 200 ml of saturated ammonium chloride aqueous solution. The reaction was then complete. The reaction mixture was extracted three times with ethyl acetate (100 ml each time), and the organic phases were combined. The organic phase was washed twice with saturated sodium bicarbonate aqueous solution (80 ml each time) and once with saturated sodium chloride aqueous solution (100 ml each time). The mixture was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by normal-phase chromatography (eluent: petroleum ether / ethyl acetate = 3 / 1, v / v) to give compound NM2-1 (2.08 g, yield 36.6%) as a yellow solid. MSESI (m / z) = 943.60 [M + H] + .

[0093] (2-2) Synthesis of compound NM2-2: Compound NM2-1 (2.08 g, 2.21 mmol, 1 eq) was dissolved in tetrahydrofuran (20 mL) at 25 °C. A 1 M tetrabutylammonium fluoride solution in tetrahydrofuran (4.4 mL, 4.42 mmol, 2 eq) was added dropwise. The mixture was purged with nitrogen three times, and the reaction solution was stirred at 0 °C under a nitrogen atmosphere for 4 hours until the reaction was complete. The reaction solution was directly concentrated to remove the solvent, and purified by normal-phase chromatography (elution: petroleum ether / ethyl acetate = 1 / 1, v / v) to give compound NM2-2 (970 mg, yield 53%) as a colorless oil. MS ESI (m / z) = 829.55 [M + H] + .

[0094] (2-3) Synthesis of compound NM2: Compound NM2-2 (970 mg, 1.17 mmol, 1 eq), which had been dehydrated three times by acetonitrile (10 ml each time), was dissolved in dichloromethane (10 ml). A solution of bis(diisopropylamino)(2-cyanoethoxy)phosphine (530.3 mg, 1.76 mmol, 1.5 eq), which had been dehydrated three times by acetonitrile (10 ml each time), in dichloromethane (10 ml) was added. 1H-imidazolium-4,5-dianitronidazole (110.5 mg, 0.94 mmol, 0.8 eq, CAS No. 1122-28-7, abbreviated as DCI) was added. The mixture was purged with argon three times. The reaction solution was stirred at 25 °C for 1 hour under an argon atmosphere until the reaction was complete. The reaction mixture was diluted with 50 ml of saturated sodium bicarbonate solution, extracted three times with 20 ml of dichloromethane each time, and the organic phases were combined. The organic phase was washed twice with 50 ml of saturated sodium chloride solution each time, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by normal-phase chromatography (elution: petroleum ether / ethyl acetate = 2 / 1, v / v) to give compound NM2 (1 g, yield 83.0%) as a colorless oil. MS ESI (m / z) = 1029.64 [M + H] + .

[0095] 1 H NMR (400 MHz, DMSO-d6) δ 11.37 (s, 1H), 7.65 (d, J = 8.1 Hz, 1H), 7.34 – 7.23 (m, 4H), 7.18 (ddd, J = 8.9, 4.1, 2.5 Hz, 5H), 6.89 – 6.80 (m,4H), 5.96 (q, J = 5.8 Hz, 1H), 5.53 (d, J = 8.0 Hz, 1H), 3.73 (s, 7H), 3.71 –3.36 (m, 10H), 3.12 – 2.92 (m, 2H), 2.70 (dt, J = 11.7, 5.9 Hz, 2H), 1.43 (s,2H), 1.22 (d, J = 10.4 Hz, 34H), 1.10 (dd, J = 6.8, 3.0 Hz, 6H), 1.04 (dd, J= 12.1, 6.7 Hz, 6H), 0.85 (t, J = 6.6 Hz, 3H). Preparation Example 3: Preparation of Reference Compound NM200 In this preparation example, the synthetic route of the reference compound NM200 is as follows: (3-1) Synthesis of compound NM200-2: Compound NM200-1 (10 g, 44.24 mmol, 1 eq, CAS No. 3736-77-4) was dissolved in N,N-dimethylformamide (100 ml, abbreviated as DMF), and imidazole (6.02 g, 88.48 mmol, 2 eq, CAS No. 288-32-4, abbreviated as IM) and tert-butyldiphenylchlorosilane (13.38 g, 48.66 mmol, 1.1 eq, CAS No. 58479-61-1, abbreviated as TBDPSCl) was added. The mixture was stirred at 25 °C for 16 hours until the reaction was complete. Add 300 ml of saturated ammonium chloride aqueous solution to the reaction mixture, extract three times with 200 ml of ethyl acetate each time, and combine the organic phases. Wash the organic phase twice with 100 ml of saturated sodium chloride aqueous solution each time, dry to anhydrous sodium sulfate, filter, concentrate, and purify by normal phase chromatography (elution: dichloromethane / methanol = 20 / 1, v / v) to give compound NM200-2 as a white solid (17.08 g, yield 83.2%). MS ESI (m / z) = 465.2 [M + H] + .

[0096] (3-2) Synthesis of compound NM200-3: At 25°C, 1-cocadiol (90.16 g, 275.96 mmol, 7.5 eq, CAS No. 661-19-8) was dissolved in diethylene glycol dimethyl ether (170 mL, CAS No. 111-96-6), and the mixture was purged with nitrogen three times. Then, a solution of trimethylaluminum (91.99 mmol, 2.5 eq, CAS No. 75-24-1) in diethylene glycol dimethyl ether (46 mL) was added under a nitrogen atmosphere. The mixture was heated to 100°C and stirred at 100°C for 30 minutes. The mixture was then cooled to 60°C, and compound NM200-2 (17.08 g, 36.79 mmol, 1 eq) was added at 60°C. The reaction system was stirred at 145°C under a nitrogen atmosphere for 16 hours until the reaction was complete. Add 300 ml of saturated ammonium chloride aqueous solution to the reaction mixture, extract three times with 200 ml of ethyl acetate each time, and combine the organic phases. Wash the organic phase twice with 100 ml of saturated sodium chloride aqueous solution each time, dry with anhydrous sodium sulfate, filter, concentrate, and purify by normal phase chromatography (elution: petroleum ether / ethyl acetate = 1 / 1, v / v) to give compound NM200-3 as a white solid (10.8 g, yield 63.46%). MS ESI (m / z) = 791.6 [M + H] + .

[0097] (3-3) Synthesis of compound NM200-4: Compound NM200-3 (10.8 g, 13.66 mmol, 1 eq) was dissolved in tetrahydrofuran (100 mL) at 25 °C, and a tetrahydrofuran solution of 1 M tetrabutylammonium fluoride (20.49 mmol, 1.5 eq) (20.5 mL) was added. The reaction mixture was stirred at 25 °C for 3 hours until the reaction was complete. The solvent was removed by rotary evaporation, and the mixture was purified by reversed-phase chromatography (eluent: water / acetonitrile = 5 / 95, v / v) to give compound NM200-4 (3.74 g, yield 49.56%) as a white solid. MS ESI (m / z) = 553.4 [M + H] + .

[0098] (3-4) Synthesis of compound NM200-5: At 25 °C, compound NM200-4 (3.26 g, 5.9 mmol, 1 eq) was dissolved in pyridine (30 ml), and triethylamine (1.19 g, 9.8 mmol, 2 eq) was added. The reaction system was cooled to 0 °C in an ice bath, and 4,4'-dimethoxytriphenylchloromethane (4 g, 9.8 mmol, 2 eq, CAS No. 40615-36-9, abbreviation DMTrCl) was added in portions at 0 °C. The reaction system was stirred at 25 °C for 16 hours, and methanol was added to quench the reaction. The reaction was then complete. The reaction mixture was evaporated to dryness to remove the solvent, diluted with ethyl acetate (100 ml), washed twice with saturated ammonium chloride aqueous solution (50 ml each time), and twice with saturated sodium chloride aqueous solution (30 ml each time). The mixture was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by normal-phase chromatography (elution: petroleum ether / ethyl acetate = 3 / 1, v / v) to give compound NM200-5 as a yellow solid (5 g, yield 99.2%). MS ESI (m / z) = 855.4 [M + H] + .

[0099] (3-5) Synthesis of compound NM200 Compound NM200-5 (4.73 g, 5.54 mmol, 1 eq) which had been dehydrated three times by acetonitrile (30 ml each time) was dissolved in dichloromethane (25 ml). A solution of bis(diisopropylamino)(2-cyanoethoxy)phosphine (2.515 g, 8.30 mmol, 1.5 eq) which had been dehydrated three times by acetonitrile (10 ml each time) in dichloromethane (25 ml) was added. 1H-imidazolium-4,5-dianitronidazole (524.2 mg, 4.43 mmol, 0.8 eq) was added. The mixture was purged with nitrogen three times. The reaction solution was stirred at 25 °C for 1 hour under a nitrogen atmosphere until the reaction was complete. The reaction mixture was diluted with 50 ml of saturated sodium bicarbonate aqueous solution, extracted three times with 30 ml of dichloromethane each time, and the organic phases were combined. The organic phase was washed twice with 30 ml of saturated sodium chloride aqueous solution each time, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by normal-phase chromatography (elution: petroleum ether / ethyl acetate = 2 / 1, v / v) to give compound NM-200 (4.87 g, yield 83.43%) as a white solid. MS ESI (m / z) = 1055.3 [M + H] + .

[0100] 1 H NMR (400 MHz, Chloroform-d) δ 7.78 (dd, J = 13.2, 8.1 Hz, 1H), 7.43 – 7.15 (m, 9H), 6.89 (ddd, J = 8.6, 6.2, 2.1 Hz, 4H), 5.80 (t, J = 3.5Hz, 1H), 5.26 (ddd, J = 14.2, 8.1, 1.8 Hz, 1H), 4.37 (ddt, J = 24.1, 10.7,5.5 Hz, 1H), 4.18 – 3.99 (m, 2H), 3.74 (d, J = 2.5 Hz, 8H), 3.56 (tdd, J =13.1, 10.2, 5.2 Hz, 5H), 3.39 – 3.33 (m, 1H), 2.80 – 2.73 (m, 1H), 2.61 (td,J = 6.0, 3.5 Hz, 1H), 1.63 – 1.43 (m, 3H), 1.22 (d, J = 3.3 Hz, 40H), 1.16 –1.01 (m, 8H), 0.98 (d, J = 6.7 Hz, 2H), 0.84 (t, J = 6.6 Hz, 3H). Preparation Example 4: Preparation of Compound NM5 In this preparation example, the synthetic route for compound NM5 is as follows: (4-1) Synthesis of compound NM5-2.

[0101] At room temperature, compound NM5-1 (2 g, 7.58 mmol, 1 eq, CAS No. 114248-23-6) was dissolved in pyridine (20 mL), cooled to 0°C in an ice bath, and DMTrCl (3.33 g, 9.85 mmol, 1.3 eq) was added in portions. The reaction mixture was stirred at 25°C for 1 hour until the reaction was complete. The reaction mixture was quenched with methanol, the solvent was removed by rotary evaporation, diluted with ethyl acetate (50 mL), washed with saturated ammonium chloride aqueous solution (2 × 30 mL) and saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 1, v / v) to give compound NM5-2 (2.5 g, yield 58.21%) as a yellow solid. MS ESI (m / z) = 567.3 [M + H]+.

[0102] (4-2) Synthesis of compound NM5 Compound NM5-2 (2.5 g, 4.41 mmol, 1 eq), which was dehydrated three times with acetonitrile (30 ml each time), was dissolved in dichloromethane (25 mL). A solution of bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.99 g, 6.62 mmol, 1.5 eq) in dichloromethane (25 mL), which was also dehydrated three times with acetonitrile (30 ml each time), was added. 1H-imidazolium-4,5-dianitronidazole (416.3 mg, 3.53 mmol, 0.8 eq) was added. The mixture was purged with nitrogen three times, and the reaction solution was stirred at 25 °C for 1 hour under a nitrogen atmosphere until the reaction was complete. The reaction mixture was diluted with 50 mL of saturated sodium bicarbonate aqueous solution, extracted with dichloromethane (3 × 30 mL), and the organic phases were combined. The organic phases were washed with saturated brine (2 × 30 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (eluent: petroleum ether / ethyl acetate = 2 / 1) to give a white solid, compound NM5 (2.86 g, yield 84.62%). MS ESI (m / z) = 767.4 [M + H]+.

[0103] Preparation Example 5: Synthesis of Compound NM6 In this preparation example, the synthetic route of compound NM6 is shown below: (5-1) Synthesis of compound NM6-2 Compound NM6-1 (3 g, 11.54 mmol, 1.0 eq, (2'R)-2'-deoxy-2'-fluoro-2'-methylurea, CAS No. 863329-66-2) and pyridine (30 ml) were added to a 500 ml reaction vessel. The mixture was cooled to 0 °C, and 4,4'-bismethoxytriphenylmethylchloro (4.29 g, 12.7 mmol, 1.1 eq) was added in portions. The mixture was purged with nitrogen three times, and the reaction system was stirred at 25 °C for 3 hours under a nitrogen atmosphere. HPLC showed no starting material. After the reaction was complete, the reaction solution was concentrated, and purified with purified water (50 ml) and ethyl acetate (50 ml) for extraction. The organic phase was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound NM6-2 (2.7 g, yield 41.7%). MS ESI (m / z) = 563.0 [M + H]+.

[0104] (5-2) Synthesis of compound NM6 Compound NM6-2 (2.7 g, 4.8 mmol, 1.0 eq) was added to a 100 mL reaction vessel. Bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.74 g, 5.76 mmol, 1.2 eq) was added in portions, followed by 4,5-dicyanimidazole (0.45 g, 3.8 mmol, 0.8 eq, abbreviated as DCI, CAS number 1122-28-7) and dichloromethane (27 mL). The mixture was purged with nitrogen three times, and the reaction system was stirred at 25 °C for 3 hours under a nitrogen atmosphere. After the reaction was complete, sodium bicarbonate aqueous solution (20 mL) was added to the reaction solution, and the organic phase was separated. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reversed-phase column chromatography (elution: acetonitrile / water = 90 / 10, v / v) to obtain compound NM6 (3.0 g). MS ESI (m / z) = 763 [M + H]+.

[0105] Preparation Example 6: Preparation of siRNA (6-1) Composition of the Justice Chain (SS) and Antisense Chain (AS): The phosphoramidite solid-phase synthesis method was used, starting the cycle with a solid support and linking nucleoside monomers one by one according to the nucleotide sequence from the 3' end to the 5' end. During the synthesis, compounds NM1, NM2, NM200, NM5, and NM6 were each considered as a nucleoside monomer.

[0106] Each connection of a nucleoside monomer involves four steps: deprotection, coupling, capping, and oxidation or sulfidation. The synthetic conditions are given below: The nucleoside monomer was prepared into an acetonitrile solution with a concentration of 0.1 M.

[0107] The deprotection reaction conditions were the same for each step. The deprotection reaction conditions were: temperature 25℃, reaction time 70 seconds, deprotection reagent was a dichloroacetic acid solution in dichloromethane (3% by volume), and the molar ratio of dichloroacetic acid to the 4,4'-dimethoxytriphenylmethyl protecting group on the solid support was 5:1.

[0108] The conditions for each coupling reaction were identical. The coupling reaction conditions were as follows: temperature 25℃, molar ratio of nucleic acid sequence to nucleoside monomer on the solid-phase support 1:10, molar ratio of nucleic acid sequence to coupling reagent on the solid-phase support 1:65, reaction time 600 seconds, coupling reagent 0.5M acetonitrile solution of 5-ethylthio-1H-tetrazole, and thioreagent 0.2mol / L acetonitrile / pyridine mixed solution of hydrogenated xanthanin (acetonitrile and pyridine volume ratio 1:1).

[0109] The conditions for each capping reaction were identical. The conditions for the capping reaction were: temperature 25℃; reaction time 2 minutes; the capping reagent solution was a 1:1 molar ratio mixture of Cap1 and Cap2, where Cap1 was a 20% (v / v) N-methylimidazole pyridine / acetonitrile mixture with a pyridine to acetonitrile volume ratio of 3:5, and Cap2 was a 20% (v / v) acetic anhydride acetonitrile solution; the molar ratio of N-methylimidazole in Cap1 and acetic anhydride in Cap2 to the nucleic acid sequence linked on the solid-phase support was 1:1:1.

[0110] The conditions for each oxidation reaction were identical. The oxidation reaction conditions were: temperature 25°C; reaction time 3 seconds; oxidizing agent concentration of 0.05M iodine solution, with a molar ratio of iodine to the nucleic acid sequence linked on the solid support in the coupling reaction of 30:1; the oxidation reaction was carried out in a water / pyridine mixed solvent (water to pyridine volume ratio 1:9). The sulfidation reaction conditions were: temperature 25°C; reaction time 360 ​​seconds; thioreagent concentration of 0.2M hydroflavin in pyridine solution, with a molar ratio of thioreagent to the nucleic acid sequence linked on the solid support in the coupling reaction of 4:1; the thioreagent reaction was carried out in a water / pyridine mixed solvent (water to pyridine volume ratio 1:9).

[0111] After the last nucleoside monomer was ligated, the nucleic acid sequence ligated on the solid-phase support was sequentially cut, deprotected, purified, and desalted, and then freeze-dried to obtain the positive strand, wherein: The cleavage and deprotection conditions were as follows: The synthesized nucleotide sequence linked to a solid-phase support was added to 25% (w / w) ammonia solution at a concentration of 0.5 mL / μmol. The reaction was carried out at 55 °C for 16 hours. The solvent was removed, and the solution was concentrated to dryness under vacuum. After ammonia treatment, the product was dissolved in 0.4 mL / μmol N-methylpyrrolidone relative to the amount of single-stranded nucleic acid. Subsequently, 0.3 mL / μmol triethylamine and 0.6 mL / μmol triethylamine trifluoride were added to remove the 2'-O-TBDMS protection from the ribose.

[0112] Purification and desalting conditions: Nucleic acid purification was performed using a preparative ion chromatography column (Source 15Q) with a NaCl gradient elution. Specifically: eluent 1 was 20 mM sodium phosphate (pH=8.1), and the solvent was a water / acetonitrile mixture (water to acetonitrile volume ratio 9:1); eluent 2 was 1.5 M sodium chloride and 20 mM sodium phosphate (pH=8.1), and the solvent was a water / acetonitrile mixture (water to acetonitrile volume ratio 9:1); the elution gradient was eluent 1: eluent 2 = (100:0) - (50:50). The product eluates were collected and combined, and desalting was performed using a reverse chromatographic purification column. Desalting conditions included using a dextran gel column (g25 packing material) and elution with deionized water.

[0113] Detection: Purity was determined using ion exchange chromatography (IEX-HPLC); molecular weight was determined using liquid chromatography-mass spectrometry (LC-MS). The measured molecular weight was compared with the theoretical value. If the measured value and the theoretical value were consistent, it indicated that the sense strand and the target antisense strand had been obtained.

[0114] (6-2) Synthesis of siRNA The sense and antisense strands synthesized in step (6-1) were mixed in an equimolar ratio, dissolved in water for injection, and heated to 95°C. The mixture was then slowly cooled to room temperature and held at room temperature for 10 minutes to allow the sense and antisense strands to form a double-stranded structure via hydrogen bonds, thereby obtaining siRNA. The siRNAs obtained in this disclosure are shown in Table 3.

[0115] Table 3 Sequence information of siRNA Unless otherwise stated, the base composition and modifications used in this disclosure have the following meanings: uppercase letters A, U, G, C, and T represent the base composition of nucleotides; lowercase letter m indicates that the nucleotide represented by the uppercase letter to its left is a nucleotide modified with 2'-O-Me; lowercase letter f indicates that the nucleotide represented by the uppercase letter to its left is a nucleotide modified with 2'-F; (moe) indicates that the nucleotide represented by the uppercase letter to its left is a nucleotide modified with 2'-O-methoxyethyl; lowercase letter s indicates that the nucleoside bond between two adjacent nucleotides on its left and right sides is a phosphate thioester bond.

[0116] VP represents 5'-terminal phosphorylated 5'-(E)-vinylphosphonate. The structural formula of VPUm is as follows: .

[0117] (NM1), (NM2), (NM200), (NM5), and (NM6) are the corresponding nucleotides. Wherein: The structural formula of (NM1) is ; The structural formula of (NM2) is ; The structural formula of (NM200) is .

[0118] The structural formula of (NM5) is: or .

[0119] The structural formula of (NM6) is: or .

[0120] Biological detection experiments: Unless otherwise stated, all reagents, consumables and instruments used in biological testing experiments in this disclosure are commercially available products.

[0121] The RNA extraction and detection procedures disclosed herein are as follows: RNA extraction: The tissue samples were taken from RNAlater and homogenized for 60 seconds in a Tissuelyser II fully automated tissue homogenizer. Total RNA was extracted from each tissue sample using the Trizol method.

[0122] Reverse transcription reaction: 1 μg of total RNA was extracted from each tissue sample and transduced using the Promega Reverse Transcription System (A3500) with Oligo (dT) reagent. 15Prepare a 20 μL reverse transcription system using the reverse transcription primers according to the kit instructions and complete the reverse transcription reaction. After the reaction, add 80 μL of RNase-free water to the reverse transcription system to obtain the cDNA solution for Real-time PCR detection.

[0123] Real-time PCR assay: Using ABI SYBR™ Select Master Mix (Catalog number: 4472908), prepare 20 μL of Real-time PCR reaction mixture per well according to the kit instructions. Each mixture contains 5 μL of cDNA template obtained from the reverse transcription reaction, 10 μL of SYBR™ Select Master Mix, 0.5 μL of 10 μM upstream primer, 0.5 μL of 10 μM downstream primer, and 4 μL of RNase-free H2O. Place the prepared reaction mixture on an ABI StepOnePlus PCR instrument and perform Real-time PCR amplification using a three-step method. The amplification program is: 95℃ pre-denaturation for 10 min, followed by 95℃ denaturation for 30 s, 60℃ annealing for 30 s, and 72℃ extension for 30 s. Repeat the denaturation, annealing, and extension process for 40 cycles. After the program is completed, calculate the gene expression difference using the ΔΔCt method.

[0124] In this real-time quantitative PCR method, the ΔΔCt method was used to calculate the relative quantitative levels and inhibition rates of target gene mRNA in each test group. The calculation method is as follows: ΔCt(test group) = Ct(target gene in test group) – Ct(internal reference gene in test group) ΔCt(control group) = Ct(target gene in control group) – Ct(internal reference gene in control group) ΔCt(test group) = ΔCt(test group) – ΔCt(control group average) ΔCt(control group) = ΔCt(control group) – ΔCt(control group average) Here, ΔCt (control group mean) is the arithmetic mean of the ΔCt (control group) values ​​of each animal that died in the control group at the same time point. Therefore, each animal in both the test group and the control group corresponds to a ΔCt value.

[0125] The relative expression level of the target gene mRNA in the test group = 2 - ΔΔCt (Test group) × 100% Using the control group as a baseline, the mRNA expression levels of the target gene in the test group were normalized. The inhibition rate of target gene mRNA expression in the test group = (1 – relative expression level of target gene mRNA in the test group) × 100% Unless otherwise stated, all in vivo activity data are expressed as X±STDEV, and all data were plotted and analyzed using GraphPadprism 8.0 software.

[0126] Example 1: Evaluation of the inhibitory activity of two lipid conjugate sequences on the target gene superoxide dismutase 1 (SOD1). In this embodiment, the inhibitory activity of double-stranded oligonucleotides R699169 (NM1 at the 4th position and 3' end of the positive strand), R699170 (NM2 at the 4th position and NM1 at the 3' end of the positive strand), R699171 (NM2 at the 4th position and 3' end of the positive strand), and the reference sequence R699120 (NM200 at the 4th position of the positive strand) on the target gene SOD1 was evaluated by subcutaneous administration.

[0127] Six- to eight-week-old C57BL / 6j mice were randomly divided into five groups of five based on body weight. Each group of mice was administered the aforementioned siRNA conjugate subcutaneously at a dose of 2 mg / kg, with a volume of 5 mL / kg. The day of administration was designated D0, and mice were sacrificed on D7. Adipose tissue (subcutaneous and gonadal fat), heart, liver, and muscle tissue were collected and preserved in RNAlater. RNA was extracted from each tissue, reverse transcribed, and quantitatively analyzed using real-time fluorescence. Differences in target gene expression were calculated using the ΔΔCt method described above.

[0128] Table 4 Primer sequence information 5. Repressive activity of the target gene in mice after administration of the siRNA conjugate described in this embodiment. The results of Example 1 showed that on day 7 after administration, the double-stranded oligonucleotides R699169 and R699170 had activities in adipose tissue (subcutaneous fat, paragonial fat) and heart that were essentially equivalent to the C22-conjugated reference sequence R699120, but showed virtually no inhibitory activity in the liver and muscle, exhibiting better tissue specificity. Figure 1 (Table 5).

[0129] The above specific embodiments are merely illustrative of the present invention and do not represent a limitation thereof. For those skilled in the art, various modifications and improvements can be made without departing from the spirit and substance of this disclosure, and these modifications and improvements are also considered to be within the scope of protection of this disclosure.

Claims

1. A double-stranded oligonucleotide, characterized in that, The double-stranded oligonucleotide comprises a sense strand and an antisense strand, each strand having 17-23 nucleotides, and the sense strand and the antisense strand are complementary or substantially complementary to form a double-stranded region. Substantialloying means that the mismatch between the sense strand and the antisense strand in the double-stranded region does not exceed 3 nucleotides. The double-stranded oligonucleotide contains at least two nucleotides of Formula I, or their tautomers, stereoisomers, or pharmaceutically acceptable salts. （Ⅰ） in," " represents the connection site between the nucleotide and the adjacent nucleotide; B is selected from substituted or unsubstituted nucleoside bases A, U, G, C or T. If B contains a substituent group, the substituent group is independently selected from halogen, C1-C3 alkyl, C1-C3 alkoxy or iminoyl. L is selected from saturated or unsaturated C. 14 -C 22 hydrocarbon group; p is selected from 1 or 2; q is selected from 1 or 2; n is selected from 1 or 2; Z is selected from hydroxyl or thiol.

2. The double-stranded oligonucleotide according to claim 1, characterized in that, The L is selected from saturated C. 16 -C 20 Hydrocarbon group; optionally, L is selected from saturated C 16 C 17 C 18 C 19 Or C 20 hydrocarbon group; n is selected from 1; p is selected from 1; q is selected from 1.

3. The double-stranded oligonucleotide according to claim 2, characterized in that, The nucleotide is selected from the structure shown in Formula II, or its tautomer, stereoisomer, or pharmaceutically acceptable salt thereof: （Ⅱ） Z is selected from hydroxyl or mercapto groups; B is selected from unsubstituted nucleoside bases A, U, G, C, or T; L is selected from saturated C. 16 C 18 Or C 20 Straight-chain alkyl groups.

4. The double-stranded oligonucleotide according to claim 3, characterized in that, Each of the nucleotides is independently selected from the structure shown in NM1 or NM2, or its tautomer, stereoisomer, or pharmaceutically acceptable salt thereof: (NM1)、 (NM2)。 5. The double-stranded oligonucleotide according to claim 1, characterized in that, The double-stranded oligonucleotide comprises two identical or different nucleotide-like structures, and the nucleotide-like structures are located on the positive strand, with the nucleotide-like structures selected from any of the following positions: 1) At least one nucleotide-like substance is located at the 5' or 3' end of the positive strand; 2) One of the nucleotide-like structures is located at the 5' end of the positive strand, and the other nucleotide-like structure is located at the 3' end of the positive strand; 3) One of the nucleotide-like molecules is located at any position in the positive strand of the double-stranded region, and the other nucleotide-like molecule is located at the 5' end of the positive strand; 4) One of the nucleotide-like molecules is located at any position in the positive strand of the double-stranded region, and the other nucleotide-like molecule is located at the 3' end of the positive strand; 5) One of the nucleotide-like positions is located at any position from the 4th to the 8th position of the positive strand, starting from the 5' end, and the other nucleotide-like position is located at the 3' end of the positive strand.

6. The double-stranded oligonucleotide according to claim 5, characterized in that, The positive strand of the double-stranded oligonucleotide contains two nucleotide-like structures, one of which is located at position 4, counting from the 5' end of the positive strand; the other is located at the 3' end of the positive strand.

7. The double-stranded oligonucleotide according to any one of claims 1-6, characterized in that, All nucleotides in the double-stranded oligonucleotide, except for those substituted by the aforementioned nucleotides, are modified, each independently selected from the following modified nucleotides: 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-O-methoxyethyl modified nucleotides, or 2',2'-disubstituted modified nucleotides.

8. The double-stranded oligonucleotide according to claim 7, characterized in that, In the double-stranded region of the double-stranded oligonucleotide, apart from the nucleotides replaced by the nucleotide-like nucleotides, at least three of the nucleotides at positions 7-10 of the positive strand, starting from the 5' end, are selected from 2'-fluoro-modified nucleotides, and the remaining nucleotides are selected from 2'-O-methyl-modified nucleotides. And, at least four of the nucleotides at positions 2, 6, 9, 12, 14 and 16 of the antisense strand, starting from the 5' end, are selected from 2'-fluoro-modified nucleotides, and the nucleotide at position 15 is selected from 2'-O-methoxyethyl or 2'-O-methyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; The double-stranded oligonucleotide also has at least one dangling end, and the dangling end is not located at the 5' end of the antisense strand; the dangling end is composed of 1-4 nucleotides; Each nucleotide constituting the overhang is independently selected from the structure shown in formula (III), or its tautomer, its stereoisomer, or its pharmaceutically acceptable salt: Where Base represents nucleoside bases A, U, G, C, or T; Z represents a hydroxyl or thiol group; R1 and R2 are each independently selected from H, halogen, optionally substituted C1-C6 alkyl or optionally substituted C1-C6 alkoxy; R1 and R2 are each independently selected from any of the following: 1) Both R1 and R2 are selected from H; 2) R1 is selected from methoxy groups, and R2 is selected from H; 3) Both R1 and R2 are selected from F; 4) R1 is selected from F, and R2 is selected from methyl; 5) R1 is selected from methoxy and R2 is selected from methyl.

9. The double-stranded oligonucleotide according to claim 8, characterized in that, The 5' end of the antisense chain contains a phosphate ester or a phosphate ester analogue selected from 5'-(E)-vinylphosphonate; the sense chain and / or the antisense chain each independently contain one or more thiophosphate bonds.

10. The double-stranded oligonucleotide according to claim 9, characterized in that, The internucleotide bonds between the following two adjacent nucleotides in the positive strand are phosphate thioester bonds: The nucleotide bond between the first and second nucleotides of the positive strand, starting at the 5' end; and, The nucleotide bond between the second and third nucleotides of the positive strand, starting from the 5' end; as well as, The internucleotide bonds between the following two adjacent nucleotides in the antisense strand are phosphate thioester bonds: The nucleotide bond between the first and second nucleotides of the antisense strand, starting at the 5' end; and, The nucleotide bond between the second and third nucleotides of the antisense strand, starting from the 5' end; and, The antisense strand consists of the nucleotide bond between the first and second nucleotides, starting at the 3' end; and, The antisense strand consists of the nucleotide bond between the second and third nucleotides starting at the 3' end; The antisense chain contains at least three thiophosphate bonds in the double-stranded region, and the sense chain contains at least two thiophosphate bonds in the double-stranded region. At least one phosphate thioester bond exists in the internucleotide bonds between the dangling end and the double-stranded region of the double-stranded oligonucleotide, and in the internucleotide bonds between nucleotides in the dangling end. The antisense strand has a nucleotide-phosphate bond between the 10th and 11th nucleotides starting from the 5' end; And / or, the nucleotide pairing between the nucleotide at position 10 (starting from the 5' end) of the sense strand and the nucleotide at position 11 (starting from the 5' end) of the antisense strand via a base complementarity is a phosphate diester bond.

11. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the double-stranded oligonucleotide as described in any one of claims 1-10, and pharmaceutically acceptable excipients.

12. The use of any of the following in the preparation of a medicament for the prevention and / or treatment of diseases associated with abnormal expression of a specific gene in a target tissue: (I) The double-stranded oligonucleotide according to any one of claims 1-10; and / or (II) The pharmaceutical composition of claim 11; The target tissue is selected from adipose tissue or myocardial tissue.

13. The use according to claim 12, characterized in that, The diseases associated with abnormal expression of specific genes in adipose tissue are selected from lipid metabolism disorders, hypertension, cardiovascular diseases, or diseases related to overweight. The diseases associated with abnormal expression of specific genes in myocardial tissue are selected from obstructive hypertrophic cardiomyopathy, familial hypertrophic cardiomyopathy, heart failure with preserved ejection fraction, atrial fibrillation, ventricular fibrillation, angina pectoris, myocardial infarction, heart failure, heart failure with reduced ejection fraction, supraventricular tachycardia, hypertrophic cardiomyopathy, dilated cardiomyopathy, arrhythmia, or congestive heart failure.

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