Double-stranded oligonucleotides, compositions and conjugates containing double-stranded oligonucleotides, and methods of making and uses

Abstract

The present disclosure provides a double-stranded oligonucleotide comprising a sense strand and an antisense strand, wherein the sense strand comprises a nucleotide sequence I consisting of 19 modified or unmodified nucleotides; the antisense strand comprises a nucleotide sequence II which is a nucleotide sequence formed by replacing at least one of the 2nd-8th nucleotides in the 5'-end-3' end direction of a nucleotide sequence A with a replacement group SN represented by formula (101), the nucleotide sequence A consisting of 19 modified or unmodified nucleotides, and at least partially reverse complement to a nucleotide sequence in mRNA expressed by a target gene; the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region. The double-stranded oligonucleotide, the pharmaceutical composition comprising the double-stranded oligonucleotide and the oligonucleotide conjugate can effectively treat and / or prevent diseases or disorders associated with the expression of the target gene, and have reduced off-target effects.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to a double-stranded oligonucleotide with reduced off-target effect, and a pharmaceutical composition and an oligonucleotide conjugate containing the same. The present disclosure also relates to a preparation method and use of the double-stranded oligonucleotide, the pharmaceutical composition and the oligonucleotide conjugate. BACKGROUND

[0002] Double-stranded oligonucleotides have been known as a pharmaceutically active ingredient. In recent years, considerable progress has been made in the development of double-stranded oligonucleotides as drugs.

[0003] In the development of double-stranded oligonucleotides as drugs, one of the important side effects or even toxicity-related effects is off-target effect. On the one hand, there has been an effort in the art to develop double-stranded oligonucleotides with good pharmaceutical activity and low off-target effect, and how to obtain double-stranded oligonucleotides meeting the requirements in both aspects still needs further exploration in the art; on the other hand, many double-stranded oligonucleotides showing excellent pharmaceutical activity in preclinical pharmaceutical research are difficult to be used in actual drug development due to the toxicity caused by their off-target effect, and therefore how to reduce the off-target effect of double-stranded oligonucleotides still has a great practical demand in the art. SUMMARY

[0004] In order to develop a double-stranded oligonucleotide with good pharmaceutical activity while showing reduced off-target effect, the inventors found that the double-stranded oligonucleotide obtained by replacing a nucleotide at a specific position in the sequence with a replacement group SN unexpectedly has significantly lower off-target effect than the double-stranded oligonucleotide without modification at the corresponding position while maintaining the pharmaceutical activity. Thus, the inventors made the following invention:

[0005] In one aspect, the present disclosure provides a double-stranded oligonucleotide, comprising a sense strand and an antisense strand, the sense strand comprising a nucleotide sequence I consisting of 19 modified or unmodified nucleotides; the antisense strand comprising a nucleotide sequence II, the nucleotide sequence II being a nucleotide sequence formed by replacing at least one of the 2nd-8th nucleotides in the nucleotide sequence A with a replacement group SN in the 5' end-3' end direction, the nucleotide sequence A consisting of 19 modified or unmodified nucleotides, and the nucleotide sequence A being at least partially reverse complementary to a first segment of nucleotide sequence in the mRNA expressed by a target gene, the first segment of nucleotide sequence being a segment of 19 nucleotides in the mRNA expressed by the target gene; the nucleotide sequence I and the nucleotide sequence II being at least partially reverse complementary to form a double-stranded region, the replacement group SN having a structure as shown in formula (101):

[0006]

[0007] wherein,

[0008] denotes the site of covalent attachment of the group;

[0009] n a and n b are each independently selected from an integer from 1 to 3, E 101 is a hydroxyl or a thiol group;

[0010] L 101 is a C1-C4 straight chain alkylene or alkyleneacyl group, wherein at least one methylene group is optionally replaced by an oxygen atom, an imide group and a sulfonyl group;

[0011] B 101 is a nucleobase, a substituted nucleobase, wherein one or more hydrogen atoms of the nucleobase are replaced by a substituent, each said substituent being independently selected from one of C1-C3 alkyl, C1-C3 alkoxy and halogen, or a base analogue selected from one of hypoxanthin-9-yl, purin-9-yl, 2-aminopurin-9-yl, 2,4-difluoro-5-methylphenyl, 5-nitroindol-1-yl, 3-nitro-pyrrol-1-yl, 4-fluoro-6-methylbenzimidazol-1-yl and 4-methylbenzimidazol-1-yl.

[0012] In another aspect, the present disclosure also provides a pharmaceutical composition containing the double-stranded oligonucleotide provided by the present disclosure and a pharmaceutically acceptable carrier.

[0013] In yet another aspect, the present disclosure also provides an oligonucleotide conjugate containing the double-stranded oligonucleotide provided by the present disclosure and a conjugate group conjugated to the double-stranded oligonucleotide, said conjugate group comprising a linker and a pharmaceutically acceptable targeting group and / or a delivery-assisting group, and, the double-stranded oligonucleotide, the linker and the targeting group or the delivery-assisting group are covalently or non-covalently linked in sequence, each said targeting group being selected from a ligand capable of binding to a cell surface receptor, and each delivery-assisting group being selected from a group capable of increasing the biocompatibility of the oligonucleotide conjugate in the target organ or tissue of delivery.

[0014] In yet another aspect, the present disclosure also provides the use of the double-stranded oligonucleotide, the pharmaceutical composition and / or the oligonucleotide conjugate of the present disclosure in the manufacture of a medicament for the treatment and / or prevention of a disease or a symptom associated with the mRNA level of a target gene expression.

[0015] In yet another aspect, the present disclosure also provides a method of treating and / or preventing a disease or a condition associated with the level of mRNA expression of a target gene, the method comprising administering to a subject in need thereof the double-stranded oligonucleotide, the pharmaceutical composition, and / or the oligonucleotide conjugate of the present disclosure.

[0016] In yet another aspect, the present disclosure also provides a method of modulating the level of expression of a target gene in a cell, the method comprising contacting an effective amount of the double-stranded oligonucleotide, the pharmaceutical composition, and / or the oligonucleotide conjugate of the present disclosure with the cell.

[0017] In addition, the present disclosure also provides a kit comprising the double-stranded oligonucleotide, the pharmaceutical composition, and / or the oligonucleotide conjugate of the present disclosure.

[0018] incorporated by reference

[0019] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually incorporated by reference.

[0020] Beneficial effects

[0021] The double-stranded oligonucleotide, the pharmaceutical composition, and / or the oligonucleotide conjugate of the present disclosure have good stability, higher target gene expression modulating activity, and have low off-target effects. The specific explanations are as follows.

[0022] First, the double-stranded oligonucleotide, the pharmaceutical composition, and / or the oligonucleotide conjugate of the present disclosure can have lower off-target effects and / or toxic reactions due to off-target effects in vitro or in vivo. For example, after administration of a reference conjugate not containing the replacement group SN at a dose of 100 mg / kg, the serum ALT and AST concentrations of mice increased; while after administration of the siRNA conjugate of the present disclosure, the serum ALT and AST concentrations were comparable to the level of the blank control group, indicating that the siRNA conjugate of the present disclosure has lower hepatotoxicity. For example, in the analysis of liver histopathology, compared with mice administered with the reference conjugate not containing the replacement group SN at a dose of 100 mg / kg, the degree and number of hepatocyte degeneration in mice administered with the siRNA conjugate of the present disclosure at the same dose were reduced, and no hepatocyte necrosis was observed. Thus, the siRNA conjugate of the present disclosure shows low toxicity due to off-target effects.

[0023] Secondly, the double-stranded oligonucleotide, the pharmaceutical composition and / or the oligonucleotide conjugate of the present disclosure show excellent target gene expression regulation activity in in vitro cell experiments. For example, the siRNA conjugate of the present disclosure shows excellent ANGPTL3 mRNA inhibition activity in C57BL / 6 mouse primary hepatocytes, with an ANGPTL3 mRNA inhibition rate of up to 85.27% at an siRNA concentration of 10 nM, showing comparable or even higher ANGPTL3 mRNA inhibition activity than the corresponding reference conjugate not comprising the replacement group SN.

[0024] Thirdly, the double-stranded oligonucleotide, the pharmaceutical composition and / or the oligonucleotide conjugate of the present disclosure can have higher stability and / or higher activity in vivo. For example, the siRNA conjugate of the present disclosure shows excellent ANGPTL3 mRNA inhibition effect in mice, with an ANGPTL3 mRNA inhibition rate of up to 96.24% at a dose of 3 mg / kg, higher mRNA inhibition rate and ANGPTL3 mRNA remaining amount reduced to less than half of the mice administered with the reference conjugate, indicating that the siRNA conjugate of the present disclosure can effectively improve the mRNA inhibition activity of siRNA on the expression of target genes. For another example, for a single administration to C57BL / 6 mice, the siRNA conjugate of the present disclosure can significantly reduce the TG and CHO levels in the serum of mice at different time points after administration, although the administration dose is only relatively low at 1 mg / kg. And the siRNA conjugate of the present disclosure can still show total cholesterol (CHO) inhibition effect for up to 71 days after a single administration, and the inhibition effect is always higher than that of the reference conjugate. And after 50 days after administration, the siRNA conjugate of the present disclosure shows higher blood lipid TG level reduction effect than the corresponding reference conjugate not comprising the stabilized modified nucleotide, with a maximum TG relative level difference of up to 24.4%.

[0025] Therefore, the double-stranded oligonucleotide, the pharmaceutical composition and / or the oligonucleotide conjugate provided by the present disclosure can have significantly lower off-target effect and toxicity reaction caused by off-target effect, while also being able to effectively regulate the expression level of target genes in vitro and in vivo, so as to effectively treat and / or prevent the disease symptoms related to the mRNA level expressed by the target gene with significantly higher safety, and has good application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 Columnar graph of relative expression level of ANGPTL3 mRNA in C57BL / 6 mouse primary hepatocytes after free uptake of conjugate 1, reference conjugate 1 or reference conjugate NC, respectively.

[0027] Figure 2 Scatter plot of relative expression level of mANGPTL3 mRNA in liver of C57BL / 6j mice after administration of 3 mg / kg (in terms of siRNA) of conjugate 1, reference conjugate 1, reference conjugate 3, reference conjugate 4, reference conjugate 5 or reference conjugate 6, and PBS, respectively.

[0028] Figure 3A and Figure 3B are line graphs showing the change of serum TG level or serum CHO level over time after administration of siRNA conjugate of the present disclosure, reference siRNA conjugate or PBS, respectively.

[0029] Figure 4A and Figure 4B are scatter plots of ALT and AST concentrations in serum of mice after administration of 100 mg / kg of conjugate 1, reference conjugate 1, reference conjugate 2, reference conjugate 3, reference conjugate 4 or PBS, respectively. DETAILED DESCRIPTION

[0030] The detailed description of the specific embodiments of the present disclosure is described below. It should be understood that the specific embodiments described herein are only for illustration and explanation of the present disclosure, and are not intended to limit the present disclosure.

[0031] In the present disclosure, unless otherwise specified, HBV gene refers to the viral gene of hepatitis B virus (HBV), for example, the gene having the sequence shown in Genbank registration number NC_003977.2, HBV mRNA refers to the mRNA transcribed from the above HBV gene; APOC3 mRNA refers to the mRNA having the sequence shown in Genbank registration number NM_000040.3, APOC3 gene refers to the gene transcribing the above APOC3 mRNA; ANGPTL3 mRNA refers to the mRNA having the sequence shown in Genbank registration number NM_014495.4, and ANGPTL3 gene refers to the gene transcribing the above ANGPTL3 mRNA.

[0032] Definitions

[0033] In the foregoing and hereinafter, capital letters C, G, U, A represent the base composition of a nucleotide; lower case letter m represents that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide; lower case letter f represents that the nucleotide adjacent to the left of the letter f is a fluoro-modified nucleotide; lower case letter s represents that the two nucleotides adjacent to the left and right of the letter s are connected by phosphorothioate group; P1 represents that the nucleotide adjacent to the right of the P1 is a 5'-phosphate nucleotide or a 5'-phosphate analogue-modified nucleotide, in some embodiments, P1 is VP, Ps or P representing specific modification, wherein the letter combination VP represents that the nucleotide adjacent to the right of the letter combination VP is a (E)-vinylphosphonate (E-VP)-modified nucleotide, the letter combination Ps represents that the nucleotide adjacent to the right of the letter combination Ps is a phosphorothioate-modified nucleotide, and the capital letter P represents that the nucleotide adjacent to the right of the letter P is a 5'-phosphate nucleotide.

[0034] In the foregoing and hereinafter, the "fluoro-modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2' position of the ribose group is replaced by fluorine; the "non-fluoro-modified nucleotide" refers to a nucleotide or a nucleotide analogue in which the hydroxyl group at the 2' position of the ribose group is replaced by a group other than fluorine. The "nucleotide analogue" refers to a group that can replace a nucleotide in a nucleic acid, but the structure is different from that of adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide or thymine deoxyribonucleotide. Such as an isonucleotide, a bridged nucleic acid (BNA) or an acyclic nucleotide. The "methoxy-modified nucleotide" refers to a nucleotide in which the 2'-hydroxyl group of the ribose group is replaced by a methoxy group.

[0035] In the context herein, the expressions "complementary" or "reverse complementary" can be used interchangeably and have the meaning well known to those skilled in the art, i.e. in a double-stranded nucleic acid molecule, the bases of one strand each pair with the bases on the other strand in a complementary manner. 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 includes 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 to be complementary to each other, and the sequence of one strand can be deduced from the sequence of its complementary strand. Accordingly, "mismatch" in the art means that the bases at the corresponding positions in a double-stranded nucleic acid do not pair in a complementary manner.

[0036] In the foregoing and hereafter, "substantially reverse complement" means that there are no more than 3 base mismatches between the two nucleotide sequences involved; "essentially reverse complement" means that there are no more than 1 base mismatch between the two nucleotide sequences; and "perfect reverse complement" means that there are no base mismatches between the two nucleotide sequences.

[0037] In the foregoing and hereafter, particularly when describing methods of making a double stranded oligonucleotide, pharmaceutical composition, or oligonucleotide conjugate of the disclosure, unless otherwise specified, the nucleoside monomers refer to modified or unmodified nucleoside phosphoramidite monomers (unmodified or modified RNA phosphoramidites, sometimes also referred to as Nucleoside phosphoramidites) used in the phosphoramidite solid phase synthesis according to the kind and order of nucleotides in the double stranded oligonucleotide or oligonucleotide conjugate to be made. Phosphoramidite solid phase synthesis is a method well known to those skilled in the art for use in RNA synthesis. All nucleoside monomers used in the present disclosure are commercially available.

[0038] Those skilled in the art will appreciate that, for any group containing one or more substituents, the group does not intend to introduce any substitution or substitution pattern that is sterically impractical, synthetically unfeasible, and / or unstable per se.

[0039] As used herein, "alkyl" refers to straight and branched chains, having the indicated number of carbon atoms, typically 1 to 20 carbon atoms, for example 1 to 10 carbon atoms, such as 1 to 8 or 1 to 6 carbon atoms. For example, C1-C6 alkyl includes straight and branched chain alkyl groups of 1 to 6 carbon atoms. When reference is made to an alkyl residue having a particular number of carbons, it is intended to encompass all branched and straight chain forms having that number of carbons; thus, for example, "butyl" is intended to include n-butyl, sec-butyl, iso-butyl, and t-butyl; "propyl" includes n-propyl and iso-propyl. Alkylene is a subset of alkyl, referring to the same residues as alkyl, but having two points of attachment.

[0040] As used herein, "alkenyl" refers to an unsaturated branched or straight-chain alkyl group having at least one carbon-carbon double bond obtained by removing one molecule of hydrogen from adjacent carbon atoms of a parent alkyl group. The group can be in either the cis- or trans-configuration about the double bond. Typical alkenyl groups include, but are not limited to, ethenyl; propenyl groups such as prop-1 -en-1 -yl, prop-1 -en-2-yl, prop-2-en-1 -yl (allyl), prop-2-en-2-yl; butenyl groups, for example, but-1 -en-1 -yl, but-1 -en-2-yl, 2-methylprop-1 -en-1 -yl, but-2-en-1 -yl, but-2-en-2-yl, but-1,3-dien-1 -yl, but-1,3-dien-2-yl, and the like. In certain embodiments, alkenyl groups have 2 to 20 carbon atoms, while in other embodiments, 2 to 10, 2 to 8, or 2 to 6 carbon atoms. Alkenylene is a subset of alkenyl, referring to the same groups as alkenyl, but with two points of attachment.

[0041] As used herein, "alkynyl" refers to an unsaturated branched or straight-chain alkyl group having at least one carbon-carbon triple bond obtained by removing two molecules of hydrogen from adjacent carbon atoms of a parent alkyl group. Typical alkynyl groups include, but are not limited to, ethynyl; propynyl groups such as prop-1 -yn-1 -yl, prop-2-yn-1 -yl; butynyl groups, for example, but-1 -yn-1 -yl, but-1 -yn-3-yl, but-3-yn-1 -yl, and the like. In certain embodiments, alkynyl groups have 2 to 20 carbon atoms, while in other embodiments, 2 to 10, 2 to 8, or 2 to 6 carbon atoms. Alkynylene is a subset of alkynyl, referring to the same groups as alkynyl, but with two points of attachment.

[0042] As used herein, "alkoxy" refers to an alkyl group of the specified number of carbon atoms attached through an oxygen bridge, for example, methoxy, ethoxy, propyloxy, isopropyloxy, n-butyloxy, sec-butyloxy, t-butyloxy, pentyloxy, 2-pentyloxy, isopentyloxy, neopentyloxy, hexyloxy, 2-hexyloxy, 3-hexyloxy, 3-methylpentyloxy, and the like. Alkoxy groups typically have 1 to 10, 1 to 8, 1 to 6, or 1 to 4 carbon atoms attached through an oxygen bridge.

[0043] As used herein, "aryl" refers to a radical derived from a mono- or polynuclear hydrocarbon ring system by removing a hydrogen atom from a ring carbon atom. The mono- or polynuclear hydrocarbon ring system contains only carbon and hydrogen atoms with 6 to 18 carbon atoms in the ring system, wherein at least one ring in the ring system is completely unsaturated, i.e., contains a cyclic, delocalized (4n+2) pi-electron system according to Hückel theory. Aryl includes, but is not limited to, phenyl, fluorenyl, and naphthyl groups, and the like. Arylene is a subset of aryl, referring to the same groups as aryl, but with two points of attachment.

[0044] "Heteroaryl" refers to a radical derived by removal of hydrogen atom from a 3- to 18- membered aromatic ring radical, containing 2 to 17 carbon atoms and 1 to 6 heteroatoms selected from nitrogen, oxygen, and sulfur. As used herein, a heteroaryl group can be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, wherein at least one ring in the ring system is completely unsaturated, i.e., contains a cyclic delocalized (4n+2) p-electron system according to Hückel theory. Heteroaryl groups include fused or bridged ring systems. In some embodiments, the heteroatoms in the heteroaryl group are oxidized heteroatoms. In some embodiments, the heteroaryl group contains one or more nitrogen atoms. In some embodiments, one or more of the nitrogen atoms in the heteroaryl group is a quaternized nitrogen atom. The heteroaryl group is attached to the rest of the molecule through any ring atom. Examples of heteroaryl groups include, but are not limited to: azepinyl, acridinyl, benzimidazolyl, benzoindolyl, 1,3-benzodioxazolyl, benzofuranyl, benzoxazolyl, benzo[d]thiazolyl, benzothiadiazolyl, benzo[b][l,4]dioxepinyl, benzo[b][l,4]oxazinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothiophenyl, benzo-thieno[3,2-d]pyrimidinyl, benzotriazolyl, benzo[4,6]imidazo[l,2-a]pyridinyl, carbazolyl, cinnolinyl, cyclopenta[d]pyrimidinyl, 6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidinyl, 5,6-dihydrobenzo[h]quinazolinyl, 5,6-dihydrobenzo[h]cinnolinyl, 6,7-dihydro-5H-benzo[6,7]cyclohepta[l,2-c]pyridazinyl, diphenofuranyl, diphenothiophenyl, furanyl, furanonyl, furopyrido[3,2-c]pyridinyl, 5,6,7,8,9,10-hexahydrocycloocta[d]pyrimidinyl, 5,6,7,8,9,10-hexahydrocycloocta[d]pyridazinyl, 5,6,7,8,9,10-hexahydrocycloocta[d]pyridinyl, isothiazolyl, imidazolyl, indazolyl, indolyl, isoindolyl, dihydroindolyl, isoindolyl, indolizinyl, isoxazolyl, 5,8-methano-5,6,7,8-tetrahydroquinazolinyl, 5,6-methano-5,6-dihydrobenzo[b][l,4]oxazinyl, 5,6-methano-5,6-dihydrobenzo[b][l,4]dioxepinyl, 5,6-methano-5,6-dihydrobenzo[b][l,4]dioxinyl, 5,6-methano-5,6-dihydrobenzo[b][l,4]oxazolyl, 5,6-methano-5,6-dihydrobenzo[b][l,4]thiazolyl, 5,6-methano-5,6-dihydrobenzo[b][l,4]thiadiazolyl, 5,6-methano-5,6-dihydrobenzo[b][l,4]cinnolinyl, 5,6-methano-5,6-dihydrobenzo[b][l,4]pyridazinyl, 5,6-methano-5,6-dihydrobenzo[b][l,4]pyridinyl, 5,6-methano-5,6-dihydrobenzo[b][l,4]thiophenyl, 5,6-methano-5,6-dihydrobenzo[b][l,4]furanyl, 5,6-methano-5,6-dihydrobenzo[b][l,4]pyranyl, 5,6-methano-5,6-dihydrobenzo[b][l,4]pyranonyl, 5,6-methano-5,6-dihydrobenzo[b][l,4]furanonyl, 5,6-methano-5,6-dihydrobenzo[b][l,4]thiophenonyl, 5,6-methano-5,6-dihydrobenzo[b][l,4]thieno[3,2-d]pyrimidinyl, 5,6-methano-5,6-dihydrobenzo[b][l,4]thieno[3,2-d]pyridazinyl, 5,6-methano-5,6-dihydrobenzo[b][l,4]thieno[3,2-d]pyridinyl, 5,6-methano-5,6-dihydrobenzo[b][l,4]thieno[3,2-d]thiophenyl, 5,6-methano-5,6-dihydrobenzo[b][l,4]thieno[3,2-d]furanyl, 5,6-methano-5,6-dihydrobenzo[b][l,4]thieno[3,2-d]pyranyl, 5,6-methano-5,6-dihydrobenzo[b][l,4]thieno[3,2-d]pyranonyl, 5,6-methano-5,6-dihydrobenzo[b][l,4]thieno[3,2-d]furanonyl, 5,6-methano-5,6-dihydrobenzo[b][l,4]thieno[3,2-d]thiophenonyl, 5,6-methano-5,6-dihydrobenzo[b][l,4]thieno[3,2-d]pyrimidinyl, 5,6-methano-5,6-dihydrobenzo[b][l,4]thieno[3,2-d]pyridazinyl, 5,6-methano-5,6-dihydrobenzo[b][l,4]thieno[3,2-d]pyridinyl, 5,6-methano-5,6-dihydrobenzo[b][l,4]thieno[3,2-d]thiophenyl, 5,6-methano-5,6-dihydrobenzo[b][l,4]thieno[3,2-d]furanyl, 5,6-methano-5,6-dihydrobenzo[b][l,4]thieno[3,2-d]pyranyl, 5,6-methano-5,6-dihydrobenzo[b][l,4]thieno[3,2-d]pyranonyl, 5,6-methano-5,6-dihydrobenzo[b][l,4]thieno[3,2-d]furanonyl, 5,6-methano-5,6-dihydrobenzo[b][l,4]thieno[3,2-d]thiophenonyl, 5,6-methano-5,6-dihydrobenzo[b][l,4]thieno[3,2-d]pyrimidinyl, 5,6-methano-5,6-dihydrobenzo[b][l,4]thieno[3,2-d]pyridazinyl, 5,6-methano-5,6-dihydrobenzo[b][l,4]thieno[3,2-d]pyridinyl, 5,6-methano-5,6-dihydrobenzo[b][l,4]thieno[3,2-d]thiophenyl, 5,6-methano-5,6-dihydrobenzo[b][l,4]thieno[3,2-d]furanyl, 5,6-methano-5,6-dihydrobenzo[b][l,4]thieno[3,2-d]pyranyl, 5,6-methano-5,6-dihydrobenzo[b][l,4]thieno8-tetrahydroquinazolinyl), naphthyridinyl, 1,6-naphthyridinonyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 5,6,6a,7,8,9,10,10a-octahydrobenzo[H]quinazolinyl, 1-phenyl-1H-pyrroleyl, phenazinyl, phenothiazinyl, phenotoxazinyl, phthalazinyl, pteridinyl, purine, pyrroleyl, pyrazolyl, pyrazolo[3,4-d]pyrimidinyl, pyridinyl, pyrido[3,2-d]pyrimidinyl, pyrido[3,4-d]pyrimidinyl Pyrimidinyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrroleyl, quinazolinyl, quinoxalinyl, quinolinyl, tetrahydroquinolinyl, 5,6,7,8-tetrahydroquinazolinyl, 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidinyl, 6,7,8,9-tetrahydro-5H-cycloheptano[4,5]thieno[2,3-d]pyrimidinyl, 5,6,7,8-tetrahydropyridano[4,5-c]pyridazinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, thieno[2,3-c]pyridinyl, thieno[3,2-d]pyrimidinyl, thieno[2,3-c]pyridinyl, and thiophenyl / thienyl. ,

[0045] Various hydroxyl protecting groups may be used in this disclosure. Generally, protecting groups make a chemical function insensitive to specific reaction conditions and can be added to and removed from that function in a molecule without substantially impairing the rest of the molecule. Representative hydroxyl protecting groups are disclosed in Beaucage et al., Tetrahedron 1992, 48, 2223-2311, and Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2ded, John Wiley & Sons, New York, 1991, all of which are incorporated herein by reference in their entirety. In some embodiments, the protecting group is stable under basic conditions but can be removed under acidic conditions. In some embodiments, non-exclusive examples of hydroxyl protecting groups that may be used herein include dimethoxytriphenylmethyl (DMT), monomethoxytriphenylmethyl, 9-phenyloxanthracene-9-yl (Pixyl), and 9-(p-methoxyphenyl)oxanthracene-9-yl (Mox). In some embodiments, non-exclusive examples of hydroxyl protecting groups that may be used herein include Tr (triphenylmethyl), MMTr (4-methoxytriphenylmethyl), DMTr (4,4'-dimethoxytriphenylmethyl), and TMTr (4,4',4”-trimethoxytriphenylmethyl).

[0046] The term “subject” as used herein refers to any animal, such as a mammal or marsupial. Subjects as described in this disclosure include, but are not limited to, humans, non-human primates (e.g., rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, rabbits, sheep, rats, and any kind of poultry.

[0047] As used herein, “treatment” refers to a method of achieving a beneficial or desired outcome, including but not limited to treatment benefits. A “treatment benefit” means the eradication or improvement of the underlying disorder being treated. Furthermore, 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, even though the subject may still be suffering from the underlying disorder.

[0048] As used herein, “prevention” refers to methods for obtaining a beneficial or desired outcome, including but not limited to preventive benefits. To obtain a “preventive benefit,” a double-stranded oligonucleotide, pharmaceutical composition, or oligonucleotide conjugate 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.

[0049] Double-stranded oligonucleotide

[0050] In one respect, this disclosure provides a double-stranded oligonucleotide that can regulate gene expression and has low off-target effects.

[0051] The first double-stranded oligonucleotide disclosed herein contains a nucleotide group as a basic structural unit. As is known to those skilled in the art, the nucleotide group contains a phosphate group, a ribose group, and a base, which will not be described in detail here.

[0052] CN102140458B discloses a siRNA that specifically inhibits the HBV gene and investigates various chemical modification strategies for this siRNA. The study found that different modification strategies have drastically different effects on the stability, bioactivity, and cytotoxicity of the siRNA. This study confirmed seven effective modification methods. Compared with unmodified siRNA, one modification method resulted in siRNA that improved blood stability while maintaining inhibitory activity essentially equivalent to that of unmodified siRNA. However, the study did not address off-target effects.

[0053] The double-stranded oligonucleotide disclosed herein comprises a sense strand and an antisense strand. The sense strand comprises a nucleotide sequence I consisting of 19 modified or unmodified nucleotides. The antisense strand comprises a nucleotide sequence II, which is a nucleotide sequence formed by replacing at least one of the 2nd to 8th nucleotides in the 5'-3' direction with a substitution group SN. The nucleotide sequence A consists of 19 modified or unmodified nucleotides, and the nucleotide sequence A is at least partially anticomplementary to a first nucleotide sequence in the mRNA expressing the target gene, the first nucleotide sequence being a nucleotide sequence of 19 nucleotides in length in the mRNA expressing the target gene. The nucleotide sequence I and the nucleotide sequence II are at least partially anticomplementary to form a double-stranded region.

[0054] The inventors of this disclosure have unexpectedly discovered that by replacing nucleotides with a substitution group SN at a specific position in the antisense strand of a double-stranded oligonucleotide, the off-target effects of double-stranded oligonucleotides, such as siRNA, can be effectively reduced or eliminated, thereby obtaining nucleic acid drugs, such as siRNA drugs, that have a good balance between the efficiency of regulating the expression level of the target gene.

[0055] The substituent group SN has a structure as shown in formula (101):

[0056]

[0057] in,

[0058] The site indicates the covalently linked group. Similar to the substituted nucleotide, the substitution group SN is linked to the 3' phosphate group and the 5' hydroxyl group of the ribose in the adjacent nucleotide via oxygen atoms and phosphate groups, respectively, forming phosphate ester bonds. Thus, the double-stranded oligonucleotide of this disclosure can reduce off-target effects by including the substitution group SN, without significantly altering the original spatial conformation of the double-stranded oligonucleotide due to the introduction of the substitution group SN, thereby maintaining high gene expression regulatory activity.

[0059] n a and n b Each integer is independently selected from 1 to 3. The inventors discovered that n with this value... a and n b This method can maintain the spatial structure of double-stranded oligonucleotides to the greatest extent possible, achieving a good balance between the activity and off-target effects of the double-stranded oligonucleotides. In some implementations, n a and n b Each can be 1 or 2 independently. In some implementations, n a and n b All are 1.

[0060] E attached to the phosphate ester group in the substituted group SN 101 It can be a hydroxyl or a thiol group. Furthermore, it is well known to those skilled in the art that the phosphate group in the double-stranded oligonucleotide can be in an acidic configuration or can form a corresponding salt. In some embodiments, the corresponding salt is a sodium salt or a potassium salt. Therefore, in the context of this disclosure, when referring to E... 101 In the structure of E, the phosphate group formed by the "hydroxyl group" and the phosphate group in the structure of formula (101) also includes the corresponding "phosphate ion" when forming a phosphate salt, and similarly, the thiophosphate group formed by the "mercapto group" and the phosphate group in the structure of formula (101) also includes the corresponding "thiophosphate ion" when forming a thiophosphate salt. In some embodiments, E 101 It is a hydroxyl group.

[0061] Linking group L 101 Its function is to add a base or its analogue group B 101 The N atom is attached to the substituted group SN. In some embodiments, L 101 It is a C1-C4 straight-chain alkylene or alkylene acyl group, wherein at least one methylene group is optionally replaced by an oxygen atom, an imide group, or a sulfonyl group. In some embodiments, L 101 It contains an acyl group, which forms an amide bond with the N atom shown in formula (101). In some embodiments, L 101 It is acetylated, propionyl, or butyryl, and L 101The acyl group in the formula (101) forms an amide bond with the N atom shown in the formula (101).

[0062] B 101 The base is a nucleic acid base, a substituted nucleic acid base, or a base analogue. A substituted nucleic acid base refers to a nucleic acid base in which one or more hydrogen atoms are replaced by a substituent, each substituent being independently selected from C1-C3 alkyl, C1-C3 alkoxy, and halogen. The base analogue is selected from hypoxanthine-9-yl, purine-9-yl, 2-aminopurine-9-yl, 2,4-difluoro-5-methylphenyl, 5-nitroindol-1-yl, 3-nitropyrrole-1-yl, 4-fluoro-6-methylbenzimidazol-1-yl, and 4-methylbenzimidazol-1-yl. In some embodiments, B... 101 It is one of the nucleic acid bases A, C, G, U, and T. In some embodiments, B... 101 The nucleic acid bases are identical to those in the nucleotides in nucleotide sequence A that are replaced by the substituted group SN.

[0063] In some embodiments, the substitution group SN is a group represented by formula (102):

[0064]

[0065] Among them, B 101 It can be A, C, G, or U.

[0066] In some embodiments, nucleotide sequence A is substantially anticomplementary, substantially anticomplementary, or completely anticomplementary to the first nucleotide sequence. In this case, in nucleotide sequence II, except for the nucleotide sequence replaced by the substitution group SN, the nucleotide sequence at the corresponding position in the first nucleotide sequence is substantially anticomplementary, substantially anticomplementary, or completely anticomplementary to the nucleotide sequence at the corresponding position. The corresponding position refers to the nucleotide position in the first nucleotide sequence where the nucleotide in nucleotide sequence II is not replaced by the substitution group SN. In some embodiments, nucleotides at positions 2-19 of nucleotide sequence A are completely anticomplementary to nucleotides at positions 1-18 of the first nucleotide sequence, following the direction from the 5' end to the 3' end. In this case, nucleotides at positions 2-19 of nucleotide sequence II that are not replaced by the substitution group SN are completely anticomplementary to the nucleotides at the corresponding positions in the first nucleotide sequence. Correspondingly, when the substitution group SN replaces nucleotide sequence N... G When the substituent group SN is counted as one nucleotide, in the double-stranded oligonucleotides of this disclosure, the second to the (N)th nucleotides in nucleotide sequence II are... G -1) nucleotide and (N) G+1) to the 19th nucleotide are completely anticomplementary to the nucleotides at the corresponding positions in the first nucleotide sequence.

[0067] In some embodiments, nucleotide sequence A is substantially anticomplementary, substantially anticomplementary, or completely anticomplementary to nucleotide sequence I. In this case, in nucleotide sequence II, the nucleotide not replaced by the substitution group SN is substantially anticomplementary, substantially anticomplementary, or completely anticomplementary to the corresponding nucleotide in nucleotide sequence I. In some embodiments, nucleotide sequence A is completely anticomplementary to nucleotide sequence I. In this case, in nucleotide sequence II, the nucleotide not replaced by the substitution group SN is completely anticomplementary to the corresponding nucleotide in nucleotide sequence I; or, a base mismatch exists between the second nucleotide in nucleotide sequence II in the 5' to 3' direction and the second nucleotide in nucleotide sequence I in the 3' to 5' direction. By including this base mismatch, the target gene expression regulatory activity of the double-stranded oligonucleotide of this disclosure can be further enhanced while maintaining low off-target effects.

[0068] In some embodiments, the double-stranded oligonucleotide of this disclosure further includes a nucleotide sequence III in the sense strand and a nucleotide sequence IV in the antisense strand. Nucleotide sequence III and nucleotide sequence IV are of equal length and consist of one, two, three, or four modified or unmodified nucleotides, respectively. Nucleotide sequence IV and nucleotide sequence III are substantially anticomplementary or completely anticomplementary. Nucleotide sequence III is attached to the 5' end of nucleotide sequence I, and nucleotide sequence IV is attached to the 3' end of nucleotide sequence II. Nucleotide sequence IV is substantially anticomplementary or completely anticomplementary to a second nucleotide sequence, which refers to a nucleotide sequence in the mRNA expressed by the target gene that is adjacent to the first nucleotide sequence and has the same length as nucleotide sequence IV. Thus, the double-stranded oligonucleotide of this disclosure may have a double-stranded complementary region of 19-23 nucleotides in length. In some embodiments, each nucleotide in nucleotide sequence III and nucleotide sequence IV is independently one of non-fluorinated modified nucleotides.

[0069] In some embodiments, the antisense strand further comprises a nucleotide sequence V, which consists of 1 to 3 modified or unmodified nucleotides attached to the 3' end of the antisense strand to form a 3' overhang of the antisense strand. Thus, the length ratio of the sense strand to the antisense strand of the double-stranded oligonucleotide provided in this disclosure can be 19 / 19, 19 / 20, 19 / 21, 19 / 22, 20 / 20, 20 / 21, 20 / 22, 20 / 23, 21 / 21, 21 / 22, 21 / 23, 21 / 24, 22 / 22, 22 / 23, 22 / 24, 22 / 25, 23 / 23, 23 / 24, 23 / 25, or 23 / 26. In some embodiments, the nucleotide sequence V consists of two non-fluorinated nucleotides, and in the direction from the 5' end to the 3' end, the nucleotide sequence V consists of two consecutive thymine deoxyribonucleotides (dTdT), two consecutive uracil ribonucleotides (UU), or two nucleotides that are completely anticomplementary to the third nucleotide sequence. The third sequence refers to a nucleotide sequence of two nucleotides in length adjacent to the 5' end of the first or second nucleotide sequence in the mRNA expressing the target gene. Therefore, in some embodiments, the sense and antisense strands of the double-stranded oligonucleotide of this disclosure each have a length of 19 / 21 nucleotides or 21 / 23 nucleotides, in which case the double-stranded oligonucleotide of this disclosure has better target gene expression regulatory activity.

[0070] As previously stated, each nucleotide in the double-stranded oligonucleotides of this disclosure is either modified or unmodified. In the context of this disclosure, the term "modified nucleotide" refers to a nucleotide or nucleotide analog formed by replacing the 2' hydroxyl group of the ribosyl group with another group, or a nucleotide whose bases are modified. The modified nucleotides do not result in a significant reduction or loss of the function of the double-stranded oligonucleotide in regulating gene expression. For example, the modified nucleotides disclosed in J.K. Watts et al., Chemically Modified siRNA: Tools and Applications. DrugDiscov Today, 2008, 13(19-20):842-55, may be selected. In some embodiments, the 2nd, 6th, 14th, and 16th nucleotides of the nucleotide sequence A are 2'-fluoromodified nucleotides in the direction from the 5' end to the 3' end. In some embodiments, all nucleotides in nucleotide sequence II are modified nucleotides; the 2nd, 6th, 14th, and 16th nucleotides of nucleotide sequence A are 2'-fluoro-modified nucleotides in the 5' to 3' direction, and the other nucleotides in nucleotide sequence A are each independently a type of non-fluoro-modified nucleotide. Correspondingly, in nucleotide sequence II, the substitution group SN is counted as one nucleotide, and the 2nd, 6th, 14th, and 16th nucleotides of nucleotide sequence II, if not substituted with SN, are 2'-fluoro-modified nucleotides; the other nucleotides in nucleotide sequence II, if not substituted with SN, are each independently a type of non-fluoro-modified nucleotide. In some embodiments, at least the 7th to 9th nucleotides of nucleotide sequence I are 2'-fluoro-modified nucleotides in the 5' to 3' direction. In some embodiments, all nucleotides in nucleotide sequence I are modified nucleotides; nucleotides 7-9 of nucleotide sequence I, arranged from the 5' end to the 3' end, are 2'-fluorinated modified nucleotides, and the other nucleotides in nucleotide sequence I are each independently a type of non-fluorinated modified nucleotide. The double-stranded oligonucleotides of this disclosure, by having the above modifications, can achieve a good balance between gene expression regulatory activity and in vivo stability.

[0071] In the context of this disclosure, a "fluorinated nucleotide" refers to a nucleotide formed by replacing the hydroxyl group at the 2' position of the ribosyl group with fluorine, having the structure shown in formula (7). A "non-fluorinated nucleotide" refers to a nucleotide or nucleotide analog formed by replacing the hydroxyl group at the 2' position of the ribosyl group with a non-fluorinated group. In some embodiments, each non-fluorinated nucleotide is independently selected from one of the nucleotides or nucleotide analogs formed by replacing the hydroxyl group at the 2' position of the ribosyl group with a non-fluorinated group.

[0072] The nucleotides formed by replacing the hydroxyl group at the 2' position of the ribosyl group with a non-fluorinated group are well known to those skilled in the art, and these nucleotides may be selected from one of the following: 2'-alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2'-amino modified nucleotides, 2'-substituted amino modified nucleotides, and 2'-deoxynucleotides.

[0073] In some embodiments, the 2'-alkoxy-modified nucleotide is a methoxy-modified nucleotide (2'-OMe), as shown in formula (8). In some embodiments, the 2'-amino-modified nucleotide (2'-NH2) is shown in formula (9). In some embodiments, the 2'-deoxynucleotide (DNA) is shown in formula (10).

[0074]

[0075] Nucleotide analogs are groups that can replace nucleotides in nucleic acids, but whose structure differs from that of adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, or thymine deoxyribonucleotides. In some embodiments, nucleotide analogs can be isonucleotides, bridged nucleic acids (BNAs), or acyclic nucleotides.

[0076] BNA refers to a restricted or inaccessible nucleotide. BNA can contain a bridging structure with a "fixed" C3'-endoglucan condensation, consisting of a five-membered, six-membered, or seven-membered ring. This bridge is typically incorporated into the 2'-, 4'-position of the ribose to provide a 2',4'-BNA nucleotide. In some embodiments, BNA can be LNA, ENA, cET BNA, etc., where LNA is shown in formula (12), ENA in formula (13), and cET BNA in formula (14).

[0077]

[0078] Acyclic nucleotides are a class of nucleotides formed by opening the sugar ring of a nucleotide. In some embodiments, acyclic nucleotides can be unblocking nucleic acids (UNA) or glycerol nucleic acids (GNA), wherein UNA is shown in formula (15) and GNA is shown in formula (16):

[0079]

[0080] In formulas (15) and (16) above, R is selected from H, OH or alkoxy (O-alkyl).

[0081] Isonucleotides are compounds formed by altering the position of a base on the ribose ring in a nucleotide. In some embodiments, an isonucleotide can be a compound formed by moving a base from the 1'-position to the 2'-position or 3'-position on the ribose ring, as shown in formula (17) or (18).

[0082] In the compounds shown in formulas (17)-(18) above, Base represents a nucleic acid base, such as A, U, G, C or T; R is selected from H, OH, F or non-fluorine groups as described above.

[0083]

[0084]

[0085] In some embodiments, the nucleotide analogue is selected from one of the following: isonucleotides, LNA, ENA, cET, UNA, and GNA. In some embodiments, each non-fluorinated nucleotide is a methoxylated nucleotide, and in the preceding and following text, the methoxylated nucleotide refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribosome with a methoxy group.

[0086] In the preceding and following text, “fluorinated nucleotides,” “2’-fluorinated nucleotides,” “nucleotides in which the 2’-hydroxyl group of the ribose group is replaced by fluorine,” and “nucleotides with a 2’-fluorinated ribose group” have the same meaning, all referring to compounds in which the 2’-hydroxyl group of the nucleotide is replaced by fluorine, resulting in compounds with the structure shown in formula (7); “methoxylated nucleotides,” “2’-methoxylated nucleotides,” “nucleotides in which the 2’-hydroxyl group of the ribose group is replaced by methoxyl,” and “nucleotides with a 2’-methoxy ribose group” have the same meaning, all referring to compounds in which the 2’-hydroxyl group of the ribose group of the nucleotide is replaced by methoxyl, resulting in compounds with the structure shown in formula (8).

[0087] In some embodiments, the double-stranded oligonucleotide of this disclosure is a double-stranded oligonucleotide having the following modifications: in the sense strand, the nucleotides at positions 7, 8, 9 or 5, 7, 8, 9 of nucleotide sequence I are fluorinated nucleotides, and the nucleotides at the remaining positions in the sense strand are methoxylated nucleotides; in the antisense strand, the nucleotides at positions 2, 6, 14, 16 or 2, 6, 8, 9, 14, 16 of nucleotide sequence II are fluorinated nucleotides, and the nucleotides at the remaining positions in the antisense strand are methoxylated nucleotides.

[0088] The modified double-stranded oligonucleotides are not only low in cost, but also less susceptible to cleavage by ribonucleases in the blood, thereby increasing their stability and making them more resistant to nuclease hydrolysis. Simultaneously, these modified double-stranded oligonucleotides exhibit higher activity in regulating target gene expression.

[0089] In some embodiments, at least one of the phosphate ester groups in the phosphate-sugar backbone of at least one single strand of the sense and antisense strands of the double-stranded oligonucleotide provided in this disclosure is a phosphate ester group with a modifying group. In some embodiments, the phosphate ester group with the modifying group is a thiophosphate ester group formed by replacing at least one oxygen atom in the phosphodiester bond of the phosphate ester group with a sulfur atom; in some embodiments, the phosphate ester group with the modifying group is a thiophosphate ester group having the structure shown in formula (121):

[0090]

[0091] This modification can stabilize the double-stranded structure of double-stranded oligonucleotides, maintaining high specificity and high affinity of base pairing.

[0092] In some embodiments, in the double-stranded oligonucleotide, the phosphate ester group having the modifying group is present at at least one of the following positions:

[0093] Between the first and second nucleotides at the 5' end of the positive strand;

[0094] Between the second and third nucleotides at the 5' end of the positive strand;

[0095] Between the first and second nucleotides at the 3' end of the positive strand;

[0096] Between the second and third nucleotides at the 3' end of the positive strand;

[0097] Between the first and second nucleotides at the 5' end of the antisense strand;

[0098] Between the second and third nucleotides at the 5' end of the antisense strand;

[0099] Between the first and second nucleotides at the 3' end of the antisense strand; and

[0100] Between the second and third nucleotides at the 3' end of the antisense strand.

[0101] In some embodiments, the 5'-terminal nucleotide of the antisense strand is a 5'-phosphate nucleotide or a nucleotide modified with a 5'-phosphate analogue. Commonly used 5'-phosphate nucleotides or 5'-phosphate analogue-modified nucleotides are well known to those skilled in the art; for example, a 5'-phosphate nucleotide may have the following structure:

[0102]

[0103] For example, Anastasia Khvorova and Jonathan K. Watts, The chemical evolution of oligonucleotide therapies of clinical utility. Nature Biotechnology, 2017, 35(3):238-48, disclosed the following four 5'-phosphate analog-modified nucleotides:

[0104]

[0105] In this context, R is selected from H, OH, methoxy, and fluorine; Base represents a nucleic acid base, selected from A, U, C, G, or T.

[0106] In some embodiments, the 5'-phosphate nucleotide is a nucleotide containing 5'-phosphate modification as shown in formula (2), the 5'-phosphate analog modified nucleotide is a nucleotide containing vinyl phosphate (5'-(E)-vinylphosphonate, E-VP) modification as shown in formula (3), or a nucleotide modified with thiophosphate as shown in formula (5).

[0107] 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 other embodiments, they can be double-stranded oligonucleotides that activate or upregulate gene expression, such as saRNA.

[0108] The double-stranded oligonucleotides modified using the present disclosure exhibit excellent lysosomal and intracellular stability, and surprisingly, while having low off-target effects, they also show excellent target gene expression regulatory activity.

[0109] The modified double-stranded oligonucleotides provided in this disclosure can be used to regulate various abnormal gene expression and treat various pathological conditions or diseases caused by abnormal gene expression. These genes 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 the modification scheme can be designed and prepared based on the mRNA expressed by the target gene. In some embodiments, the mRNA expressed by the target gene is selected from the mRNA transcribed from the following genes: 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. In some embodiments, the double-stranded oligonucleotide is siRNA, and the mRNA expressing the target gene is selected from the mRNA expressed by the hepatitis B virus gene (HBV), the mRNA expressed by the angiopoietin-like protein 3 (ANGPTL3) gene, or the mRNA expressed by the apolipoprotein C3 (ApoC3) gene.

[0110] In some embodiments, the double-stranded oligonucleotide of this disclosure may be, for example, one of the siRNAs shown in Table 1:

[0111] Table 1. siRNA sequences disclosed herein

[0112]

[0113]

[0114] In this context, uppercase letters C, G, U, and A represent the base composition of a nucleotide; lowercase letter m indicates that the nucleotide adjacent to the left of m is methoxylated; lowercase letter f indicates that the nucleotide adjacent to the left of f is fluorinated; and underlined uppercase letter combinations... SN This indicates the letter combination SN The nucleotide adjacent to the left is replaced by a substitution group SN with the corresponding nucleic acid base, and the lowercase letter 's' indicates that the two nucleotides to the left and right of this letter are linked by a phosphate thioester group; P1 indicates that the nucleotide adjacent to the right of P1 is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide. In some embodiments, SN It indicates a specific substituent group, for example snbThe underlined letter combination snb Indicates that in the letter combination snb The nucleotide adjacent to the left is replaced by the substitution group SN of formula (102) with the corresponding nucleic acid base. In some embodiments, P1 represents VP, Ps, or P, where the letter combination VP indicates that the nucleotide adjacent to the right of the letter combination VP is a nucleotide modified with vinyl phosphate (5'-(E)-vinylphosphonate, E-VP), the letter combination Ps indicates that the nucleotide adjacent to the right of the letter combination Ps is a nucleotide modified with thiophosphate, and the capital letter P indicates that the nucleotide adjacent to the right of the letter P is a 5'-phosphate nucleotide. Furthermore, each U in the above sequence can be replaced by T without significantly reducing the gene expression regulatory activity and / or off-target effect inhibition ability of the double-stranded oligonucleotide.

[0115] In the double-stranded oligonucleotides described in this disclosure and in the pharmaceutical compositions or oligonucleotide conjugates described below, each adjacent nucleotide is linked by a phosphodiester bond or a thiophosphate diester bond. The non-bridging oxygen or sulfur atom in the phosphodiester bond or thiophosphate diester bond carries a negative charge and can exist in the form of a hydroxyl or mercapto group. The hydrogen ion in the hydroxyl or mercapto group can also be partially or completely replaced by a cation. The cation can be any cation, such as a metal cation, ammonium ion (NH4+), etc. + The cation is one of the organic ammonium cations. For improved solubility, in one embodiment, the cation is selected from one or more of alkali metal ions, ammonium cations formed from tertiary amines, and quaternary ammonium cations. The alkali metal ion may be K... + and / or Na + The cation formed by the tertiary amine can be an ammonium ion formed from triethylamine and / or an ammonium ion formed from N,N-diisopropylethylamine. Therefore, the double-stranded oligonucleotides or oligonucleotide conjugates described in this disclosure can exist at least partially in the form of salts. In one embodiment, the non-bridging oxygen or sulfur atom in the phosphodiester bond or thiophosphodiester bond is at least partially bonded to a sodium ion, and the double-stranded oligonucleotides or oligonucleotide conjugates described in this disclosure exist in the form of a sodium salt or a partially sodium salt.

[0116] 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.

[0117] The phosphoramidite monomer corresponding to the substituted group SN is commercially available to those skilled in the art or can be readily synthesized. In some embodiments, the phosphoramidite monomer has the structure shown in formula (201):

[0118]

[0119] Among them, L 101 B 101 n a n b The definition and selection range are the same as described above; R 201 It is a hydroxyl protecting group; R 202 Selected from one of C1-C5 alkyl, cyanoethyl, cyanopropyl, and cyanobutyl; each R 203 Independently selected from C1-C5 alkyl groups. In some embodiments, R 201 Selected from Tr (triphenylmethyl), MMTr (4-methoxytriphenylmethyl), DMTr (4,4'-bismethoxytriphenylmethyl), or TMTr (4,4',4”-trimethoxybenzyl). In some embodiments, R 201 It is DMTr. In some implementations, R 202 Selected from cyanoethyl or cyanopropyl. In some embodiments, each R 203 It is independently selected from isopropyl or tert-butyl.

[0120] The phosphorus amide monomer represented by formula (201) is commercially available or can be prepared by those skilled in the art through a reasonable synthetic route. In some embodiments, the phosphorus amide monomer represented by formula (201) can be prepared by a method comprising contacting the compound represented by formula (202) with the phosphorus diamine represented by formula (203) in an organic solvent under condensation reaction conditions and in the presence of an activator and a condensation reaction aid, thereby isolating the compound represented by formula (201).

[0121]

[0122] Among them, L 101B 101 n a n b R 201 R 202 R 203 The definition and selection range are the same as those mentioned above.

[0123] The phosphorylated diamine compound represented by formula (203) is commercially available or can be synthesized by those skilled in the art through a reasonable process route. In some embodiments, the compound represented by formula (203) is a commercially available bis(diisopropylamino)(2-cyanoethoxy)phosphine. Each R 203 It is isopropyl, R 202 It is 2-cyanoethyl. The molar ratio of the compound shown in formula (203) to the compound shown in formula (202) can be 0.5:1-5:1, for example, 0.5:1-3:1.

[0124] The condensation reaction conditions include a reaction temperature of 0-100°C and a reaction time of 1-10 hours. In some embodiments, the condensation reaction conditions include a reaction temperature of 10-40°C, such as room temperature, and a reaction time of 2-6 hours. The reaction can be carried out under various pressures commonly used in laboratories, such as atmospheric pressure, and the reaction is terminated after complete monitoring by TLC.

[0125] The organic solvent may be one or more selected from epoxy solvents, ether solvents, haloalkane solvents, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropylethylamine. The epoxy solvent may be, for example, dioxane and / or tetrahydrofuran; the ether solvent may be, for example, diethyl ether and / or methyl tert-butyl ether; and the haloalkane solvent may be, for example, one or more selected from dichloromethane, trichloromethane, and 1,2-dichloroethane. In some embodiments, the organic solvent is dichloromethane. The amount of the organic solvent used relative to the compound shown in formula (202) may be 3-50 L / mol, for example, 5-20 L / mol.

[0126] The activator can be a C2-C4 carboxylate or a halocarboxylate of an organic base, such as pyridinium trifluoroacetate. The amount of the activator relative to the compound shown in formula (202) can be 0.1:1-5:1, for example, 0.5:1-3:1.

[0127] The condensation reaction auxiliary can be imidazole or N-methylimidazole, for example, N-methylimidazole. The amount of the reaction auxiliary relative to the compound shown in formula (202) can be 0.1:1-5:1, for example, 0.5:1-3:1.

[0128] The compound of formula (201) can be separated from the reaction mixture using any suitable separation method. In some embodiments, the compound of formula (201) can be separated by evaporation to remove the solvent, followed by chromatographic separation, for example, using the following chromatographic conditions: normal phase purification silica gel: 200-300 mesh silica gel packing, eluted with ethyl acetate: dichloromethane = 5:1; reverse phase purification: C18, C8 reverse phase packing, eluted with a gradient of methanol: acetonitrile = 0.1:1-1:0.1.

[0129] The compound represented by formula (202) is commercially available or can be prepared by a person skilled in the art using a reasonable synthetic route. In some embodiments, the compound represented by formula (202) can be prepared by a method comprising contacting the compound represented by formula (203) with a deprotection reaction auxiliary in an organic solvent under deprotection reaction conditions to isolate the compound represented by formula (202).

[0130]

[0131] Among them, L 101 B 101 n a n b R 201 The definition and selection range are the same as described above. R 204 Is with R 201 Different hydroxyl protecting groups. In some embodiments, R 204 It is a silane protecting group, such as TMS (trimethylsilyl), TES (triethylsilyl), TIPS (triisopropylsilyl), TBDPS (tert-butyldiphenylsilyl), or TBS (tert-butyldimethylsilyl). In some embodiments, R 204 It is a TBS protection base.

[0132] The deprotection reaction conditions include a reaction temperature of 0-50°C and a reaction time of 0.5-5 hours. In some embodiments, the condensation reaction conditions include a reaction temperature of 15-35°C, such as room temperature, and a reaction time of 1-3 hours. The reaction can be carried out under various pressures commonly used in the laboratory, such as atmospheric pressure, and the reaction is terminated after complete monitoring by TLC.

[0133] The organic solvent is one or more selected from epoxy solvents, ether solvents, haloalkane solvents, dimethyl sulfoxide, N,N-dimethylformamide, and organic bases. The epoxy solvent may be, for example, dioxane and / or tetrahydrofuran; the ether solvent may be, for example, diethyl ether and / or methyl tert-butyl ether; the haloalkane solvent may be, for example, one or more selected from dichloromethane, trichloromethane, and 1,2-dichloroethane; and the organic base may be, for example, pyridine or N,N-diisopropylethylamine. In some embodiments, the organic solvent is tetrahydrofuran. The amount of the organic solvent used relative to the compound represented by formula (203) is 0.1-20 L / mol, preferably 0.2-10 L / mol.

[0134] The deprotecting agent is an ammonium salt solution containing fluoride ions, such as a solution containing pyridinium fluoride, triethylamine fluoride, tetrabutylammonium fluoride, tetraoctylammonium fluoride, tetramethylammonium fluoride, tetraethylammonium fluoride, or benzyltrimethylammonium fluoride; preferably, the deprotecting agent is a tetrabutylammonium fluoride (TBAF) solution. The molar ratio of the deprotecting agent to the compound shown in formula (203) is 1:1-10:1-1:1-5:1.

[0135] The compound represented by formula (202) can be separated from the reaction mixture using any suitable separation method. In some embodiments, the solvent can be removed by evaporation, and the compound represented by formula (202) can be separated by chromatography. For example, column chromatography can be used to separate the compound represented by formula (202) produced in the reaction, with separation conditions such as normal-phase silica gel packing and gradient elution with dichloromethane:methanol = 100:1–20:1 (V:V). In some embodiments, the solvent can be removed directly to obtain a crude product of the compound represented by formula (202), which can be used directly in subsequent reactions.

[0136] The compound represented by formula (203) is commercially available or can be prepared by a person skilled in the art using a reasonable synthetic route. In some embodiments, the compound represented by formula (203) can be prepared by a method comprising contacting the compound represented by formula (204) and the compound represented by formula (205) in an organic solvent under condensation reaction conditions and in the presence of a condensation reaction activator and an organic base, thereby separating the compound represented by formula (203).

[0137]

[0138] Among them, L 101 B 101 n a n b R 201 R 204The definition and selection range are the same as described above. X 205 It is a leaving group. In some embodiments, L 101 It is a C2-C4 alkylene acyl group, X 205 It is a hydroxyl group, a C2-C4 acyl group, or a halogen.

[0139] The ratio of the compound shown in formula (205) to the compound shown in formula (204) can be 1:5 to 5:1, for example, 1:2 to 2:1. In some embodiments, considering production costs, the ratio is 1:1 to 1.5:1.

[0140] According to L 101 and X 205 The structure of L determines the condensation reaction conditions. In some embodiments, L 101 It is a C2-C4 alkylene acyl group, and the condensation reaction conditions are amidation reaction conditions. In some embodiments, X 205 The hydroxyl group is used. The amidation reaction conditions include a reaction temperature of 0-100°C and a reaction time of 0.5-20 hours. In some embodiments, the amidation reaction conditions are a reaction temperature of 10-40°C (e.g., room temperature) and a reaction time of 2-10 hours. The reaction can be carried out under various pressures commonly used in the laboratory, such as atmospheric pressure, and the reaction is terminated after complete monitoring by TLC.

[0141] The organic solvent may be one or more selected from epoxy solvents, ether solvents, haloalkane solvents, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropylethylamine. The epoxy solvent may be, for example, dioxane and / or tetrahydrofuran; the ether solvent may be, for example, diethyl ether and / or methyl tert-butyl ether; and the haloalkane solvent may be, for example, one or more selected from dichloromethane, trichloromethane, and 1,2-dichloroethane. In some embodiments, the organic solvent is N,N-dimethylformamide. The amount of organic solvent used relative to the compound shown in formula (204) may be 2-20 L / mol, for example, 4-10 L / mol.

[0142] The condensation activator can be selected according to the reaction type. In some embodiments, the condensation reaction is an amidation reaction, and the condensation activator can be one of 3-diethoxyphosphoryloxy-1,2,3-benzozolium-4(3H)-one, O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU), 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethylurea hexafluorophosphate, or dicyclohexylcarbodiimide, for example, HBTU. The amount of the condensation activator relative to the compound shown in formula (204) can be 0.1:1 to 10:1, for example, 1:1 to 5:1.

[0143] The organic base may be, for example, a tertiary amine. In some embodiments, the tertiary amine may be one of triethylamine, tripropylamine, tributylamine, and diisopropylethylamine, for example, triethylamine. The amount of the tertiary amine organic base relative to the compound shown in formula (204) may be 1:1 to 20:1, for example, 2:1 to 10:1.

[0144] The compound represented by formula (203) can be separated from the reaction mixture using any suitable separation method. In some embodiments, the solvent can be removed by evaporation, and the compound represented by formula (203) can be separated by chromatography. For example, column chromatography can be used to separate the compound represented by formula (203) produced in the reaction, with separation conditions, for example, using normal-phase silica gel and eluting with a gradient of dichloromethane:methanol = 50:1–20:1 (V:V). In some embodiments, the solvent can be removed directly to obtain a crude product of the compound represented by formula (203), which can be used directly in subsequent reactions.

[0145] The compound represented by formula (204) is commercially available or can be synthesized by those skilled in the art through a reasonable process route. In some embodiments, the compound represented by formula (204) can be obtained by reacting a secondary alcoholamine sequentially with different hydroxyl protecting agents using methods known in the art, followed by separation. For example, in some embodiments, R 201 It is a triphenylmethyl protecting group, R 204 It is a silane protecting group. The compound shown in formula (204) can be obtained by reacting and separating a secondary alkanolamine, such as diethanolamine, with a triphenylmethyl protecting agent, such as DMTrCl, and a silane protecting agent, such as TBSCl, respectively. These reactions are well known to and readily achievable by those skilled in the art.

[0146] The compound represented by formula (205) is commercially available or can be synthesized by those skilled in the art using known methods. In some embodiments, the compound represented by formula (205) can be prepared by contacting the compound represented by formula (206) and the compound represented by formula (207) in a solvent under condensation reaction conditions and in the presence of a base, thereby separating the compound represented by formula (205).

[0147]

[0148] HB 101

[0149] Equation (207)

[0150] Among them, L 101 B 101 X 205The definition and selection range are the same as described above. X 206 Is with X 205 Different leaving groups, such as halogens. In some embodiments, L 101 It is a C2-C4 alkylene acyl group, X 205 It is a hydroxyl group, X 206 It is Cl or Br.

[0151] The compound shown in formula (206) is commercially available or can be prepared by those skilled in the art via a reasonable route. In some embodiments, X 205 For hydroxyl group, L 101 It is an imide group, X 206 The compound represented by formula (206) is Br, which is bromoacetic acid or sodium bromoacetate, which are readily available commercially.

[0152] The compound shown in formula (207) is based on B 101 The structure is defined and is commercially available or can be prepared by those skilled in the art through a reasonable route. In some embodiments, B 101 The base is a nucleic acid base, and the compound represented by formula (207) is a readily available commercial adenine, guanine, cytosine, uracil, or thymine.

[0153] The ratio of the compound shown in formula (206) to the compound shown in formula (207) can be 1:5 to 5:1, for example, 1:2 to 2:1. In some embodiments, considering production costs, the ratio is 1:1 to 1.5:1.

[0154] According to B 101 and X 206 The structure of B is selected to determine the condensation reaction conditions. In some embodiments, B 101 It is a nucleic acid base, X 206 The condensation reaction is carried out under halogen conditions, including a reaction temperature of 0-100°C and a reaction time of 4-30 hours. In some embodiments, the condensation reaction conditions are a reaction temperature of 40-80°C and a reaction time of 10-20 hours. The reaction can be carried out under various pressures commonly used in the laboratory, such as atmospheric pressure, and the reaction is terminated after complete monitoring by TLC.

[0155] The solvent may be water or a polar organic solvent, such as an alcohol solvent and / or an ester solvent. For example, one or more of methanol, ethanol, and ethyl acetate; in some embodiments, the solvent is water. The amount of solvent used relative to the compound shown in formula (207) may be 0.5-10 L / mol, for example, 1-5 L / mol.

[0156] The base can be a strong organic base or an inorganic base, such as a sodium alkoxide or an alkali metal hydroxide, such as potassium hydroxide or sodium hydroxide. The amount of the tertiary amine organic base relative to the compound shown in formula (207) can be 1:1 to 10:1, for example, 1.5:1 to 3:1.

[0157] The compound of formula (205) can be separated from the reaction mixture using any suitable separation method. In some embodiments, excess base in the reaction mixture can be removed by adding an excess acid (such as an inorganic acid like hydrochloric acid) to precipitate the compound of formula (205) from the solvent, followed by filtration, washing, and evaporation to remove the remaining solvent, thereby obtaining the compound of formula (205). In some embodiments, the crude product of the compound of formula (203) can be obtained by directly removing the solvent after adding an excess acid; this crude product can be used directly in subsequent reactions.

[0158] Those skilled in the art will appreciate that modified nucleotide groups can be introduced into the double-stranded oligonucleotides described herein using appropriately modified nucleoside monomers. Methods for preparing appropriately modified nucleoside monomers and for introducing modified nucleotide groups into double-stranded oligonucleotides are also well known to those skilled in the art. All modified nucleoside monomers are commercially available or prepared using known methods.

[0159] The modified double-stranded oligonucleotides provided in this disclosure can be used alone, or in combination with a pharmaceutically acceptable carrier to form a pharmaceutical composition, or in combination with a conjugated molecule to form an oligonucleotide conjugate, or in other forms. Contacting cells with an effective amount of the double-stranded oligonucleotide, the pharmaceutical composition, or the oligonucleotide conjugate can modulate the expression of a target gene, or administering the double-stranded oligonucleotide, the pharmaceutical composition, or the conjugate to a subject can modulate the expression of a target gene to achieve the purpose of treating a pathological condition or disease related to the level of target gene expression.

[0160] The blood stability, targeting ability, and in vivo delivery issues of the disclosed double-stranded oligonucleotides can be further improved by forming drug compositions with suitable carriers or oligonucleotide conjugates with suitable conjugate molecules. For double-stranded oligonucleotides, carriers or conjugate molecules that can confer or enhance targeting are highly advantageous, as this greatly improves the efficiency of the double-stranded oligonucleotides in regulating target gene expression and reduces potential side effects. Furthermore, after introducing a targeting carrier or conjugate molecule, the double-stranded oligonucleotide also needs to be able to function at the target site; that is, the encapsulation / conjugation of the carrier or conjugate molecule should not affect the activity of the double-stranded oligonucleotide itself (e.g., in the case of siRNA, it should not affect the RNAi machine, i.e., the RISC complex, into which the siRNA is loaded into the cell). In addition, these targeting carriers or conjugate molecules are required to have good biocompatibility and minimal toxicity.

[0161] The pharmaceutical composition can be systematically distributed throughout the body or selectively enriched in specific parts of the body. The conjugate is generally targeted, and the type of conjugate molecule can be adaptively modified according to the expression and distribution of the target gene in the human or animal body to deliver the double-stranded oligonucleotide to the relevant site. For example, the conjugate molecule can be a conjugate molecule targeting the liver, lungs, kidneys, or cancer cells.

[0162] Pharmaceutical composition

[0163] In another aspect, this disclosure provides a pharmaceutical composition comprising the double-stranded oligonucleotide provided in this disclosure and a pharmaceutically acceptable carrier.

[0164] The pharmaceutically acceptable carrier can be a carrier conventionally used in the field of double-stranded oligonucleotide drug delivery, such as, but not limited to, magnetic nanoparticles (e.g., Fe3O4 or Fe2O3-based nanoparticles), carbon nanotubes, mesoporous silicon, calcium phosphate nanoparticles, polyethylenimine (PEI), polyamidoamine (PAMAM) dendrimer, poly(L-lysine) (PLL), chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), poly(D&L-lactic / glycolic acid) copolymer (PLGA), and poly(2-aminoethyl ethylene) phosphate. One or more of the following: phosphate), PPEEA, and poly(2-dimethylaminoethylmethacrylate), PDMAEMA, and their derivatives.

[0165] In some embodiments, there are no particular requirements for the content of double-stranded oligonucleotides and pharmaceutically acceptable carriers in the pharmaceutical composition. In some embodiments, the weight ratio of double-stranded oligonucleotides to pharmaceutically acceptable carriers can be 1:(1-500), and in some embodiments, the weight ratio is 1:(1-50).

[0166] In some embodiments, the pharmaceutical composition may also contain other pharmaceutically acceptable excipients, which may be one or more of a variety of formulations or compounds conventionally used in the art. For example, the other pharmaceutically acceptable excipients may include at least one of pH buffers, protectants, and osmotic pressure regulators.

[0167] The pH buffer solution can be a tris(hydroxymethyl)aminomethane hydrochloride buffer with a pH of 7.5-8.5 and / or a phosphate buffer with a pH of 5.5-8.5, for example, a phosphate buffer with a pH of 5.5-8.5.

[0168] The protective agent may be at least one selected from inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose, and glucose. Based on the total weight of the pharmaceutical composition, the content of the protective agent may be 0.01-30% by weight.

[0169] The osmotic pressure regulator may be sodium chloride and / or potassium chloride. The content of the osmotic pressure regulator results in an osmotic pressure of 200-700 milliosm / kg (mOsm / kg) for the pharmaceutical composition. The content of the osmotic pressure regulator can be readily determined by those skilled in the art based on the desired osmotic pressure. In some embodiments, the dosage of the formulation made from the pharmaceutical composition may be adjusted during administration depending on the route of administration.

[0170] In some embodiments, the pharmaceutical composition may be a liquid formulation, such as an injection; or it may be a lyophilized powder for injection, which is mixed with liquid excipients to form a liquid formulation for administration. The liquid formulation may be used, but is not limited to, for subcutaneous, intramuscular, or intravenous administration, or may be delivered via, but is not limited to, aerosol administration to the lungs, aerosol administration to other organs (such as the liver), or oral inhalation, or nasal administration. In some embodiments, the pharmaceutical composition is used for aerosol administration.

[0171] In some embodiments, the pharmaceutical composition may be in the form of a liposomal formulation. In some embodiments, the pharmaceutically acceptable carrier used in the liposomal formulation comprises an amine-containing transfection compound (hereinafter also referred to as an organic amine), a cofactor lipid, and / or a polyethylene glycol-modified lipid. The organic amine, cofactor lipid, and polyethylene glycol-modified lipid may be selected from one or more of the amine-containing transfection compounds or their pharmaceutically acceptable salts or derivatives, cofactor lipids, and polyethylene glycol-modified lipids described in Chinese patent application CN103380113A (which is incorporated herein by reference in its entirety).

[0172] In some embodiments, the organic amine may be a compound of formula (201) as described in Chinese patent application CN103380113A, or a pharmaceutically acceptable salt thereof:

[0173]

[0174] in:

[0175] X 101 and X 102 Each can be independently O, S, NA, or CA, where A is hydrogen or C1-C. 20 hydrocarbon chain;

[0176] Y 101 and Z 101Each can be independently C=O, C=S, S=O, CH-OH, or SO2;

[0177] R 101 R 102 R 103 R 104 R 105 R 106 and R 107 Each is independently hydrogen, cyclic or acyclic, substituted or unsubstituted, branched or straight aliphatic group, cyclic or acyclic, substituted or unsubstituted, branched or straight heteroaliphatic group, substituted or unsubstituted, branched or straight acyl group, substituted or unsubstituted, branched or straight aryl group, substituted or unsubstituted, branched or straight heteroaryl group;

[0178] x is an integer from 1 to 10;

[0179] n is an integer from 1 to 3, m is an integer from 0 to 20, and p is 0 or 1; where, if m = p = 0, then R 102 It is hydrogen;

[0180] Furthermore, if at least one of n or m is 2, then R 103 The nitrogen in formula (201) forms a structure as shown in formula (202) or formula (203):

[0181]

[0182] In this context, g, e, and f are each an integer from 1 to 6, "HCC" represents a hydrocarbon chain, and each *N represents a nitrogen atom in formula (201).

[0183] In some implementations, R 103 It is a polyamine. In other embodiments, R 103 It is a ketal. In some embodiments, R in formula (201) 101 and R 102 Each of them is independently an arbitrary substituted or unsubstituted, branched or straight-chain alkyl or alkenyl group having 3 to 20 carbon atoms, such as 8 to 18 carbon atoms, and 0 to 4 double bonds, such as 0 to 2 double bonds.

[0184] In some implementations, if each of n and m independently has a value of 1 or 3, then R 103 It can be any one of the following equations (204)-(213):

[0185]

[0186] In equations (204)-(213), g, e, and f are each independent integers from 1 to 6, each "HCC" represents a hydrocarbon chain, and each * indicates R. 103 Possible connection points with nitrogen atoms in equation (201), wherein each H at any * position can be replaced to achieve connection with nitrogen atoms in equation (201).

[0187] Those skilled in the art can obtain the compound represented by formula (201) by any reasonable method. In some embodiments, the compound represented by formula (201) can be prepared according to the description in Chinese patent application CN103380113A.

[0188] In some embodiments, the organic amine is an organic amine as shown in formula (214) and / or an organic amine as shown in formula (215):

[0189]

[0190] The auxiliary lipid is cholesterol, cholesterol analogues and / or cholesterol derivatives;

[0191] The PEGylated lipid is 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)]-2000.

[0192] In some embodiments, the molar ratio of the organic amine, the auxiliary lipid, and the polyethylene glycol-modified lipid in the pharmaceutical composition is (19.7-80):(19.7-80):(0.3-50), for example, (50-70):(20-40):(3-20).

[0193] In some embodiments, the pharmaceutical composition particles formed from the double-stranded oligonucleotide of the present disclosure and the above-mentioned amine-containing transfection reagent have an average diameter of about 30 nm to about 200 nm, typically about 40 nm to about 135 nm, and more typically, the average diameter of the liposome particles is about 50 nm to about 120 nm, about 50 nm to about 100 nm, about 60 nm to about 90 nm, or about 70 nm to about 90 nm. For example, the average diameter of the liposome particles is about 30, 40, 50, 60, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, 150, or 160 nm.

[0194] In some embodiments, in the pharmaceutical composition formed from the double-stranded oligonucleotide of this disclosure and the above-mentioned amine-containing transfection reagent, the weight ratio (weight / weight ratio) of the double-stranded oligonucleotide to all lipids (e.g., organic amines, auxiliary lipids and / or polyethylene glycol-modified lipids) is in the range of about 1:1 to about 1:50, about 1:1 to about 1:30, about 1:3 to about 1:20, about 1:4 to about 1:18, about 1:5 to about 1:17, about 1:5 to about 1:15, about 1:5 to about 1:12, about 1:6 to about 1:12 or about 1:6 to about 1:10. For example, the weight ratio of the double-stranded oligonucleotide of this disclosure to all lipids is about 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17 or 1:18.

[0195] In some embodiments, the components of the pharmaceutical composition may exist independently when sold, and may be in liquid form when used. In some embodiments, the pharmaceutical composition formed by the double-stranded oligonucleotide provided in this disclosure and the above-described pharmaceutically acceptable carrier can be prepared according to various known methods, simply by replacing existing double-stranded oligonucleotides with the double-stranded oligonucleotide provided in this disclosure; in some embodiments, it can be prepared according to the following method:

[0196] An organic amine, auxiliary lipid, and polyethylene glycol-modified lipid are suspended in an alcohol at the above molar ratio and mixed to obtain a lipid solution. The amount of alcohol used is such that the total mass concentration of the resulting lipid solution is 2-25 mg / mL, for example, 8-18 mg / mL. The alcohol is selected from pharmaceutically acceptable alcohols, such as alcohols that are liquid near room temperature, for example, one or more of ethanol, propylene glycol, benzyl alcohol, glycerol, polyethylene glycol 200, polyethylene glycol 300, and polyethylene glycol 400, for example, ethanol.

[0197] The double-stranded oligonucleotide provided in this disclosure is dissolved in a buffer salt solution to obtain an aqueous solution of the double-stranded oligonucleotide. The concentration of the buffer salt solution is 0.05-0.5M, for example, 0.1-0.2M. The pH of the buffer salt solution is adjusted to 4.0-5.5, for example, 5.0-5.2. The amount of buffer salt solution used is such that the concentration of the double-stranded oligonucleotide does not exceed 0.6 mg / mL, for example, 0.2-0.4 mg / mL. The buffer salt is selected from one or more of soluble acetate and soluble citrate, for example, sodium acetate and / or potassium acetate.

[0198] The lipid solution and the aqueous solution of the double-stranded oligonucleotide are mixed, and the resulting product is incubated at 40-60°C for at least 2 minutes, for example, 5-30 minutes, to obtain the incubated liposome formulation. The volume ratio of the lipid solution to the aqueous solution of the double-stranded oligonucleotide is 1:(2-5), for example, 1:4.

[0199] The incubated liposome formulation is concentrated or diluted, impurities are removed, and sterilization is performed to obtain the pharmaceutical composition provided in this disclosure. Its physicochemical parameters are: pH value of 6.5-8, encapsulation efficiency of not less than 80%, particle size of 40-200 nm, polydispersity index of not more than 0.30, and osmotic pressure of 250-400 mOsm / kg; for example, the physicochemical parameters can be: pH value of 7.2-7.6, encapsulation efficiency of not less than 90%, particle size of 60-100 nm, polydispersity index of not more than 0.20, and osmotic pressure of 300-400 mOsm / kg.

[0200] Concentration or dilution can be performed before, after, or simultaneously with impurity removal. Impurity removal can be achieved using various existing methods, such as ultrafiltration at 100 kDa using a tangential flow system or hollow fiber column, with the ultrafiltration exchange solution being phosphate-buffered saline (PBS) at pH 7.4. Sterilization can be achieved using various existing methods, such as filtration sterilization through a 0.22 μm filter.

[0201] Oligonucleotide conjugate

[0202] In another aspect, this disclosure provides an oligonucleotide conjugate comprising a double-stranded oligonucleotide provided in this disclosure and a conjugating group conjugated to the double-stranded oligonucleotide. In some embodiments, the conjugating group comprises a linker and a pharmaceutically acceptable targeting group and / or a delivery aid group, and the double-stranded oligonucleotide, the linker, and the targeting group or the delivery aid group are sequentially covalently or non-covalently linked, each targeting group being selected from ligands capable of binding to cell surface receptors, and each delivery aid group being selected from groups capable of increasing the biocompatibility of the oligonucleotide conjugate in the target organ or tissue.

[0203] In the context of this disclosure, unless otherwise stated, "conjugation" refers to the covalent connection between two or more chemical moieties, each having a specific function; correspondingly, "conjugated compound" refers to a compound formed by the covalent connection of these chemical moieties. Further, "oligonucleotide conjugated compound" refers to a compound formed by the covalent attachment of one or more chemical moieties having a specific function to an oligonucleotide. Oligonucleotide conjugated compounds should be understood, depending on the context, as a collective term for multiple oligonucleotide conjugated compounds or an oligonucleotide conjugated compound represented by a particular chemical formula. In the context of this disclosure, "conjugated molecule" should be understood as a specific compound that can be reactively conjugated to an oligonucleotide to ultimately form the oligonucleotide conjugated compounds of this disclosure.

[0204] 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 one embodiment, there are 1-6 target groups. In another 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 specific 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.

[0205] 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, the conjugation group is usually attached to a phosphate group of the nucleotide; when the conjugation group is attached to the inner sequence of a double-stranded oligonucleotide, the conjugation group is usually attached to a ribose ring or a base. Various connection methods can be found in: Muthiah Manoharan et al. siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo inhepatocytes. ACS Chemical biology, 2015, 10(5): 1181-7.

[0206] 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.

[0207] The targeting group can be linked to the double-stranded oligonucleotide molecule via a suitable adapter. Those skilled in the art can select a suitable adapter based on the specific type of the targeting group. For details on these adapters, types of targeting groups, and the methods of linking them to the double-stranded oligonucleotide, please refer to the disclosure of WO2015006740A2, the entire contents of which are incorporated herein by reference.

[0208] In some embodiments, the targeting group may be a ligand commonly 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.

[0209] In some embodiments, at least one or each of the target groups is selected from ligands capable of binding to cell surface receptors expressing the target gene.

[0210] In some embodiments, at least one or each of the target groups is selected from ligands capable of binding to receptors on the surface of mammalian hepatocytes. In some embodiments, each of the target groups is independently a ligand with affinity for desialyl glycoprotein receptors on the surface of mammalian hepatocytes. In some embodiments, each of the target groups is independently a desialyl glycoprotein or a sugar. In some embodiments, each of the target groups is independently selected from D-mannose pyranoyl ... α-D-Furfural, β-D-Furfural, α-D-Fructose, α-D-Galactopyranose, α-D-Galactopyranose, β-D-Galactopyranose, α-D-Galactopyranose, β-D-Galactopyranose, Glucosamine, Sialic acid, Galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-butyrylgalactosamine, N-isobutyrylgalactosamine, 2 -Amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-carboxamido-2,3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfonamido-D-glucopyranose, N-ethanolyl-α-neuraminic acid, 5-thio-β-D-glucopyranose, 2,3,4 One of the following: tri-O-acetyl-1-thio-6-O-triphenylmethyl-α-D-glucopyranoside methyl ester, 4-thio-β-D-galactopyranose, 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-glucopyranoside ethyl ester, 2,5-dehydrated-D-alosulfonyl, ribose, D-ribose, D-4-thioribose, L-ribose, and L-4-thioribose. In some embodiments, at least one or each of the target groups is galactose or N-acetylgalactosamine.

[0211] In some embodiments, at least one or each of the target groups is selected from ligands capable of binding to receptors on the surface of lung epithelial cells. In some embodiments, each of the target groups is selected from groups targeting integrin αvβ6 or groups targeting integrin αvβ3. In some embodiments, each of the target groups is independently a peptide or a small molecule ligand.

[0212] In some embodiments, at least one or each of the delivery aid groups is selected from groups capable of increasing the biocompatibility of the oligonucleotide conjugate in the central nervous system. In some embodiments, at least one or each of the delivery aid groups is selected from lipophilic molecules. In some embodiments, each of the delivery aid groups is C5-C. 18 Straight-chain hydrocarbon groups or steroidal compounds.

[0213] In some embodiments, the linkers in the oligonucleotide conjugates of this disclosure have a structure as shown in formula (301):

[0214]

[0215] Where k is an integer from 1 to 3;

[0216] L A Having a structure containing amide bonds as shown in formula (302), L B Having a structure containing N-acylpyrrolidine as shown in formula (303), containing a carbonyl group and an oxygen atom, L C It is a linking group based on hydroxymethylaminomethane, dihydroxymethylaminomethane, or trihydroxymethylaminomethane;

[0217]

[0218] Where, n 302 q 302 and p 302 Each is an independent integer from 2 to 6; optionally, n 302 q 302 and p 302 Each is independently 2 or 3; n 303 n is an integer between 4 and 16, optionally n 303 For integers between 8 and 12, This indicates the site where the group is covalently linked.

[0219] In the aforementioned connector, each L A Each of the target groups is connected via an ether bond and via L C The oxygen atom of the hydroxyl group in some of the middle groups is related to L. C Partially linked by ether bonds; L B Through the carbonyl group in formula (303) and L C The nitrogen atom of the amino group in some of the oligonucleotides forms an amide bond and is linked to the double-stranded oligonucleotide through the oxygen atom in formula (303) by forming a phosphate ester bond or a thiophosphate ester bond.

[0220] In some embodiments, the oligonucleotide conjugates provided in this disclosure have a structure as shown in formula (305):

[0221]

[0222] Nu represents the double-stranded oligonucleotide provided in this disclosure.

[0223] In some embodiments, the linkers in the oligonucleotide conjugates of this disclosure have the structure shown in formula (306):

[0224]

[0225] Where, n 306 For each p, the integer is between 0 and 3. 306 Independently, integers from 1 to 6. The site indicates a covalently linked group; the linking group is connected to the target group by an ether bond formed by an oxygen atom marked with *; the linking group is connected to the double-stranded oligonucleotide by at least one of the oxygen atoms marked with # forming a phosphate ester bond or a thiophosphate ester bond, and the remaining oxygen atoms marked with # are connected to hydrogen atoms to form hydroxyl groups, or connected to C1-C3 alkyl groups to form C1-C3 alkoxy groups;

[0226] In some embodiments, the oligonucleotide conjugates of this disclosure have a structure as shown in formula (307):

[0227]

[0228] Nu represents the double-stranded oligonucleotide provided in this disclosure.

[0229] In some embodiments, the oligonucleotide conjugates of this disclosure have the structure shown in formula (308):

[0230]

[0231] in,

[0232] n1 is an integer selected from 1 to 3, and n3 is an integer selected from 0 to 4;

[0233] Each m1, m2, or m3 is an independent integer selected from 2 to 10;

[0234] R 10 R 11 R 12 R 13 R 14 or R 15 Each is independently H, or selected from the group consisting of C1-C. 10 Alkyl, C1-C 10 Halogenated alkyl groups and C1-C 10 Alkoxy;

[0235] R3 has the structure shown in Equation A59:

[0236]

[0237] Wherein, E1 is OH, SH or BH2, and Nu represents the double-stranded oligonucleotide provided in this disclosure;

[0238] R2 is a straight-chain alkylene group with a length of 1-20 carbon atoms, wherein one or more carbon atoms are optionally replaced by any one or more groups selected from the group consisting of: C(O), NH, O, S, CH=N, S(O)2, C2-C 10 alkenyl, C2-C 10 Ethyne group, C6-C 10 Aromatic, C3-C 18 Heterocyclic groups and C5-C 10 Heteroaryl; and wherein R2 may optionally have any one or more substituents from the group consisting of: C1-C 10 Alkyl, C6-C 10 Aryl, C5-C 10 heteroaryl, C1-C 10 Halogenated alkyl, -OC1-C 10 Alkyl, OC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-OH, -OC1-C 10 Halogenated alkyl, -SC1-C 10 Alkyl, -SC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-SH, -SC1-C 10 Halogenated alkyl groups, halogenated substituents, -OH, -SH, -NH2, -C1-C 10 Alkyl-NH2,-N(C1-C) 10 Alkyl) (C1-C 10 alkyl), -NH(C1-C 10 Alkyl), -N(C1-C 10 Alkyl) (C1-C 10 alkylphenyl), NH(C1-C 10 Alkylphenyl), cyano, nitro, -CO2H, -C(O)O(C1-C 10 Alkyl), -CON (C1-C) 10 Alkyl) (C1-C 10 Alkyl), -CONH (C1-C) 10 Alkyl groups, -CONH2, -NHC(O) (C1-C) 10 alkyl), -NHC(O)(phenyl), -N(C1-C 10Alkyl)C(O)(C1-C 10 Alkyl), -N(C1-C 10 alkyl)C(O)(phenyl), C(O)C1-C 10 Alkyl, C(O)C1-C 10 Alkylphenyl, C(O)C1-C 10 Haloalkyl, -OC(O)Cl-C 10 Alkyl group, -SO2 (C1-C) 10 alkyl), -SO2 (phenyl), -SO2 (C1-C 10 Halogenated alkyl groups), -SO2NH2, -SO2NH (C1-C 10 alkyl), -SO2NH (phenyl), -NHSO2 (C1-C 10 Alkyl), -NHSO2 (phenyl) and -NHSO2 (C1-C) 10 (halogenated alkyl);

[0239] Each L1 is independently a straight-chain alkylene group with a length of 1-70 carbon atoms, wherein one or more carbon atoms are optionally replaced by any one or more groups selected from the group consisting of: C(O), NH, O, S, CH=N, S(O)2, C2-C 10 alkenyl, C2-C 10 Ethyne group, C6-C 10 Aromatic, C3-C 18 Heterocyclic groups and C5-C 10 Heteroaryl; and wherein L1 may optionally have any one or more substituents from the group consisting of: C1-C 10 Alkyl, C6-C 10 Aryl, C5-C 10 heteroaryl, C1-C 10 Halogenated alkyl, -OC1-C 10 Alkyl, OC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-OH, -OC1-C 10 Halogenated alkyl, -SC1-C 10 Alkyl, -SC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-SH, -SC1-C 10 Halogenated alkyl groups, halogenated substituents, -OH, -SH, -NH2, -C1-C 10 Alkyl-NH2,-N(C1-C) 10 Alkyl) (C1-C 10 alkyl), -NH(C1-C 10 Alkyl), -N(C1-C 10Alkyl) (C1-C 10 alkylphenyl), NH(C1-C 10 Alkylphenyl), cyano, nitro, -CO2H, -C(O)O(C1-C 10 Alkyl), -CON (C1-C) 10 Alkyl) (C1-C 10 Alkyl), -CONH (C1-C) 10 Alkyl group), -CONH2, -NHC(O) (C1-C 10 alkyl), -NHC(O)(phenyl), -N(C1-C 10 Alkyl)C(O)(C1-C 10 Alkyl), -N(C1-C 10 alkyl)C(O)(phenyl), C(O)C1-C 10 Alkyl, C(O)C1-C 10 Alkylphenyl, C(O)C1-C 10 Haloalkyl, -OC(O)Cl-C 10 Alkyl group, -SO2 (C1-C) 10 alkyl), -SO2 (phenyl), -SO2 (C1-C 10 Halogenated alkyl groups), -SO2NH2, -SO2NH (C1-C 10 alkyl), -SO2NH (phenyl), -NHSO2 (C1-C 10 Alkyl), -NHSO2 (phenyl) and -NHSO2 (C1-C) 10 (halogenated alkyl);

[0240] Indicates the site where groups are covalently linked;

[0241] M1 represents a targeting group, the definition of which and the range of possible selections are the same as described above. In some embodiments, each M1 is independently selected from a group of ligands that have affinity for desialylate glycoprotein receptors on the surface of mammalian liver cells.

[0242] Those skilled in the art will understand that, although L1 is defined as a linear alkyl group for convenience, it may not be a linear group or may have a different name, such as an amine or alkenyl group resulting from the above substitutions and / or replacements. For the purposes of this disclosure, the length of L1 is the number of atoms in the chain connecting the two attachment points. For this purpose, the ring (such as a heterocyclic or heteroaryl group) obtained by replacing a carbon atom of the linear alkylene group is counted as one atom.

[0243] When M1 is a ligand with affinity for the desialyl glycoprotein receptor on the surface of mammalian liver cells, in some embodiments, n1 can be an integer from 1 to 3, and n3 can be an integer from 0 to 4, ensuring that the number of M1 ligands in the conjugate is at least 2; in some embodiments, n1 + n3 ≥ 2, which ensures that the number of M1 ligands is at least 3, making it easier for the M1 ligand to bind to the desialyl glycoprotein receptor on the liver surface, thereby promoting the conjugate's entry into the cell via endocytosis. Experiments show that when the number of M1 ligands is greater than 3, the ease of binding of the M1 ligand to the desialyl glycoprotein receptor on the liver surface does not increase significantly. Therefore, considering factors such as ease of synthesis, structural / process cost, and delivery efficiency, in some embodiments, n1 is an integer from 1 to 2, n3 is an integer from 0 to 1, and n1 + n3 = 2 - 3.

[0244] In some embodiments, when m1, m2, and m3 are independently selected from integers of 2 to 10, the spatial positions between the multiple M1 ligands can be adapted to the binding of the M1 ligand to the liver surface desialylate glycoprotein receptor. In order to make the conjugates provided in this disclosure simpler, easier to synthesize, and / or reduce costs, in some embodiments, m1, m2, and m3 are each independently an integer of 2 to 5, and in some embodiments, m1 = m2 = m3.

[0245] Those skilled in the art will understand that when R 10 R 11 R 12 R 13 R 14 and R 15 Each is independently selected from H, C1-C 10 Alkyl, C1-C 10 Halogenated alkyl groups and C1-C 10 Using one of the alkoxy groups will not change the properties of the conjugates disclosed herein, and the objectives of this disclosure can still be achieved. In some embodiments, R 10 R 11 R 12 R 13 R 14 and R 15 Each is independently selected from H, methyl, and ethyl. In some embodiments, R 10 R 11 R 12 R 13 R 14 and R 15 All are H.

[0246] According to the oligonucleotide conjugates provided in this disclosure, R3 is a group with the structure shown in Formula A59, wherein E1 is OH, SH or BH2. In some embodiments, E1 is OH or SH, based on the consideration of the availability of raw materials.

[0247] In some implementations, R2 is chosen to enable the connection between N and A59 on the nitrogen-containing framework. In the context of this disclosure, "nitrogen-containing framework" refers to a framework connected to R. 10 R 11 R 12 R 13 R 14 and R 15 The chain structure in which carbon atoms are interconnected with nitrogen (N) is used. Therefore, R2 can be any linking group capable of connecting the A59 group to the N on the nitrogen-containing backbone in a suitable manner. In some embodiments, when the oligonucleotide conjugates of this disclosure are prepared by solid-phase synthesis, the R2 group needs to contain both a linking site for the N on the nitrogen-containing backbone and a linking site for the P in R3. In some embodiments, the site in R2 that links to the N on the nitrogen-containing backbone forms an amide bond with N, and the site that links to the P on R3 forms a phosphate ester bond with P. In some embodiments, R2 is B5, B6, B5', or B6'.

[0248]

[0249] in, This indicates the site where a group is covalently bonded.

[0250] The value of q2 can be an integer from 1 to 10. In some implementations, q2 is an integer from 1 to 5.

[0251] The function of L1 is to link the M1 ligand to the N on the nitrogen-containing backbone, providing targeting functionality for the oligonucleotide conjugates of this disclosure. In some embodiments, L1 is selected from one or more linkage combinations of groups of formulas A1-A26. In some embodiments, L1 is selected from one or more linkage combinations of A1, A4, A5, A6, A8, A10, A11, and A13; in some embodiments, L1 is selected from a linkage combination of at least two of A1, A4, A8, A10, and A11; in some embodiments, L1 is selected from a linkage combination of at least two of A1, A8, and A10.

[0252]

[0253]

[0254] Where j1 is an integer from 1 to 20; R' is an integer from 1 to 20; 10Alkyl group; Ra is selected from one of the groups of formula A27-A45:

[0255]

[0256]

[0257] Rb is C1-C 10 Alkyl groups.

[0258] In some embodiments, the length of L1 can be 3-25 atoms, 3-20 atoms, 4-15 atoms, or 5-12 atoms. In some embodiments, the length of L1 is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, or 60 atoms.

[0259] In some embodiments, j1 is an integer from 2 to 10, and in some embodiments, j1 is an integer from 3 to 5. In some embodiments, j2 is an integer from 2 to 10, and in some embodiments, j2 is an integer from 3 to 5. R' is a C1-C4 alkyl group, and in some embodiments, R' is one of methyl, ethyl, and isopropyl. Ra is one of A27, A28, A29, A30, and A31, and in some embodiments, Ra is A27 or A28. Rb is a C1-C5 alkyl group, and in some embodiments, Rb is one of methyl, ethyl, isopropyl, and butyl. In some embodiments, j1, j2, R', Ra, and Rb in formulas A1-A26 are selected respectively to achieve N-linking of the M1 ligand to the nitrogen-containing backbone and to make the spatial positions between the M1 ligands more suitable for the binding of the M1 ligand to the liver surface desialyl glycoprotein receptor.

[0260] In some embodiments, the oligonucleotide conjugates of this disclosure have the structures shown in formulas (403), (404), (405), (406), (407), (408), (409), (410), (411), (412), (413), (414), (415), (416), (417), (418), (419), (420), (421), or (422):

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268] In some embodiments, P in Formula A59 can be attached to any possible position in the double-stranded oligonucleotide sequence; for example, P in Formula A59 can be attached to any nucleotide of the sense or antisense strand of the double-stranded oligonucleotide. In some embodiments, P in Formula A59 is attached to any nucleotide of the sense strand of the double-stranded oligonucleotide. In some embodiments, P in Formula A59 is attached to the end of the sense or antisense strand of the double-stranded oligonucleotide; in some embodiments, P in Formula A59 is attached to the end of the sense strand of the double-stranded oligonucleotide. The end refers to the first four nucleotides of the sense or antisense strand counting from one end. In some embodiments, P in Formula A59 is attached to the end of the sense or antisense strand of the double-stranded oligonucleotide; in some embodiments, P in Formula A59 is attached to the 3' end of the sense strand of the double-stranded oligonucleotide. When attached to the above-described positions on the sense strand of the double-stranded oligonucleotide, the conjugate provided in this disclosure, upon entering the cell, can release the individual antisense strand of the double-stranded oligonucleotide upon unwinding to regulate target gene expression.

[0269] The P in Formula A59 can be attached to any possible position on the nucleotide in the double-stranded oligonucleotide, such as the 5' position, 2' position, 3' position, or base of the nucleotide. In some embodiments, the P in Formula A59 can be attached to the 2', 3', or 5' position of the nucleotide in the double-stranded oligonucleotide by forming a phosphodiester bond. In some embodiments, the P in Formula A59 is attached to the oxygen atom formed by the dehydrogenation of the 3'-hydroxyl group of the 3'-terminal nucleotide of the positive strand of the double-stranded oligonucleotide, or the P in Formula A59 is attached to the nucleotide by substituting a hydrogen atom in the 2'-hydroxyl group of one nucleotide in the positive strand of the double-stranded oligonucleotide, or the P in Formula A59 is attached to the nucleotide by substituting a hydrogen atom in the 5'-hydroxyl group of the 5'-terminal nucleotide of the positive strand of the double-stranded oligonucleotide.

[0270] In some embodiments, the double-stranded oligonucleotides contained in the oligonucleotide conjugates of this disclosure may be siRNAs, in which case the oligonucleotide conjugates of this disclosure are also referred to as siRNA conjugates. In some embodiments, the double-stranded oligonucleotides contained in the oligonucleotide conjugates of this disclosure may be, for example, siRNAs listed in Table 1. Oligonucleotide conjugates containing these siRNAs exhibit low off-target effects and high mRNA repressive activity against target gene expression.

[0271] Preparation of the disclosed oligonucleotide conjugates

[0272] The aforementioned oligonucleotide conjugates can be synthesized using methods already described in detail in the prior art. For example, WO2015006740A2 describes in detail various methods for preparing siRNA conjugates. When the double-stranded oligonucleotide is siRNA, the oligonucleotide conjugates disclosed herein can also be obtained using methods well known to those skilled in the art. For example, WO2014025805A1 describes a method for preparing the structure shown in formula (305), and Rajeev et al. describe a method for preparing the structure shown in formula (307) in ChemBioChem 2015, 16, 903-908. Chinese patent application CN110959011A also discloses in detail a method for preparing the oligonucleotide conjugate shown in formula (308). The contents of the above-mentioned documents are incorporated herein by reference in their entirety.

[0273] The oligonucleotide conjugates disclosed herein may also be used in combination with other pharmaceutically acceptable excipients, which may be one or more of a variety of formulations or compounds conventionally used in the art, as detailed in the above description of the pharmaceutical compositions of this disclosure.

[0274] Use of the disclosed double-stranded oligonucleotides, pharmaceutical compositions, and oligonucleotide conjugates

[0275] In some embodiments, this disclosure provides the use of the double-stranded oligonucleotides, pharmaceutical compositions, and / or oligonucleotide conjugates provided herein in medicaments for treating and / or preventing diseases or symptoms associated with mRNA levels of a target gene expression. In some embodiments, the specific gene is a gene aberrantly expressed in hepatocytes. In some embodiments, the specific gene is an endogenous gene expressed in the liver. In some embodiments, the specific gene is a pathogen gene that proliferates in the liver. In some embodiments, the specific gene is a gene expressed in lung epithelial cells. In some embodiments, the specific gene is a gene expressed in the central nervous system. In some embodiments, the specific gene is a gene expressed in tumor cells. In some embodiments, the mRNA expressing the target gene is selected from one of the following mRNAs transcribed from genes: 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. In some embodiments, the mRNA expressing the target gene is selected from mRNA expressed by the hepatitis B virus gene (HBV), mRNA expressed by the angiopoietin-like protein 3 (ANGPTL3) gene, or mRNA expressed by the apolipoprotein C3 (ApoC3) gene. In some embodiments, the disease or symptom associated with the mRNA level of the target gene expression is chronic liver disease, hepatitis, liver fibrosis, hepatoproliferative disorders, and / or dyslipidemia. In some embodiments, the disease or symptom associated with the mRNA level of the target gene expression is hepatitis B or dyslipidemia. In some embodiments, the dyslipidemia is hypercholesterolemia, hypertriglyceridemia, or atherosclerosis.

[0276] In some embodiments, this disclosure provides a method for treating and / or preventing diseases or symptoms associated with the level of mRNA expression of a target gene, the method comprising administering to a subject in need an effective amount of a double-stranded oligonucleotide, pharmaceutical composition, and / or oligonucleotide conjugate provided in this disclosure. In some embodiments, the mRNA expressing the target gene is selected from mRNAs transcribed from the following genes: 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. In some embodiments, the mRNA expressed by the target gene is selected from mRNA expressed by the hepatitis B virus (HBV) gene, mRNA expressed by the angiopoietin-like protein 3 (ANGPTL3) gene, or mRNA expressed by the apolipoprotein C3 (ApoC3) gene. In some embodiments, the disease or symptom associated with the level of the target gene's expressed mRNA is chronic liver disease, hepatitis, liver fibrosis, hepatoproliferative disorders, and / or dyslipidemia. In some embodiments, the disease or symptom associated with the level of the target gene's expressed mRNA is hepatitis B or dyslipidemia. In some embodiments, the dyslipidemia is hypercholesterolemia, hypertriglyceridemia, or atherosclerosis.

[0277] In some embodiments, the conjugates provided in this disclosure can also be used to treat other liver diseases, including diseases characterized by unwanted cell proliferation, hematologic disorders, metabolic disorders, and diseases characterized by inflammation. Proliferative liver diseases can be benign or malignant, such as cancer, hepatocellular carcinoma (HCC), liver metastases, or hepatoblastoma. Hematologic or inflammatory liver diseases can involve clotting factors, complement-mediated inflammation, or fibrosis. Metabolic liver diseases include dyslipidemia and irregularities in glucose regulation. In one embodiment, the disease is treated by administering one or more double-stranded oligonucleotides having a sequence highly homologous to the gene sequence involved in the disease.

[0278] In some embodiments, this disclosure provides a method for regulating the expression level of a target gene in cells, the method comprising contacting the cells with an effective amount of the double-stranded oligonucleotide, pharmaceutical composition, and / or oligonucleotide conjugate provided in this disclosure. In some embodiments, the mRNA expressing the target gene is selected from the mRNAs transcribed from the following genes: 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. In some embodiments, the regulation refers to inhibiting the expression of a target gene in the cell, wherein the mRNA expressing the target gene is selected from the mRNA expressed by the hepatitis B virus gene (HBV), the mRNA expressed by the angiopoietin-like protein 3 (ANGPTL3) gene, or the mRNA expressed by the apolipoprotein C3 (ApoC3) gene.

[0279] By administering the double-stranded oligonucleotides, pharmaceutical compositions, and / or oligonucleotide conjugates provided in this disclosure to subjects in need, the prevention and / or treatment of pathological conditions or diseases caused by the expression of specific genes in cells can be achieved through mechanisms that regulate gene expression. Therefore, the double-stranded oligonucleotides, pharmaceutical compositions, and / or oligonucleotide conjugates provided in this disclosure can be used for the prevention and / or treatment of said pathological conditions or diseases, or for the preparation of medicaments for the prevention and / or treatment of the pathological conditions or diseases described herein.

[0280] As used herein, the term "administration" refers to the placement of a double-stranded oligonucleotide, pharmaceutical composition, and / or oligonucleotide conjugate into a subject by means of a method or route that at least partially targets a desired site to produce a desired effect. Routes of administration suitable for the methods of this disclosure include local administration and systemic administration. Generally, local administration results in the delivery of more double-stranded oligonucleotides, pharmaceutical compositions, and / or oligonucleotide conjugates to a specific site compared to the entire body of the subject; while systemic administration results in the delivery of said double-stranded oligonucleotides, pharmaceutical compositions, and / or oligonucleotide conjugates to substantially the entire body of the subject.

[0281] The medication may be administered to the subject via any suitable route known in the art, including but not limited to: oral or parenteral routes, such as intravenous administration, intramuscular administration, subcutaneous administration, transdermal administration, airway administration (aerosol), pulmonary administration, nasal administration, rectal administration, and local administration (including oral and sublingual administration). Administration frequency may be once or more daily, weekly, bi-weekly, bi-weekly, monthly, or annually.

[0282] The dosages of the double-stranded oligonucleotides, pharmaceutical compositions, and / or oligonucleotide conjugates described in this disclosure are conventional dosages in the art, which can be determined based on various parameters, particularly the age, weight, and sex of the subject. Toxicity and efficacy can be determined in cell culture or laboratory animals using standard pharmaceutical procedures, such as determining the LD50 (the dose that causes 50% of the population to die) and ED50 (the dose that elicits 50% of the maximum response intensity in a quantitative response, and the dose that elicits a positive response in 50% of the subjects in a qualitative response). Ranges of human dosages can be derived based on data obtained from cell culture analysis and animal studies.

[0283] When administering the double-stranded oligonucleotides, pharmaceutical compositions, and / or oligonucleotide conjugates described in this disclosure, for example, to male or female C57BL / 6J or C3H / HeNCrlVr mice, 6-12 weeks old, weighing 18-25 g, the amount of double-stranded oligonucleotides in the double-stranded oligonucleotides, pharmaceutical compositions, and / or oligonucleotide conjugates may be 0.001-100 mg / kg body weight, in some embodiments 0.01-50 mg / kg body weight, in further embodiments 0.05-20 mg / kg body weight, in even further embodiments 0.1-15 mg / kg body weight, and in still further embodiments 0.1-10 mg / kg body weight. The above amounts are preferred when administering the double-stranded oligonucleotides, pharmaceutical compositions, and / or oligonucleotide conjugates described in this disclosure.

[0284] Furthermore, by introducing the double-stranded oligonucleotides, pharmaceutical compositions, and / or oligonucleotide conjugates disclosed herein into cells with abnormal gene expression, the expression of that specific gene in the cells can also be inhibited through gene expression regulation mechanisms. In some embodiments, the cells are hepatocytes. In some embodiments, the hepatocytes may be cells selected from hepatocellular carcinoma cell lines such as Hep3B, HepG2, and Huh7, or isolated primary hepatocytes; in some embodiments, the hepatocytes are primary hepatocytes.

[0285] The methods provided in this disclosure for inhibiting the expression of specific genes in cells involve double-stranded oligonucleotides, pharmaceutical compositions, and / or oligonucleotide conjugates, the amount of which is readily determined by those skilled in the art based on the desired effect. For example, in some embodiments, the double-stranded oligonucleotides, pharmaceutical compositions, and / or oligonucleotide conjugates are siRNA conjugates, and the amount of siRNA in the provided siRNA conjugate is sufficient to reduce the expression of the target gene and result in an extracellular concentration of 1 pM to 1 μM, or 0.01 nM to 100 nM, or 0.05 nM to 50 nM, or about 5 nM at the surface of the target cells. The amount required to achieve this local concentration will vary depending on various factors, including the delivery method, delivery site, the number of cell layers between the delivery site and the target cells or tissue, and whether the delivery is local or systemic. The concentration at the delivery site can be significantly higher than the concentration at the surface of the target cells or tissue.

[0286] Kit

[0287] This disclosure provides a kit comprising the double-stranded oligonucleotide, pharmaceutical composition and / or oligonucleotide conjugate provided in this disclosure.

[0288] In some embodiments, the kit described herein may provide double-stranded oligonucleotides, pharmaceutical compositions, and / or conjugates in a single container. In some embodiments, the kit described herein may include a container providing pharmaceutically acceptable excipients. In some embodiments, the kit may also contain other components, such as stabilizers or preservatives. In some embodiments, the kit described herein may contain at least one other therapeutic agent in a container other than the one providing the double-stranded oligonucleotides, pharmaceutical compositions, and / or conjugates described herein. In some embodiments, the kit may include instructions for mixing the double-stranded oligonucleotides, pharmaceutical compositions, and / or conjugates with pharmaceutically acceptable carriers and / or excipients or other components (if any).

[0289] In the kits disclosed herein, the double-stranded oligonucleotides and pharmaceutically acceptable carriers and / or excipients, as well as the pharmaceutical compositions and / or conjugates, and / or pharmaceutically acceptable excipients, may be provided in any form, such as liquid, dry, or lyophilized. In some embodiments, the double-stranded oligonucleotides and pharmaceutically acceptable carriers and / or excipients, as well as the pharmaceutical compositions and / or conjugates and optional pharmaceutically acceptable excipients, are substantially pure and / or sterile. In some embodiments, sterile water may be provided in the kits disclosed herein.

[0290] The present disclosure will be further illustrated by the following examples, but the present disclosure is not limited thereto.

[0291] Without intending to be limiting, the invention is further described in detail below in the embodiments and in exemplary embodiments relating to small interfering RNA (siRNA) in pharmaceutical compositions and / or oligonucleotide conjugates of this disclosure. In this context, the double-stranded oligonucleotides, pharmaceutical compositions, and oligonucleotide conjugates of this disclosure are siRNA, siRNA-containing pharmaceutical compositions, and siRNA conjugates, respectively. In the context of this disclosure, for ease of description, the siRNA, siRNA-containing pharmaceutical compositions, and siRNA conjugates in these embodiments are also referred to as the siRNA, pharmaceutical compositions, and siRNA conjugates of this disclosure. This does not mean that the double-stranded oligonucleotides of this disclosure can only be siRNA; rather, the double-stranded oligonucleotides can be other variants disclosed herein or known to those skilled in the art, such as small activating RNA (saRNA), etc. It is contemplated that, based on the detailed description of siRNA, siRNA-containing pharmaceutical compositions, and siRNA conjugates, other double-stranded oligonucleotides will similarly function when used alone or in the formation of the pharmaceutical compositions and / or oligonucleotide conjugates described in this disclosure.

[0292] Example

[0293] Unless otherwise specified, the reagents and culture media used in the following examples are all commercially available products, and the nucleic acid electrophoresis, real-time PCR and other operations used are all performed in accordance with the methods described in Molecular Cloning (Cold Spring Harbor LBboratory Press (1989)).

[0294] Preparation Example 1: Synthesis of the siRNA conjugates disclosed herein

[0295] 1-1 Synthesis of the monomer SN201 with a substitute group

[0296] First, the monomer SN201 with the substituted group shown in the following formula was synthesized according to the following method:

[0297] 1-1-1 Synthesis of SNB-1

[0298]

[0299] 10.5 g of diethanolamine (100 mmol, 1.0 eq) was dissolved in 100 mL of pyridine, and then 35.6 g of 4,4'-dimethyltriphenylmethyl chloride (DMTrCl) was added. The mixture was reacted with stirring for 6 h. 20 mL of methanol was added to the reaction mixture, and after concentration to remove the solvent, 10 mL of saturated sodium bicarbonate aqueous solution was added. The mixture was extracted with 5 mL of dichloromethane, and the aqueous phase was extracted once more with 5 mL of dichloromethane. The organic phases were combined and washed once with 5 mL of saturated NaCl aqueous solution. The organic phase was dried over anhydrous sodium sulfate and concentrated to remove the solvent. Subsequently, the mixture was subjected to normal-phase silica gel column chromatography with a gradient elution of dichloromethane:methanol = 100:1-50:1 (V:V). The eluent was collected, concentrated to remove the solvent, and 15 g of compound SNB-1 was obtained as a yellow oily liquid. 1 H NMR(500MHz,Chloroform-d)δ7.47–7.41(m,2H),7.37–7.30(m,2H),7.30–7.24(m,1H),7.16–7.10(m,4H),6.99–6.93(m ,4H),3.80–3.71(m,8H),3.67(t,J=3.9Hz,2H),3.45(tt,J=5.5,4.0Hz,1H),3.28(t,J=5.7Hz,1H),2.95–2.88(m,4H).

[0300] 1-1-2 Synthesis of SNB-2

[0301]

[0302] 15 g of compound SNB-1 prepared in step (1-1-1) was dissolved in 150 mL of pyridine, and 6.1 g of tert-butyldimethylchlorosilane (TBSCl) was added. The mixture was reacted with stirring for 6 h. The solvent was removed by concentration, and the mixture was dissolved in 50 mL of dichloromethane. The solution was washed once with 10 mL of saturated sodium bicarbonate aqueous solution and once with saturated NaCl aqueous solution. The organic phase was dried with anhydrous sodium sulfate and the solvent was removed by concentration. The mixture was then subjected to normal-phase silica gel column chromatography, using a mixed solvent of dichloromethane:methanol = 50:1 (V:V) as the mobile phase. The eluent was collected, concentrated to remove the solvent, and 18 g of compound SNB-2 was obtained as a brown oily liquid. 1H NMR(500MHz,Chloroform-d)δ7.47–7.41(m,2H),7.34(ddd,J=7.1,6.2,0.9Hz,2H),7.30–7.24(m,1H),7.13(dd,J=8.3,1.6Hz,3H),6.99–6.93( m,4H),3.78(s,4H),3.66(td,J=4.2,2.8Hz,3H),3.60(t,J=4.5Hz,2H), 2.92(q,J=4.0Hz,2H),2.87(q,J=4.5Hz,2H),0.89(s,7H),0.03(s,4H).

[0303] Synthesis of UP-1 (1-1-3)

[0304]

[0305] 20 g of uracil was added to a saturated aqueous solution containing 20 g of KOH, followed by 29.7 g of bromoacetic acid. The reaction was carried out at 60 °C for 16 h. The pH was adjusted to pH = 2 with concentrated hydrochloric acid, and the mixture was stirred for 2 h. The filtrate was removed by filtration, and the filter cake was washed twice with 50 mL of water each time. The filter cake was dried to obtain 24 g of white solid UP-1. 1 H NMR (500MHz, Chloroform-d) δ9.76 (s, 1H), 7.49 (d, J = 7.5Hz, 1H), 5.72 (d, J = 7.5Hz, 1H), 4.32 (s, 2H).

[0306] 1-1-4 Synthesis of SNB-3

[0307]

[0308] 18 g of compound SNB-2 prepared in step 1-1-2 was dissolved in 200 mL of N,N-dimethylformamide (DMF), and 7.1 g of compound UP-1 prepared in step 1-1-3, 15.7 g of O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU) and 10.4 g of triethylamine were added. The mixture was reacted at room temperature for 3 h. The reaction mixture was extracted with 200 mL of ethyl acetate and 100 mL of saturated sodium bicarbonate aqueous solution. The aqueous phase was extracted twice with 100 mL of ethyl acetate each time. The combined organic phases were washed successively with 20 mL of saturated sodium bicarbonate aqueous solution and 20 mL of saturated NaCl aqueous solution. The organic phase was dried over anhydrous sodium sulfate, concentrated to remove the solvent, and dried to obtain 20 g of white solid SNB-3. 1H NMR(500MHz,Chloroform-d)δ9.79(s,1H),7.47–7.37(m,4H),7.34(ddd,J=7.1,6.2,0.9Hz,2H),7.30–7.22(m,1H),7.13(dd,J=8.3, 1.6Hz,4H),6.99–6.93(m,4H),4.51(s,2H),3.78(s,5H),3.71(td,J=6.2,4.8Hz,5H),3.53–3.41(m,5H),0.89(s,8H),0.03(s,5H).

[0309] 1-1-5 Synthesis of SNB-4

[0310]

[0311] 20 g of compound SNB-3 obtained in step 1-1-4 was dissolved in 12 mL of tetrahydrofuran (THF), and 45 mL of 1 M tetrabutylammonium fluoride (TBAF) aqueous solution was added. The mixture was stirred at room temperature for 2 h. 150 mL of ethyl acetate and 150 mL of saturated sodium bicarbonate aqueous solution were added to the reaction mixture, and the mixture was extracted. The aqueous phase was extracted once with 100 mL of ethyl acetate. The organic phases were combined, washed with 20 mL of saturated NaCl aqueous solution, concentrated to remove the solvent, and dried to obtain 16 g of pale yellow solid SNB-4. 1 H NMR(500MHz,Chloroform-d)δ9.79(s,1H),7.47–7.37(m,3H),7.34(ddd,J=7.1,6.2,0.9Hz,2H),7.30–7.24(m,1H),7.16–7.10(m,4H),6. 99–6.93(m,4H),5.72(d,J=7.5Hz,1H),4.51(s,2H),3.78(s,6H),3.74–3.65(m,4H),3.51(t,J=6.3Hz,1H),3.44(dt,J=9.7,6.0Hz,4H).

[0312] Synthesis of SN201, a monomer with 1-1-6 substituent groups

[0313]

[0314] 6.0 g of compound SNB-4, prepared in step 1-1-5, was dissolved in 120 mL of dichloromethane (DCM). 1.1 g of N-methylimidazolium, 2.5 g of pyridinium trifluoroacetate, and 6.5 g of bis(diisopropylamino)(2-cyanoethoxy)phosphine were added sequentially, and the mixture was stirred at room temperature for 2 h under nitrogen protection. The reaction mixture was extracted with 100 mL of saturated sodium bicarbonate aqueous solution, followed by extraction with 20 mL of dichloromethane. The combined organic phases were washed sequentially with 20 mL of saturated sodium bicarbonate aqueous solution and 20 mL of saturated NaCl aqueous solution. The organic phase was dried over anhydrous sodium sulfate and concentrated to remove the solvent. The mixture was then subjected to normal-phase silica gel column chromatography, eluting with ethyl acetate:dichloromethane = 5:1 (V:V). The eluent was collected, concentrated to remove the solvent, and 5.9 g of compound SN201 was obtained as a white solid. 1 H NMR(500MHz,Chloroform-d)δ9.79(s,1H),7.47–7.37(m,3H),7.34(ddd,J=7.1,6.2,1.0Hz,2H) ,7.30–7.24(m,1H),7.13(dd,J=8.3,1.6Hz,4H),6.99–6.93(m,4H),5.72(d,J=7.5Hz,1H),4.51( s,2H),3.96(dt,J=7.9,6.8Hz,2H),3.83(dt,J=8.0,5.7Hz,2H),3.78(s,6H),3.71(t,J=5.9Hz,2 H), 3.47–3.41 (m, 4H), 3.13 (hept, J = 7.5Hz, 2H), 2.68 (t, J = 5.7Hz, 2H), 1.12 (d, J = 7.5Hz, 12H). 31 The purity was determined by P NMR to be 95%, and the mass spectrometry m / z (M⁻¹) was 758.34 (theoretical) and 758.0 (actual), indicating that the obtained monomer is phosphoridamide SN₂₀₁ with the structure shown in formula (201), where n a and n b Both are 2, R 201 As a DMTr protecting base, R 202 For cyanoethyl, each R 203 All are isopropyl, L 101 It is an imide group, B 101 It is 1-uracilyl.

[0315] Next, following the preparation method described in Preparation Example 1 of CN110959011A, conjugate 1 as shown in Table 2 was prepared, with the only difference being that the sense and antisense strands of the siRNA contained in conjugate 1 are as shown in Table 2. According to the nucleic acid sequences corresponding to the siRNA conjugates numbered 1 in Table 2, the sense and antisense strands of the siRNA were synthesized respectively. Specifically, when connecting U... snb When the substituted group SN is used, the phosphoramidite monomer SN201 prepared by step 1-1 above is used instead of the nucleoside phosphoramidite monomer for synthesis. The conditions and reagents used in the four-step reaction of deprotection, coupling, capping, and oxidation are the same as those in the corresponding steps of nucleic acid phosphoramidite synthesis. Conjugate 1 was diluted to a concentration of 0.2 mg / mL (based on siRNA) using ultrapure water (Milli-Q ultrapure water system, resistivity 18.2 MΩ*cm (25℃)) and the molecular weight was determined using liquid chromatography-mass spectrometry (LC-MS, Liquid Chromatography-Mass Spectrometry, purchased from Waters Corporation, model: LCT Premier). The measured value is consistent with the theoretical value, indicating that the synthesized conjugate 1 is the target double-stranded siRNA conjugate. Conjugate 1 has the structure shown in formula (403), and the siRNA contained in this siRNA conjugate has the siRNA sequence corresponding to conjugate 1 in Table 2.

[0316] Table 2. siRNA sequences in siRNA conjugates

[0317]

[0318] In this context, "SS" and "AS" indicate that the nucleic acid sequence in that row is the sense / antisense strand sequence of the corresponding siRNA conjugate, respectively. Uppercase letters C, G, U, and A represent the base composition of the nucleotides. Lowercase letter m indicates that the nucleotide adjacent to the left of m is 2'-methoxy modified; lowercase letter f indicates that the nucleotide adjacent to the left of f is 2'-fluorine modified; lowercase letter s indicates that the connection between the two nucleotides adjacent to s is a phosphate thioester linkage; U snb The substitution group SN shown in formula (102), wherein B 101 U; Ugn2-Ugn6 represent substitution groups having the following formulas (GN2-GN6), respectively.

[0319]

[0320] Comparative preparation examples 1-7: Synthesis of reference siRNA conjugates

[0321] Following the preparation method described in Preparation Example 1, reference siRNA conjugates numbered 1-6 and NC in Table 2 were prepared, with the only difference being that the sense and antisense strands of the siRNA contained in each reference siRNA conjugate are shown in Table 2. The sense and antisense strands of the siRNA were synthesized according to the nucleic acid sequences of the siRNAs numbered 1-6 and NC in Table 2. When GN2-GN6 were linked, the phosphoramidite monomer corresponding to that substituent was used instead of phosphoramidite monomer SN201. Each reference siRNA conjugate was diluted to a concentration of 0.2 mg / mL (based on siRNA) using ultrapure water (Milli-Q ultrapure water system, resistivity 18.2 MΩ*cm (25℃)), and the molecular weight was determined using liquid chromatography-mass spectrometry (LC-MS, Waters Corporation, model: LCT Premier). The measured values ​​are consistent with the theoretical values, indicating that the synthesized reference conjugates 1-6 and reference conjugate NC each possess the target designed double-stranded nucleic acid sequences. Each reference siRNA conjugate has the structure shown in formula (403), and the siRNA contained therein has the siRNA sequences corresponding to reference conjugates 1-6 and reference conjugate NC in Table 2, respectively.

[0322] Experimental Example 1: Target mRNA inhibitory activity of siRNA conjugates in primary mouse hepatocytes

[0323] Primary hepatocytes were obtained from fresh liver tissue of C57BL / 6 mice (6-8 weeks old, purchased from Spefair Pharmaceuticals). The primary hepatocytes were cultured in DMEM medium (MACGENE, catalog number CM15019) and the cell density was adjusted to 1×10⁻⁶. 5 Primary mouse liver cell suspension was obtained by adding the mouse primary liver cell suspension at a density of 1 mL / well to different wells of a 24-well plate, and the mouse primary liver cells were then seeded into the wells. 5 Cells / pores.

[0324] Prepare a 4 μM (based on siRNA) working solution for each of the following siRNA conjugates using DEPC-treated water. The siRNA conjugates used are conjugate 1, reference conjugate 1, or reference conjugate NC, respectively.

[0325] In each well containing a primary mouse liver cell suspension as described above, the working solution of each conjugate siRNA conjugate was added and mixed thoroughly at a volume of 2.5 μL / well. Each siRNA conjugate was transfected into three wells to obtain a transfection mixture containing siRNA (final concentration of 10 nM), which was designated as the test group. The primary mouse liver cell suspension in the other three wells was designated as the blank control group.

[0326] Each siRNA-containing transfection mixture and the blank control group were placed in an incubator with an air atmosphere containing 5% CO2 and cultured at 37°C for 24 hours.

[0327] Subsequently, total RNA was extracted from the cells in each well using TRIZOL (purchased from SIGMA, catalog number T9424) according to the method described in the instructions, and an aqueous solution of total RNA was obtained.

[0328] For each well of cells, a total RNA aqueous solution containing 1 μg of total RNA was taken and used to reverse transcriptase the cells using the Goldenstar reverse transcription kit. TM The reagents provided in the RT6cDNA Synthesis Kit (purchased from Beijing Qingke Xinyue Biotechnology Co., Ltd., catalog number TSK301M) included Goldenstar reagents. TM Oligo(dT) 17 As primers, a 20 μL reverse transcription reaction system was prepared according to the reverse transcription procedure in the kit instructions, and the total RNA from each well was reverse transcribed. The reverse transcription conditions were as follows: for each reverse transcription reaction system, the system was incubated at 50°C for 50 min, then at 85°C for 5 min, and finally at 4°C for 5 min. After the reaction was completed, 80 μL of DEPC water was added to the reverse transcription reaction system to obtain a solution containing cDNA.

[0329] For each reverse transcription reaction system, 5 μL of the above-mentioned cDNA-containing solution was used as a template. A 20 μL qPCR reaction system was prepared using the reagents provided in the SYBR Select Master Mix kit (purchased from Life Technologies, catalog number 4472908). The PCR primer sequences for amplifying the target gene mANGPTL3 and the internal reference gene GAPDH are shown in Table 3, with a final concentration of 0.25 μM for each primer. Each qPCR reaction system was placed on an ABI StepOnePlus Real-Time PCR instrument and amplified using a three-step method. The amplification program was: 95°C pre-denaturation for 10 min, followed by 95°C denaturation for 30 s, 60°C annealing for 25 s, and 72°C extension for 25 s. This denaturation, annealing, and extension process was repeated 40 times to obtain product W containing amplified target gene mANGPTL3 and internal reference gene mGAPDH. In the context of this disclosure, the mANGPTL3 gene and mGAPDH gene refer to the mouse-derived ANGPTL3 gene and mouse-derived GAPDH gene, respectively. Product W was then incubated sequentially at 95℃ for 1 min, 55℃ for 30 s, and 95℃ for 30 s. The melting curves of the target gene mANGPTL3 and the internal reference gene mGAPDH in product W were collected by real-time quantitative PCR instrument, and the Ct values ​​of the target gene mANGPTL3 and the internal reference gene mGAPDH were obtained.

[0330] Table 3 Primer sequence information

[0331]

[0332] The relative expression level and inhibition rate of the target gene mANGPTL3 in each test group were calculated using the Ct(ΔΔCt) method. The calculation method is as follows:

[0333] ΔCt(test group) = Ct(target gene in test group) – Ct(internal reference gene in test group)

[0334] ΔCt(control group) = Ct(target gene in control group) – Ct(internal reference gene in control group)

[0335] ΔCt(test group) = ΔCt(test group) - ΔCt(control group average)

[0336] ΔCt(control group) = ΔCt(control group) - ΔCt(control group average)

[0337] Here, ΔCt (control group average) is the arithmetic mean of ΔCt (control group) for each of the three culture wells in the control group. Thus, each culture well in both the test group and the control group corresponds to a ΔCt value.

[0338] Using the control group as a baseline, the expression level of mANGPTL3 mRNA in the test group was normalized, and the expression level of mANGPTL3 mRNA in the blank control group was defined as 100%.

[0339] The relative expression level of mANGPTL3 mRNA in the test group was 2. -ΔΔCt(测试组) ×100%

[0340] The inhibition rate of mANGPTL3 mRNA in the test group = (1 - the relative expression level of mANGPTL3 mRNA in the test group) × 100%

[0341] Therefore, the relative expression level and inhibition rate of ANGPTL3 mRNA in primary mouse hepatocytes were calculated after obtaining free uptake of each siRNA conjugate. The results are as follows: Figure 1 As shown.

[0342] Figure 1 The bar chart shows the relative expression levels of ANGPTL3 mRNA in primary hepatocytes of C57BL / 6 mice after free uptake of conjugate 1, reference conjugate 1, or reference conjugate NC, respectively. Furthermore, the inhibition rates of each siRNA conjugate or reference siRNA conjugate on ANGPTL3 mRNA are summarized in Table 4.

[0343] Table 4. Inhibition of ANGPTL3 mRNA in primary mouse hepatocytes

[0344]

[0345] Depend on Figure 1 As shown in Table 4, the siRNA conjugates disclosed herein exhibit excellent ANGPTL3 mRNA inhibitory activity in C57BL / 6 mouse primary hepatocytes. At a siRNA concentration of 10 nM, the ANGPTL3 mRNA inhibition rate can reach 85.27%, demonstrating ANGPTL3 mRNA inhibitory activity comparable to or even higher than that of the corresponding reference conjugate 1 without the substitution group.

[0346] Experimental Example 2: Inhibition of ANGPTL3 mRNA by siRNA conjugates in mice (in vivo)

[0347] C57BL / 6j mice (8-10 weeks old, purchased from Spiefer Pharmaceuticals) were randomly divided into groups of five, all female. Each group was administered conjugate 1, reference conjugate 1, reference conjugate 3, reference conjugate 4, reference conjugate 5, reference conjugate 6, and a PBS control. Dosage was calculated based on body weight, and all mice were administered a single subcutaneous injection. The dosage (based on siRNA content) for each siRNA conjugate was 3 mg / kg body weight, with a volume of 5 ml / kg. Each siRNA conjugate was provided in PBS aqueous solution, and the required concentration was calculated based on the dosage and volume. An additional group of mice was administered 1×PBS at a volume of 5 ml / kg as a blank control.

[0348] Day 1 was designated as the time of drug administration. Animals were sacrificed on day 8, and liver tissue was collected from each mouse. The liver tissue was preserved using RNA later (Sigma Aldrich). 1 mL of Trizol (Sigma) was added to each liver tissue sample, and the tissue was homogenized three times for 30 seconds each time using a Tissuelyset II automated tissue homogenizer to obtain a liver tissue homogenate. 0.2 mL of chloroform was added, and the mixture was incubated for 3 minutes. The homogenate was centrifuged at 12000 rpm for 10 minutes at 4°C, and 0.4 mL of the supernatant was collected. 0.5 mL of isopropanol was added to the supernatant, and the mixture was incubated at room temperature for 10 minutes. The mixture was then centrifuged at 12000 rpm for 10 minutes at 4°C, and the supernatant was discarded. 1 mL of 75% ethanol was added to the precipitate to wash it, and the mixture was centrifuged at 12000 rpm for 5 minutes at 4°C, and the supernatant was discarded. 30 μL of DEPC-diluted water was added to the precipitate to obtain the extracted total RNA solution. RNA concentration was determined using a NANO DROP 2000 (Thermo Scientific) according to the instructions.

[0349] For total RNA from the liver tissue of each mouse, a total RNA aqueous solution containing 1 μg of total RNA was prepared, with a solution volume of 1000 μL / RNA concentration (ng / μL). Using the Reverse Transcription System (purchased from Promega), a 20 μL reverse transcription reaction system was prepared according to the reverse transcription procedure in the kit instructions. The reverse transcription conditions were as follows: for each reverse transcription reaction system, the system was incubated at 50°C for 50 min, then at 85°C for 5 min, and finally at 4°C for 5 min to obtain a solution containing cDNA.

[0350] For each reverse transcription reaction system, 1 μL of the above-mentioned cDNA-containing solution was used as a template. A 20 μL qPCR reaction system was prepared using the reagents provided by the 2×UltraSYBR Mixture (with ROX) kit (purchased from Beijing Kangwei Century Co., Ltd.). The PCR primer sequences used to amplify the target gene mANGPTL3 and the internal reference gene mGAPDH are shown in Table 3, with a final concentration of 0.25 μM for each primer. Each qPCR reaction system was placed on an ABI StepOnePlus Real-Time PCR instrument and amplified using a three-step method. The amplification program was: 95℃ pre-denaturation for 10 min, followed by 95℃ denaturation for 30 s, 60℃ annealing for 30 s, and 72℃ extension for 30 s. This denaturation, annealing, and extension process was repeated 40 times to obtain product W containing amplified target gene mANGPTL3 and internal reference gene mGAPDH. Product W was then incubated sequentially at 95℃ for 1 min, 55℃ for 30 s, and 95℃ for 30 s. The melting curves of the target gene mANGPTL3 and the internal reference gene mGAPDH in product W were collected by real-time quantitative PCR instrument, and the Ct values ​​of the target gene mANGPTL3 and the internal reference gene mGAPDH were obtained.

[0351] The relative expression level and inhibition rate of the target gene mANGPTL3 in each test group were calculated using the Ct(ΔΔCt) method. The calculation method is as follows:

[0352] ΔCt(test group) = Ct(target gene in test group) – Ct(internal reference gene in test group)

[0353] ΔCt(control group) = Ct(target gene in control group) – Ct(internal reference gene in control group)

[0354] ΔCt(test group) = ΔCt(test group) - ΔCt(control group average)

[0355] ΔCt(control group) = ΔCt(control group) - ΔCt(control group average)

[0356] Here, ΔCt (control group mean) is the arithmetic mean of the ΔCt (control group) values ​​of each of the five mice in the control group. Thus, each mouse in both the test group and the control group corresponds to a ΔCt value.

[0357] Using the control group as a baseline, the expression level of mANGPTL3 mRNA in the test group was normalized, and the expression level of mANGPTL3 mRNA in the blank control group was defined as 100%.

[0358] The relative expression level of mANGPTL3 mRNA in the test group was 2. -ΔΔCt(测试组) ×100%

[0359] The inhibition rate of mANGPTL3 mRNA in the test group = (1 - the relative expression level of mANGPTL3 mRNA in the test group) × 100%

[0360] The results are shown in Figure 2 middle. Figure 2 A scatter plot shows the relative expression levels of mANGPTL3 mRNA in the liver of C57BL / 6j mice after administration of 3 mg / kg (based on siRNA) of conjugate 1, reference conjugate 1, reference conjugate 3, reference conjugate 4, reference conjugate 5, or reference conjugate 6, and PBS, respectively. The inhibition rates of each siRNA conjugate on mANGPTL3 mRNA are summarized in Table 5.

[0361] Table 5. Inhibition of mANGPTL3 mRNA in mice

[0362]

[0363] Depend on Figure 2 As shown in Table 5, the siRNA conjugates disclosed herein exhibit excellent mANGPTL3 mRNA inhibition in mice. At a dose of 3 mg / kg, conjugate 1 showed a mANGPTL3 mRNA inhibition rate as high as 96.24%, which is significantly higher than that of reference conjugate 1 without the SN substitution group. Furthermore, the residual mANGPTL3 mRNA level was reduced to less than half that of mice given reference conjugate 1, indicating that the siRNA conjugates disclosed herein can effectively enhance the inhibitory activity of siRNA on the mRNA expression of the target gene. In contrast, the mRNA inhibition rates of other reference conjugates were all lower than that of reference conjugate 1, with the largest difference being approximately 13%.

[0364] Example 3: The effect of siRNA conjugates on lowering blood lipids in mice.

[0365] Human APOC3 transgenic mice (Tg(APOC3)3707Bres, purchased from Jackson Laboratories, USA) with serum TG levels >2 mmol / L were randomly divided into groups of six, half male and half female. Each group of mice was administered conjugate 1, reference conjugate 1, reference conjugate 2, reference conjugate 3, and reference conjugate 5, respectively. Dosage was calculated based on body weight, and all mice were administered a single subcutaneous injection. The dosage of each siRNA conjugate (based on siRNA content) was 1 mg / kg mouse body weight, with an administration volume of 5 ml / kg. Each siRNA conjugate was provided in PBS aqueous solution, and the required concentration of the conjugate was calculated based on the dosage and administration volume. An additional group of mice was administered 1×PBS at an administration volume of 5 ml / kg as a blank control group.

[0366] Blood samples (100 μL each time) were collected from the orbital venous plexus of mice on days 1, 8, 15, 22, 29, 36, 43, 50, 57, and 71, with the administration date designated as day 1. After collection, the blood was left at room temperature for 30 min, then centrifuged at 3000 rpm for 15 min at 4°C to obtain serum. The levels of total cholesterol (CHO) and triglycerides (TG) in the serum were further determined using a PM1P000 / 3 fully automated serum biochemistry analyzer (SABA, Italy).

[0367] Standardized blood lipid level = (blood lipid level in the test group after drug administration / blood lipid level in the test group before drug administration) × 100%.

[0368] The inhibition rate of blood lipid levels = (1 - blood lipid content of the test group after administration / blood lipid content of the test group before administration) × 100%.

[0369] Blood lipids refer to total cholesterol (CHO) or triglycerides (TG).

[0370] Figure 3A and Figure 3B Line graphs showing the changes in serum TG or serum CHO levels over time after administration of the siRNA conjugate, reference siRNA conjugate, or PBS. Further, the inhibition rates of serum TG and serum CHO in mice at each time point after administration of each siRNA conjugate or PBS are summarized in Tables 6A and 6B below:

[0371] Table 6A. Serum TG inhibition rate of siRNA conjugates in transgenic mice

[0372]

[0373]

[0374] Table 6B Serum CHO inhibition rate of siRNA conjugates in transgenic mice

[0375]

[0376] Figure 3A , Figure 3BThe results in Tables 6A and 6B indicate that, at different time points after administration, despite a low dose of 1 mg / kg, conjugate 1 significantly reduced serum TG and CHO levels in mice. Furthermore, it maintained an inhibitory effect on total cholesterol (CHO) for up to 71 days after a single administration, consistently exceeding that of reference conjugate 1. At 50 days post-administration, conjugate 1 showed a higher reduction in TG levels compared to the corresponding reference conjugate 1 without the stabilizing nucleotide modification, with a maximum relative difference of 24.4%. In contrast, reference conjugates 2, 3, and 5 showed a more rapid return to pre-administration lipid levels after administration, indicating a weakened or even lost ability to reduce lipids within a short period after administration.

[0377] Experimental Example 11: Toxicity of siRNA conjugates in mice

[0378] Conjugate 1, reference conjugate 1, reference conjugate 2, reference conjugate 3, and reference conjugate 4 were each dissolved in PBS to prepare a 10 mg / ml solution (based on siRNA conjugates). ICR mice (6-8 weeks old, purchased from Spiering Biotechnology) were randomly divided into groups of 6 mice (half male and half female) and numbered accordingly. Each mouse was administered the above siRNA conjugate solution via subcutaneous injection in the neck and back, with an administration volume of 10 mL / kg, serving as the test group. Another group of mice was given PBS, with an administration volume of 10 mL / kg, serving as the blank control group.

[0379] Taking the drug administration point as day 1, on day 8, blood was collected from the orbital sinus of each mouse in both the test group and the blank control group. The blood volume was 0.6 mL. After collection, the blood was incubated at 37°C for 60 min, and then centrifuged at 3000 rpm for 15 min at 4°C to obtain serum. The concentrations of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum were further detected using a PM1P000 / 3 fully automated serum biochemistry analyzer (SABA, Italy). The results are shown below. Figure 4A , Figure 4B .

[0380] Figure 4A and Figure 4B Scatter plots showing the concentrations of ALT and AST in mouse serum after administration of conjugate 1 (100 mg / kg), reference conjugate 1, reference conjugate 2, reference conjugate 3, reference conjugate 4, or PBS. Figure 4A and Figure 4BAs can be seen, compared with the blank control group, administration of reference conjugate 1 (without the substituted SN group) increased the serum ALT and AST concentrations in mice; while administration of the siRNA conjugate of this disclosure resulted in serum ALT and AST concentrations comparable to those in the blank control group, indicating that the siRNA conjugate of this disclosure has low hepatotoxicity. On the other hand, administration of reference conjugate 3 or reference conjugate 4 significantly increased the serum ALT and AST concentrations in mice, to a similar degree as reference conjugate 1, suggesting that these reference conjugates may be less effective in reducing hepatotoxicity.

[0381] Furthermore, after blood collection on day 8, mice given conjugate 1, reference conjugate 1, reference conjugate 2, and PBS were euthanized and necropsy was performed. The tissue samples were fixed in 10% neutral buffered formalin and prepared for pathological sections. The severity of hepatocyte degeneration in the pathological sections was evaluated and graded, and relative comparisons were made.

[0382] Pathological section results showed that, compared with the blank control, among the six mice given reference conjugate 1 without the substitution group SN, four mice showed moderate to severe hepatocyte degeneration, specifically manifested as widespread loose cytoplasm of hepatocytes, ballooning degeneration of many hepatocytes, cell swelling, vacuolated cytoplasm, localized necrosis of a small number of hepatocytes, and nuclear condensation or fragmentation. One mouse showed mild hepatocyte degeneration, specifically manifested as loose and pale cytoplasm of many hepatocytes, showing more severe hepatic steatosis than the blank control group.

[0383] On the other hand, among the six mice given reference conjugate 2, three showed severe hepatocyte degeneration, and one showed severe cellular degeneration, specifically characterized by widespread ballooning degeneration of hepatocytes, cell swelling, and vacuolated cytoplasm. It also showed more severe hepatic steatosis than the control group.

[0384] In contrast, among the six mice administered conjugate 1, two showed moderate hepatocellular degeneration, two showed mild hepatocellular degeneration, and no severe or higher hepatocellular degeneration was observed. The main manifestations were reduced and milder numbers of hepatocellular cytoplasmic loosening, ballooning degeneration, and cell swelling compared to mice administered the reference conjugate, and no hepatocellular necrosis was observed. These results indicate that, compared to the reference conjugate, the disclosed siRNA conjugate can effectively reduce hepatotoxicity due to off-target effects, thus demonstrating significantly higher safety in the preparation of drugs for the treatment and / or prevention of HBV disease or symptoms, and showing excellent development potential.

[0385] Some embodiments of this disclosure have been described in detail above. However, this disclosure is not limited to the specific details of the above embodiments. Within the scope of the technical concept of this disclosure, various simple modifications can be made to the technical solutions of this disclosure, and these simple modifications all fall within the protection scope of this disclosure.

[0386] It should also be noted that the various specific technical features described in the above embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, this disclosure will not describe the various possible combinations separately.

[0387] Furthermore, various different embodiments of this disclosure can be combined in any way, as long as they do not violate the spirit of this disclosure, they should also be regarded as the content disclosed in this disclosure. sequence list <110> Suzhou Ribobio Biotechnology Co., Ltd., Beijing RiboKaituo Biotechnology Co., Ltd. <120> Double-stranded oligonucleotides, compositions and conjugates containing double-stranded oligonucleotides, their preparation methods and uses <130> CP1220382-210705 / CB <150> 202110835762.3 <151> 2021-7-23 <160> 48 <170> SIPOSequenceListing 1.0 <210> 1 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> The positive chain of siRNAa1M1 <400> 1 ccaagagcac caagaacua 19 <210> 2 <211> twenty three <212> RNA <213> Artificial Sequence <220> <223> antisense strand of siRNAa1M1 <220> <221> misc-feature <222> (8)..(9) <223> sn is a substituent group, and the substituent group has the structure shown in formula (101). <400> 2 uaguucusnu ggugcucuug gcu 23 <210> 3 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> The positive chain of siRNAa1M2 <400> 3 ccaagagcac caagaacua 19 <210> 4 <211> twenty three <212> RNA <213> Artificial Sequence <220> <223> antisense strand of siRNAa1M2 <220> <221> misc-feature <222> (7)..(8) <223> sn is a substituent group, and the substituent group has the structure shown in formula (101). <400> 4 uaguucsnuu ggugcucuug gcu 23 <210> 5 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> The positive chain of siRNAa1M3 <400> 5 ccaagagcac caagaacua 19 <210> 6 <211> twenty three <212> RNA <213> Artificial Sequence <220> <223> antisense strand of siRNAa1M3 <220> <221> misc-feature <222> (9) (10) <223> sn is a substituent group, and the substituent group has the structure shown in formula (101). <400> 13 uaguucuusn ggugcucuug gcu 23 <210> 7 <211> twenty one <212> RNA <213> Artificial Sequence <220> <223> The positive chain of siRNAa2M1 <400> 7 agccaagagc accaagaacu a 21 <210> 8 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> antisense strand of siRNAa2M1 <220> <221> misc-feature <222> (8)..(9) <223> sn is a substituent group, and the substituent group has the structure shown in formula (101). <400> 8 uaguucusnu ggugcucuug gcuug 25 <210> 9 <211> twenty one <212> RNA <213> Artificial Sequence <220> <223> The justice chain of siRNAa2M2 <400> 9 agccaagagc accaagaacu a 21 <210> 10 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> antisense strand of siRNAa2M2 <220> <221> misc-feature <222> (7)..(8) <223> sn is a substituent group, and the substituent group has the structure shown in formula (101). <400> 10 uaguucsnuuggugcucuug gcuug 25 <210> 11 <211> twenty one <212> RNA <213> Artificial Sequence <220> <223> The positive chain of siRNAa2M3 <400> 11 agccaagagc accaagaacu a 21 <210> 12 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> antisense strand of siRNAa2M3 <220> <221> misc-feature <222> (9) (10) <223> sn is a substituent group, and the substituent group has the structure shown in formula (101). <400> 12 uaguucuusnggugcucuug gcuug 25 <210> 13 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> The justice chain of siRNAa1M1S <400> 13 ccaagagcac caagaacua 19 <210> 14 <211> twenty three <212> RNA <213> Artificial Sequence <220> <223> antisense strand of siRNAa1M1S <220> <221> misc-feature <222> (8)..(9) <223> sn is a substituent group, and the substituent group has the structure shown in formula (101). <400> 14 uaguucusnu ggugcucuug gcu 23 <210> 15 <211> 18 <212> RNA <213> Artificial Sequence <220> <223> The justice chain of siRNAa1M2S <400> 15 ccagagcacc aagaacua 18 <210> 16 <211> twenty three <212> RNA <213> Artificial Sequence <220> <223> antisense strand of siRNAa1M2S <220> <221> misc-feature <222> (7)..(8) <223> sn is a substituent group, and the substituent group has the structure shown in formula (101). <400> 16 uaguucsnuu ggugcucuug gcu 23 <210> 17 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> The justice chain of siRNAa1M3S <400> 17 ccaagagcac caagaacua 19 <210> 18 <211> twenty three <212> RNA <213> Artificial Sequence <220> <223> antisense strand of siRNAa1M3S <220> <221> misc-feature <222> (9) (10) <223> sn is a substituent group, and the substituent group has the structure shown in formula (101). <400> 18 uaguucuusn ggugcucuug gcu 23 <210> 19 <211> twenty one <212> RNA <213> Artificial Sequence <220> <223> The justice chain of siRNAa2M1S <400> 19 agccaagagc accaagaacu a 21 <210> 20 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> antisense strand of siRNAa2M1S <220> <221> misc-feature <222> (8)..(9) <223> sn is a substituent group, and the substituent group has the structure shown in formula (101). <400> 20 uaguucusnu ggugcucuug gcuug 25 <210> twenty one <211> twenty one <212> RNA <213> Artificial Sequence <220> <223> The justice chain of siRNAa2M2S <400> twenty one agccaagagc accaagaacu a 21 <210> twenty two <211> 25 <212> RNA <213> Artificial Sequence <220> <223> antisense strand of siRNAa2M2S <220> <221> misc-feature <222> (7)..(8) <223> sn is a substituent group, and the substituent group has the structure shown in formula (101). <400> twenty two uaguucsnuuggugcucuug gcuug 25 <210> twenty three <211> twenty one <212> RNA <213> Artificial Sequence <220> <223> The justice chain of siRNAa2M3S <400> twenty three agccaagagc accaagaacu a 21 <210> twenty four <211> 25 <212> RNA <213> Artificial Sequence <220> <223> antisense strand of siRNAa2M3S <220> <221> misc-feature <222> (9) (10) <223> sn is a substituent group, and the substituent group has the structure shown in formula (101). <400> twenty four uaguucuusnggugcucuug gcuug 25 <210> 25 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> The positive chain of siRNAa1M1SP1 <400> 25 ccaagagcac caagaacua 19 <210> 26 <211> twenty three <212> RNA <213> Artificial Sequence <220> <223> antisense strand of siRNAa1M1SP1 <220> <221> misc-feature <222> (8)..(9) <223> sn is a substituent group, and the substituent group has the structure shown in formula (101). <400> 26 uaguucusnu ggugcucuug gcu 23 <210> 27 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> The positive chain of siRNAa1M2SP1 <400> 27 ccaagagcac caagaacua 19 <210> 28 <211> twenty three <212> RNA <213> Artificial Sequence <220> <223> antisense strand of siRNAa1M2SP1 <220> <221> misc-feature <222> (7)..(8) <223> sn is a substituent group, and the substituent group has the structure shown in formula (101). <400> 28 uaguucsnuu ggugcucuug gcu 23 <210> 29 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> The positive chain of siRNAa1M3SP1 <400> 29 ccaagagcac caagaacua 19 <210> 30 <211> twenty three <212> RNA <213> Artificial Sequence <220> <223> antisense strand of siRNAa1M3SP1 <220> <221> misc-feature <222> (9) (10) <223> sn is a substituent group, and the substituent group has the structure shown in formula (101). <400> 30 uaguucuusn ggugcucuug gcu 23 <210> 31 <211> twenty one <212> RNA <213> Artificial Sequence <220> <223> The positive chain of siRNAa2M1SP1 <400> 31 agccaagagc accaagaacu a 21 <210> 32 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> antisense strand of siRNAa2M1SP1 <220> <221> misc-feature <222> (8)..(9) <223> sn is a substituent group, and the substituent group has the structure shown in formula (101). <400> 32 uaguucusnu ggugcucuug gcuug 25 <210> 33 <211> twenty one <212> RNA <213> Artificial Sequence <220> <223> The justice chain of siRNAa2M2SP1 <400> 33 agccaagagc accaagaacu a 21 <210> 34 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> antisense strand of siRNAa2M2SP1 <220> <221> misc-feature <222> (7)..(8) <223> sn is a substituent group, and the substituent group has the structure shown in formula (101). <400> 34 uaguucsnuuggugcucuug gcuug 25 <210> 35 <211> twenty one <212> RNA <213> Artificial Sequence <220> <223> The justice chain of siRNAa2M3SP1 <400> 35 agccaagagc accaagaacu a 21 <210> 36 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> antisense strand of siRNAa2M3SP1 <220> <221> misc-feature <222> (9) (10) <223> sn is a substituent group, and the substituent group has the structure shown in formula (101). <400> 36 uaguucuusnggugcucuug gcuug 25 <210> 37 <211> twenty four <212> RNA <213> Artificial Sequence <220> <223> antisense chain of conjugate 1 <220> <221> misc-feature <222> (8)..(10) <223> The substitution group SN shown in formula (102) of usnb <400> 37 uaguucusnb uggugcucuu ggcu 24 <210> 38 <211> twenty one <212> RNA <213> Artificial Sequence <220> <223> antisense chain of reference conjugate 1 <400> 38 uaguucuugg ugcucuuggc u 21 <210> 39 <211> twenty three <212> RNA <213> Artificial Sequence <220> <223> antisense chain of reference conjugate 2 <220> <221> misc-feature <222> (7)..(9) <223> ugn represents ugn2, and ugn2 indicates a substituent group having the formula (GN2). <400> 39 uaguucugnu ggugcucuug gcu 23 <210> 40 <211> twenty three <212> RNA <213> Artificial Sequence <220> <223> antisense chain of reference conjugate 3 <220> <221> misc-feature <222> (7)..(9) <223> ugn represents ugn3, where ugn3 indicates a substituent group having the formula (GN3). <400> 40 uaguucugnu ggugcucuug gcu 23 <210> 41 <211> twenty three <212> RNA <213> Artificial Sequence <220> <223> antisense chain of reference conjugate 4 <220> <221> misc-feature <222> (7)..(9) <223> ugn represents ugn4, where ugn4 indicates a substituent group having the formula (GN4). <400> 41 uaguucugnu ggugcucuug gcu 23 <210> 42 <211> twenty three <212> RNA <213> Artificial Sequence <220> <223> antisense chain of reference conjugate 5 <220> <221> misc-feature <222> (7)..(9) <223> ugn represents ugn5, and ugn5 indicates a substituent group having the formula (GN5). <400> 42 uaguucugnu ggugcucuug gcu 23 <210> 43 <211> twenty three <212> RNA <213> Artificial Sequence <220> <223> antisense chain of reference conjugate 6 <220> <221> misc-feature <222> (7)..(9) <223> ugn represents ugn6, where ugn6 indicates a substituent group having the formula (GN6). <400> 43 uaguucugnu ggugcucuug gcu 23 <210> 44 <211> twenty one <212> RNA <213> Artificial Sequence <220> <223> antisense chain of reference conjugate NC <400> 44 uaguucuugg ugcucuuggc u 21 <210> 45 <211> twenty three <212> DNA <213> Artificial Sequence <220> <223> upstream primer of mANGPTL3 <400> 45 gaggagcagc taaccaactt aat 23 <210> 46 <211> twenty three <212> DNA <213> Artificial Sequence <220> <223> downstream primers of mANGPTL3 <400> 46 tctgcatgtg ctgttgactt aat 23 <210> 47 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> upstream primer of mGAPDH <400> 47 tgcaccacca actgcttag 19 <210> 48 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> downstream primers for mGAPDH <400> 48 ggatgcaggg atgatgttc 19

Claims

1. A double-stranded oligonucleotide comprising a sense strand and an antisense strand, wherein the sense strand comprises a nucleotide sequence I consisting of 19 modified or unmodified nucleotides; the antisense strand comprises a nucleotide sequence II, wherein the nucleotide sequence II is a nucleotide sequence formed by replacing one of the 2nd to 8th nucleotides in the 5'-3' direction with a substitution group SN, wherein the nucleotide sequence A consists of 19 modified or unmodified nucleotides, and the nucleotide sequence A is at least partially anticomplementary to a first nucleotide sequence in the mRNA of a target gene expression, the first nucleotide sequence being a nucleotide sequence of 19 nucleotides in length in the mRNA of the target gene expression; wherein the nucleotide sequence I and the nucleotide sequence II are at least partially anticomplementary to form a double-stranded region, and the substitution group SN has a structure as shown in formula (101): in, Indicates the site where groups are covalently linked; n a and n b Both are 2, E 101 It is a hydroxyl group; L 101 It is an imide group, and L 101 The acyl group in the formula forms an amide bond with the N atom shown in formula (101); B 101 It is one of the nucleic acid bases A, C, G, U, and T; or B 101 The nucleic acid bases are identical to those in the nucleotides in nucleotide sequence A that are replaced by the substituted group SN.

2. The double-stranded oligonucleotide of claim 1, wherein, The nucleotide sequence II is a nucleotide sequence formed by replacing the 6th, 7th, or 8th nucleotide in nucleotide sequence A with the substitution group SN in the 5'-3' direction.

3. The double-stranded oligonucleotide as described in claim 1 or 2, wherein, The substitution group SN is the group shown in formula (102): Among them, B 101 It can be A, C, G, or U.

4. The double-stranded oligonucleotide according to any one of claims 1-3, wherein, In the nucleotide sequence II, except for the nucleotide sequence replaced by the substitution group SN, which is substantially anticomplementary, substantially anticomplementary, or completely anticomplementary to the nucleotide sequence at the corresponding position in the first nucleotide sequence, the corresponding position refers to the nucleotide position in the first nucleotide sequence of the nucleotide sequence II that was not replaced by the substitution group SN.

5. The double-stranded oligonucleotide of claim 4, wherein, In the nucleotide sequence II, the nucleotides not replaced by the substitution group SN at positions 2-19 are completely inversely complementary to the nucleotides at the corresponding positions in the first nucleotide sequence II.

6. The double-stranded oligonucleotide according to any one of claims 1-5, wherein, In the nucleotide sequence II, the nucleotides not replaced by the substitution group SN are substantially anticomplementary, substantially anticomplementary, or completely anticomplementary to the nucleotides at the corresponding positions in the nucleotide sequence I.

7. The double-stranded oligonucleotide of claim 6, wherein, In the nucleotide sequence II, the nucleotide not replaced by the substitution group SN is completely anticomplementary to the nucleotide at the corresponding position in the nucleotide sequence I, or there is a base mismatch between the second nucleotide in the nucleotide sequence II and the second nucleotide in the nucleotide sequence I in the direction from the 5' end to the 3' end.

8. The double-stranded oligonucleotide according to any one of claims 1-7, wherein, Following the direction from the 5' end to the 3' end, the 2nd, 6th, 14th, and 16th nucleotides of the nucleotide sequence A are 2'-fluorinated nucleotides.

9. The double-stranded oligonucleotide of claim 8, wherein, All nucleotides in nucleotide sequence II are modified nucleotides; the substitution group SN is counted as one nucleotide in the direction from the 5' end to the 3' end; the 2nd, 6th, 14th, and 16th nucleotides of nucleotide sequence II, if not the substitution group SN, are 2'-fluoro-modified nucleotides; and the other nucleotides in nucleotide sequence II are each independently a type of non-fluoro-modified nucleotide.

10. The double-stranded oligonucleotide according to any one of claims 1-9, wherein, Following the direction from the 5' end to the 3' end, the 7th to 9th nucleotides of nucleotide sequence I are 2'-fluorinated nucleotides.

11. The double-stranded oligonucleotide of claim 10, wherein, All nucleotides in nucleotide sequence I are modified nucleotides; nucleotides 7-9 of nucleotide sequence I are 2'-fluoro modified nucleotides in the direction from the 5' end to the 3' end, and the other nucleotides in nucleotide sequence I are each independently a type of non-fluoro modified nucleotide.

12. The double-stranded oligonucleotide according to any one of claims 1-11, wherein, The sense strand further contains nucleotide sequence III, and the antisense strand further contains nucleotide sequence IV. Nucleotide sequence III and nucleotide sequence IV are of equal length and are composed of 1, 2, 3, or 4 modified or unmodified nucleotides, respectively. Nucleotide sequence IV and nucleotide sequence III are substantially anticomplementary or completely anticomplementary. Nucleotide sequence III is attached to the 5' end of nucleotide sequence I, and nucleotide sequence IV is attached to the 3' end of nucleotide sequence II. Nucleotide sequence IV is substantially anticomplementary or completely anticomplementary to a second nucleotide sequence, which is a nucleotide sequence in the mRNA expressed by the target gene that is adjacent to the first nucleotide sequence and has the same length as nucleotide sequence IV.

13. The double-stranded oligonucleotide of claim 12, wherein, Each nucleotide in nucleotide sequence III and nucleotide sequence IV is independently one of non-fluorinated modified nucleotides.

14. The double-stranded oligonucleotide according to any one of claims 1-13, wherein, The antisense strand further includes a nucleotide sequence V, which consists of 1 to 3 modified or unmodified nucleotides, attached to the 3' end of the antisense strand to form the 3' overhang of the antisense strand.

15. The double-stranded oligonucleotide of claim 14, wherein, The nucleotide sequence V consists of two non-fluorinated nucleotides, and in the direction from the 5' end to the 3' end, the nucleotide sequence V consists of two consecutive thymine deoxyribonucleotides, two consecutive uracil ribonucleotides, or two nucleotides that are completely inversely complementary to the third nucleotide sequence. The third sequence refers to a nucleotide sequence of two nucleotides in length in the mRNA expressed by the target gene that is adjacent to the 5' end of the first or second nucleotide sequence.

16. The double-stranded oligonucleotide according to any one of claims 1-15, wherein, Each non-fluorinated modified nucleotide is independently selected from one of the nucleotides or nucleotide analogs formed by replacing the hydroxyl group at the 2' position of the ribosyl group of a nucleotide with a non-fluorinated group.

17. The double-stranded oligonucleotide of claim 16, wherein, Each non-fluorinated nucleotide is a methoxylated nucleotide, which refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribosome with a methoxy group.

18. The double-stranded oligonucleotide according to any one of claims 1-17, wherein, At least one of the phosphate ester groups in the phosphate-sugar backbone of at least one single strand of the sense chain and the antisense chain is a phosphate ester group with a modifying group, wherein the phosphate ester group with the modifying group is present at least at one of the following positions: Between the first and second nucleotides at the 5' end of the positive strand; Between the second and third nucleotides at the 5' end of the positive strand; Between the first and second nucleotides at the 3' end of the positive strand; Between the second and third nucleotides at the 3' end of the positive strand; Between the first and second nucleotides at the 5' end of the antisense strand; Between the second and third nucleotides at the 5' end of the antisense strand; Between the first and second nucleotides at the 3' end of the antisense strand; and Between the second and third nucleotides at the 3' end of the antisense strand.

19. The double-stranded oligonucleotide according to any one of claims 1-18, wherein, The 5' terminal nucleotide of the antisense strand is a 5'-phosphate nucleotide or a nucleotide modified with a 5'-phosphate analogue.

20. The double-stranded oligonucleotide according to any one of claims 1-19, wherein, The double-stranded oligonucleotide is saRNA or siRNA.

21. The double-stranded oligonucleotide according to any one of claims 1-20, wherein, The mRNA expressed by the target gene is selected from one of the following mRNAs transcribed from genes: 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.

22. The double-stranded oligonucleotide of claim 21, wherein, The double-stranded oligonucleotide is siRNA, and the mRNA expressing the target gene is selected from the mRNA expressed by the HBV gene, the mRNA expressed by the ANGPTL3 gene, or the mRNA expressed by the ApoC3 gene.

23. The double-stranded oligonucleotide of claim 22, wherein, The double-stranded oligonucleotide is one of siRNAa1M1, siRNAa1M2, siRNAa1M3, siRNAa2M1, siRNAa2M2, siRNAa2M3, siRNAa1M1S, siRNAa1M2S, siRNAa1M3S, siRNAa2M1S, siRNAa2M2S, siRNAa2M3S, siRNAa1M1SP1, siRNAa1M2SP1, siRNAa1M3SP1, siRNAa2M1SP1, siRNAa2M2SP1, and siRNAa2M3SP1.

24. A pharmaceutical composition comprising the double-stranded oligonucleotide as described in any one of claims 1-23 and a pharmaceutically acceptable carrier.

25. An oligonucleotide conjugate comprising a double-stranded oligonucleotide as described in any one of claims 1-23 and a conjugating group conjugated to the double-stranded oligonucleotide, the conjugating group comprising a linker and a pharmaceutically acceptable targeting group and / or a delivery aid group, wherein the double-stranded oligonucleotide, the linker, and the targeting group or the delivery aid group are covalently or non-covalently linked in sequence, each of the targeting groups being selected from ligands capable of binding to cell surface receptors, and each delivery aid group being selected from groups capable of increasing the biocompatibility of the oligonucleotide conjugate in a target organ or tissue.

26. Use of the double-stranded oligonucleotide of any one of claims 1-23, and / or the pharmaceutical composition of claim 24 and / or the oligonucleotide conjugate of claim 25 in the preparation of a medicament for treating and / or preventing diseases or symptoms associated with the mRNA level of a target gene expression.

27. The use as described in claim 26, wherein, The mRNA expressed by the target gene is selected from one of the following mRNAs transcribed from genes: 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.

28. The use as described in claim 26, wherein, The mRNA expressing the target gene is selected from the mRNA expressed by the HBV gene, the mRNA expressed by the ANGPTL3 gene, or the mRNA expressed by the ApoC3 gene.

29. The use as described in any one of claims 26-28, wherein, The diseases or symptoms associated with the mRNA levels of the target gene expression are hepatitis B or dyslipidemia.

30. A kit comprising the double-stranded oligonucleotide of any one of claims 1-23, and / or the pharmaceutical composition of claim 24 and / or the oligonucleotide conjugate of claim 25.

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