Nucleic acid chimeras comprising multiple double-stranded oligonucleotide molecules, compositions and uses thereof

Abstract

The invention provides a nucleic acid chimera containing multiple double-stranded oligonucleotide molecules, and belongs to the field of nucleic acid drugs. The nucleic acid chimera comprises a chimera structure composed of at least two oligonucleotide molecules, and can simultaneously regulate the same or different gene expressions through interaction with different target sites.

Description

Nucleic acid chimera comprising multiple double-stranded oligonucleotide molecules, composition and use thereof

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. 202311265471.0 filed with the State Intellectual Property Office of China on September 27, 2023, entitled “Nucleic acid chimeras, compositions and uses thereof comprising multiple double-oligonucleotide molecules,” the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention belongs to the field of nucleic acid drugs, and in particular relates to a nucleic acid chimera comprising multiple double-stranded oligonucleotide molecules, a composition and uses thereof. Background Art

[0004] Double-stranded oligonucleotides can regulate gene expression through RNA interference and gene-targeting RNA. While conventional techniques typically utilize a single double-stranded oligonucleotide molecule for gene regulation, chimeric molecules composed of multiple double-stranded oligonucleotides can regulate gene expression through different mechanisms. For example, two double-stranded oligonucleotides can regulate the expression of one or more genes by targeting different genes or different locations within the mRNA of the same gene.

[0005] Therefore, it is necessary to provide a novel nucleic acid chimera, including a chimera structure composed of two or more oligonucleotide molecules, which can regulate the expression of different genes by interacting with different targets.

[0006] Summary of the Invention

[0007] The present disclosure provides a nucleic acid chimera comprising at least two double-stranded oligonucleotide molecules, a composition, and uses thereof.

[0008] In a first aspect, the present disclosure provides a nucleic acid chimera, comprising a nucleotide unit I, wherein the nucleotide unit I has a structure represented by formula (101):

[0009] Each X1, X2, and X3 are independently selected from Each M1 independently comprises a ligand capable of binding to a cell receptor; each M2 is independently selected from modified or unmodified nucleotides; each m is independently selected from 1, 2 or 3, and each n is independently selected from an integer from 0 to 3;

[0010] Wherein, X1, X2, and X3 may be the same or different;

[0011] Wherein, each Y1 and Y2 independently represents a sense strand of a double-stranded oligonucleotide, and each sense strand may correspond to the same target gene or different target genes; the correspondence means that the sense strand is identical or substantially identical to a continuous stretch of nucleotides in the mRNA of the target gene;

[0012] j1, j2, j3 are independently selected from integers of 0-3, and j2≠0;

[0013] In some embodiments, the nucleic acid chimera further comprises a nucleotide unit II, wherein the nucleotide unit II comprises an antisense strand that is complementary or substantially complementary to the sense strand in the double-stranded oligonucleotide, the number of the antisense strands is equal to the number of the sense strands, and each of the antisense strands is at least partially complementary to its corresponding sense strand to form a duplex region; wherein the antisense strand is complementary or at least partially complementary to the mRNA of at least one target gene.

[0014] In other embodiments of the present disclosure, the present disclosure provides a nucleic acid chimera comprising a nucleotide unit III, wherein the nucleotide unit III has a structure represented by formula (301), or an isomer thereof, or a pharmaceutically acceptable salt thereof:

[0015] Each M1 independently contains a ligand capable of binding to a cell receptor.

[0016] wherein Y3 is selected from the sense strand and Y4 is selected from the sense strand, or, Y3 is selected from the antisense strand and Y4 is selected from the sense strand;

[0017] j4, j5, j6, and j7 are each independently selected from an integer of 0 to 3, and at least one of j6 and j7 is not 0; when Y3 is selected from the sense chain, at least one of j4 and j5 is not 0;

[0018] In a second aspect, the present disclosure provides a composition comprising the nucleic acid chimera described in the present disclosure and a physiologically acceptable excipient.

[0019] In a third aspect, the present disclosure provides the nucleic acid chimera for use in treating a disease or condition caused by dysregulated expression of at least one gene.

[0020] In a fourth aspect, the present disclosure provides use of the nucleic acid chimera in the preparation of a drug for treating a disease or condition caused by dysregulated expression of at least one gene.

[0021] In a fifth aspect, the present disclosure provides a method for treating a disease or disorder, comprising administering the nucleic acid chimera of the present disclosure to an individual in need of treatment.

[0022] In some embodiments, the nucleic acid chimera of the present disclosure is administered to the individual subcutaneously or intravenously.

[0023] In some embodiments, after in vivo administration, the nucleic acid chimeras described herein disassemble to yield at least two independent double-stranded oligonucleotide molecules, each targeting a portion of an mRNA transcribed from one or more target genes, which may be the same or different. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Relative expression levels of the target gene SOD1 / ANGPTL3 in mouse primary hepatocytes after administration of nucleic acid chimeras.

[0025] Figure 2 IC of the target gene SOD1 in primary mouse hepatocytes after administration of nucleic acid chimeras 50 curve.

[0026] Figure 3 IC of the target gene ANGPTL3 in mouse primary hepatocytes after administration of nucleic acid chimeras 50 curve.

[0027] Figure 4 Relative expression levels of the target gene SOD1 in mice after administration of nucleic acid chimeras

[0028] FIG5 shows the relative expression level of the target gene ANGPTL3 in mice after administration of nucleic acid chimeras.

[0029] FIG6 shows the relative expression levels of the target gene SOD1 / ANGPTL3 in primary mouse hepatocytes after administration of nucleic acid chimeras.

[0030] FIG7 shows the relative expression levels of the target gene SOD1 / ANGPTL3 in mouse primary hepatocytes after administration of nucleic acid chimeras.

[0031] FIG8 shows the relative expression level of the target gene SOD1 in mice after administration of nucleic acid chimeras.

[0032] FIG9 shows the relative expression level of the target gene ANGPTL3 in mice after administration of nucleic acid chimeras.

[0033] FIG10 shows the relative expression level of the target gene SOD1 in mice after administration of nucleic acid chimeras.

[0034] FIG11 shows the relative expression level of the target gene ANGPTL3 in mice after administration of nucleic acid chimeras. DETAILED DESCRIPTION

[0035] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts are within the scope of protection of the present invention.

[0036] [definition]

[0037] The term "aliphatic ring" refers to a ring having a carbon skeleton structure, such as a monocyclic ring, a spirocyclic ring, a bridged ring, etc. The term "4- to 8-membered aliphatic ring" refers to a ring having 4 to 8 carbon atoms in the carbon skeleton structure.

[0038] The term "saturated aliphatic ring" refers to a cyclic carbon skeleton structure having only carbon-carbon single bonds and no carbon-carbon double bonds or carbon-carbon triple bonds.

[0039] The general structural formula of the term "alkane" is The chain alkyl group may be a straight chain alkyl group or a branched chain alkyl group. The term "C1-C6 chain alkyl group" refers to a chain alkyl group having 1 to 6 carbon atoms.

[0040] The general structural formula of the term "alkylene" is The chain alkylene group may be a linear alkylene group or a branched alkylene group.

[0041] The term "alkoxy" has the general structural formula

[0042] The term "NH" refers to an imino group, which has the formula

[0043] The term "CO" refers to a carbonyl group, which has the structural formula

[0044] The term "CN" refers to a cyano group, which has the structural formula

[0045] The structural formula of the term "trityl" is:

[0046] The structural formula of the term "4-methoxytrityl" is

[0047] The structural formula of the term "4,4'-dimethoxytrityl" is

[0048] The structural formula of the term "4,4',4"-trimethoxytriphenyl" is

[0049] the term The symbol indicates the site where a group is attached via a covalent bond.

[0050] In the chemical structure of a ligand or compound, the bond Indicates that the configuration is not specified. If chiral isomers exist in the chemical structure, the bond Can be or include both and Although all of the above structural formulae are drawn as certain isomers for simplicity, the present disclosure may include all isomers, such as tautomers, rotational isomers, geometric isomers, diastereomers, racemates, and enantiomers.

[0051] In the chemical structure of a ligand or compound, the bond Indicates that the configuration is not specified. If there are cis-trans isomers in the chemical structure, the bond The configuration can be E-type, Z-type, or both E and Z configurations.

[0052] Unless otherwise stated, "selected from . . . and . . . " in the present disclosure means that at least one of them can be selected.

[0053] Unless otherwise stated, "for... or..." in the present disclosure means that any one of them can be selected.

[0054] Unless otherwise indicated, the "compound," "ligand," "nucleic acid ligand conjugate," and "nucleic acid" of the present disclosure may independently exist in the form of a salt, a mixed salt, or a non-salt form (e.g., a free acid or a free base). When present in the form of a salt or a mixed salt, it may be a pharmaceutically acceptable salt.

[0055] The term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0056] The term "pharmaceutically acceptable acid addition salt" refers to salts formed with inorganic or organic acids that retain the biological effectiveness of the free base without other side effects. Inorganic acid salts include, but are not limited to, hydrochlorides, hydrobromides, sulfates, nitrates, and phosphates; organic acid salts include, but are not limited to, formate, acetate, 2,2-dichloroacetate, trifluoroacetate, propionate, hexanoate, octanoate, decanoate, undecylenate, glycolate, gluconate, lactate, sebacate, adipate, glutarate, malonate, oxalate, maleate, succinate, fumarate, tartrate, citrate, palmitate, stearate, oleate, cinnamate, laurate, malate, glutamate, pyroglutamate, aspartate, benzoate, methanesulfonate, benzenesulfonate, p-toluenesulfonate, alginate, ascorbate, salicylate, 4-aminosalicylate, and naphthalene disulfonate. These salts can be prepared by methods known in the art.

[0057] The term "pharmaceutically acceptable base addition salt" refers to salts formed with inorganic or organic bases that retain the biological effectiveness of the free acid without other side effects. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts, and the like. Preferred inorganic salts are ammonium salts, sodium salts, potassium salts, calcium salts, and magnesium salts, with sodium salts being preferred. Salts derived from organic bases include, but are not limited to, primary amines, secondary amines, and tertiary amines. Substituted amines include natural substituted amines, cyclic amines, and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. These salts can be prepared by methods known in the art.

[0058] The term "oligonucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), typically composed of 10 to 50 nucleotides. Oligonucleotides can regulate gene expression through a range of processes, including RNA interference, ribonuclease-mediated target degradation, splicing regulation, noncoding RNA inhibition, gene activation, and programmed gene editing.

[0059] The term "antisense oligonucleotides (ASOs)" refers to single-stranded oligonucleotides, typically consisting of 10 to 50 nucleotides. Once inside cells, ASOs bind to their complementary target mRNA through base pairing under the action of ribonuclease H1, inhibiting the expression of the target gene.

[0060] The term "target gene" refers to a gene encoding a target protein.

[0061] The term "target nucleic acid" or "target gene" nucleic acid refers to any nucleic acid molecule whose expression or activity can be modulated by a double-stranded oligonucleotide compound. Target nucleic acids include, but are not limited to, RNA (including but not limited to pre-mRNA and mRNA or portions thereof) transcribed from DNA encoding a target protein, and cDNA derived from such RNA and miRNA. For example, a target nucleic acid can be a cellular gene (or mRNA transcribed from such a gene) whose expression is associated with a particular condition or disease state.

[0062] "Gene inhibition" by an RNA interference molecule refers to a reduction in the mRNA level of a target gene in a cell by at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, and any integer therebetween.

[0063] The term "modulating gene expression" means that the expression of a gene, or the level of an RNA molecule or equivalent RNA molecule encoding one or more proteins or protein subunits, is upregulated or downregulated such that the expression, level or activity is greater or less than that observed in the absence of regulation.

[0064] The term "complementary" refers to the ability of a nucleic acid to form hydrogen bonds with another nucleic acid sequence via traditional Watson-Crick or other non-traditional types. "Complementary" or fully complementary refers to the ability of all consecutive residues of a nucleic acid sequence to form hydrogen bonds with the same number of consecutive residues in a second nucleic acid sequence. Imperfect complementarity refers to the ability of some (but not all) of the nucleoside units of the two chains to hydrogen bond with each other. "Substantially complementary" refers to polynucleotide chains exhibiting complementarity that differs by no more than three nucleotides, excluding regions of the polynucleotide chains selected as non-complementary, such as overhangs.

[0065] The terms "chimera" or "construct" or "fusion" have the same meaning and refer to a compound comprising at least two oligonucleotide molecules, which may be the same or different.

[0066] The term "targeting" or "targeted to" refers to the association of the antisense strand of an siRNA with a specific target nucleic acid molecule or a specific nucleotide region within a target nucleic acid molecule.

[0067] The term "2'-modified" or "2'-substituted" refers to sugars that contain a substituent other than H or OH at the 2' position. 2'-modified monomers include, but are not limited to, BNAs and monomers with 2'-substituents (eg, nucleosides and nucleotides).

[0068] The term "optionally substituted" means that the group may or may not have a substituent. Examples of substituents include, but are not limited to, C1-C3 alkyl, C1-C3 alkoxy, amino, hydroxy, halogen, and aryl. Preferred substituents are C1-C3 alkyl, C1-C3 alkoxy, and fluorine.

[0069] Unless otherwise indicated, the terms "multi-target siRNA conjugate", "multi-target siRNA conjugate", "siRNA chimera" and "nucleic acid chimera" as used herein may be used interchangeably.

[0070] Unless otherwise specified, in the sequences disclosed herein, base composition and modification have the following meanings: capital letters A, U, G, C, and T represent the base composition of the nucleotides; lowercase letter m represents that the nucleotide represented by the capital letter adjacent to the left of the letter m is a 2'-methoxy-modified nucleotide; lowercase letter f represents that the nucleotide represented by the capital letter adjacent to the left of the letter f is a 2'-fluoro-modified nucleotide; lowercase letter d represents that the nucleotide represented by the capital letter adjacent to the left of the letter d is a 2'-deoxy-modified nucleotide; the combination symbol (moe) represents that the nucleotide represented by the capital letter adjacent to the left of the combination symbol (moe) is a 2'-O-methoxyethyl-modified nucleotide; the structural formula of (CR01008) is A lowercase letter "s" indicates that the nucleotide represented by the capital letter to the left of the letter "s" or (CR01008) is linked to the nucleotide represented by the capital letter to the right of the letter "s" or (CR01008) by a phosphorothioate bond. VP indicates that the nucleotide following the letter is 5'-phosphate modified to contain vinyl phosphate.

[0071] Among them, the structural formula of VPUm is

[0072] Nucleic acid chimeras

[0073] In a first aspect, the present disclosure provides a nucleic acid chimera, comprising a nucleotide unit I, wherein the nucleotide unit I has a structure represented by formula (101):

[0074] Each X1, X2, and X3 are independently selected from Each M1 independently comprises a ligand capable of binding to a cell receptor; each M2 is independently selected from modified or unmodified nucleotides; each m is independently selected from 1, 2 or 3, and each n is independently selected from an integer from 0 to 3;

[0075] Wherein, X1, X2, and X3 may be the same or different;

[0076] Wherein, each Y1 and Y2 independently represents a sense strand of a double-stranded oligonucleotide, and each sense strand may correspond to the same target gene or different target genes; the correspondence means that the sense strand is identical or substantially identical to a continuous stretch of nucleotides in the mRNA of the target gene;

[0077] j1, j2, j3 are independently selected from integers of 0-3, and j2≠0;

[0078] k is selected from integers of 1 to 5.

[0079] In some embodiments, n is independently selected from an integer from 0 to 2; for example, each n is independently selected from 0 or 1.

[0080] In some embodiments, k is selected from 1;

[0081] In some embodiments, j1 is selected from 0, j2 is selected from 3, and j3 is selected from 0;

[0082] In some embodiments, j1 is selected from 0, j2 is selected from 3, and j3 is selected from 3;

[0083] In some embodiments, j1 is selected from 1, j2 is selected from 1, j3 is selected from 1;

[0084] In some embodiments, j1 is selected from 2, j2 is selected from 2, j3 is selected from 2;

[0085] In some embodiments, j1 is selected from 3, j2 is selected from 3, and j3 is selected from 0;

[0086] In some embodiments, j1 is selected from 3, j2 is selected from 0, and j3 is selected from 3;

[0087] In some embodiments, m is selected from 1 or 2 or 3, and each n is independently selected from 0 or 1;

[0088] In some embodiments, m is selected from 1 and n is selected from 0; alternatively, m is selected from 1 and n is selected from 1.

[0089] In some embodiments, the nucleic acid chimera further comprises a nucleotide unit II, wherein the nucleotide unit II comprises an antisense strand that is complementary or substantially complementary to the sense strand in the double-stranded oligonucleotide, the number of the antisense strands is equal to the number of the sense strands, and each of the antisense strands is at least partially complementary to its corresponding sense strand to form a duplex region; wherein the antisense strand is complementary or at least partially complementary to the mRNA of at least one target gene.

[0090] In some embodiments, the antisense strand is complementary or at least partially complementary to the mRNA of two or at least two target genes.

[0091] In other embodiments, there is one antisense strand that is complementary or at least partially complementary to a portion of the mRNA of target gene A; and there is another antisense strand that is complementary or at least partially complementary to a portion of the mRNA of target gene B, and the target gene A and target gene B may be the same or different.

[0092] In some embodiments, the sense strand and the antisense strand of the double-stranded oligonucleotide each comprise at least partially modified nucleotides; preferably, the nucleotides in both the sense strand and the antisense strand are modified nucleotides.

[0093] In some embodiments, the sense strand and / or the antisense strand comprises at least one phosphorothioate bond; preferably, the nucleotides at positions 1 and 2 from the 5' end of the sense strand and the antisense strand are linked to adjacent nucleotides via a phosphorothioate bond.

[0094] In some embodiments, the sense strand and the antisense strand comprise at least 15 consecutive nucleotides, wherein the antisense strand has complementarity with the mRNA of the target gene.

[0095] In some embodiments, one or more unmodified nucleotides are present in X2. Alternatively, unmodified nucleotides may be present as structural units that promote cleavage or hydrolysis of the chimera.

[0096] After in vivo administration, the chimeras disclosed herein can optionally be cleaved at an intermediate position, such as X2, or at one or more unmodified nucleotide positions, thereby generating two or more independent double-stranded oligonucleotides.

[0097] For example, in some embodiments, the chimera is cleaved at X2 or at a position of connection to X2.

[0098] In some embodiments, the chimera can be decomposed into at least two independent double-stranded oligonucleotide parts after in vivo administration, and the independent double-stranded oligonucleotides respectively target the same or different target genes, thereby being able to regulate the expression of the same or different target genes.

[0099] In other embodiments, the chimera can be decomposed (including complete decomposition or partial decomposition) after in vivo administration to obtain two independent parts containing double-stranded oligonucleotides; wherein, the first double-stranded oligonucleotide targets the mRNA portion of target gene A, and the second double-stranded oligonucleotide targets the mRNA portion of target gene B, and: the first double-stranded oligonucleotide can regulate the expression of the target gene A, and the second double-stranded oligonucleotide can regulate the expression of the target gene B.

[0100] In some embodiments, when the chimera is partially resolved, the unresolved chimera can modulate the expression of the target gene associated with either double-stranded oligonucleotide.

[0101] In some embodiments, the nucleic acid chimera comprises a first double-stranded oligonucleotide (siRNA) molecule and a second double-stranded oligonucleotide molecule; optionally, wherein the sense strand of the first double-stranded oligonucleotide and / or the second double-stranded oligonucleotide comprises a single-stranded overhang at its 3'-end; optionally, the antisense strand of the first and / or second double-stranded oligonucleotide comprises an overhang at its 3'-end, wherein the nucleic acid sequence of the single-stranded overhang of the sense strand is complementary or substantially complementary to the nucleotide sequence of the single-stranded overhang of the antisense strand, and the first double strand and the second double strand are each conjugated to at least one ligand.

[0102] In some embodiments, the overhang is 1-3 nucleotides or 1-2 nucleotides. Optionally, the overhang comprises all DNA, all RNA, or a mixture of DNA and RNA nucleotides, and the DNA and RNA can be natural or modified.

[0103] In some embodiments, the first double-stranded oligonucleotide modulates expression of a first target gene and the second double-stranded oligonucleotide modulates expression of a second target gene, the first target gene and the second target gene are not the same.

[0104] In some embodiments, the nucleic acid chimera independently modulates the expression level of their respective target genes by at least 70%, at least 75%, or at least 80% relative to when the first and second double-stranded oligonucleotides are not linked together.

[0105] In some embodiments, the nucleic acid chimera comprises at least partially modified nucleotides. In other embodiments, the double-stranded oligonucleotide in the nucleic acid chimera comprises fully modified nucleotides.

[0106] wherein the modified nucleotides are independently selected from abasic nucleotides, 2'-halogenated nucleotides, 2'-deoxy-modified nucleotides or 2'-O-(CH2) n2 -R1 modified nucleotide, or quasi-nucleotide; the quasi-nucleotide is selected from one or more of peptide nucleic acid (PNA), morpholino (MNA), bridged nucleic acid (BNA), locked nucleic acid (LNA), glycol nucleic acid / glycerol nucleic acid (GNA), threose nucleic acid (TNA) and unlocked nucleic acid (UNA); wherein n2 is selected from 0, 1 or 2; R1 is selected from optionally substituted C 1-6 Alkyl, optionally substituted C 1-6 Alkoxy or -Si(R 1a )3, each R 1a Independently selected from optionally substituted C 1-6 Alkyl or optionally substituted C 1-6 Alkoxy, the substitution means that one or more hydrogen atoms on the alkyl or alkoxy group are replaced by a substituent; optionally, the substituent is independently selected from C1-C3 alkyl, C1-C3 alkoxy or halogen.

[0107] In some embodiments, 2'-O-(CH2)n2 -R1 is selected from 2'-O-CH3, 2'-O-CH2-CH3, 2'-O-TBDMS, 2'-O-TIPS, 2'-O-TOM, 2'-O-CH2-O-CH2-CH3, 2'-O-CH2-O-CH2-CF3 or 2'-O-CH2-CH2-O-CH3.

[0108] In some embodiments, the sense strand and the antisense strand of the double-stranded oligonucleotide are complementary or substantially complementary to form a duplex, as shown in the following formula:

[0109] SS:5'-(N)a'-(X)p'-(N)b'-(X)q'-(N)c'-(X)r'-(N)d'-3'

[0110] AS:3'-(N)a-(X)p-(N)b-(X)q-(N)c-5',

[0111] Wherein, SS represents the sense strand, and AS represents the antisense strand; all nucleotides of the sense strand and the antisense strand are modified nucleotides;

[0112] Each of the Ns independently represents a modified nucleotide: a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, or a 2'-deoxy modified nucleotide;

[0113] The Xs are each independently a 2'-O-methoxyethyl modified nucleotide, a 2'-O-methyl modified nucleotide or a 2'-O(CH2) n3 OR3 substituted modified nucleotides; wherein n3 is 1 or 2, R is selected from substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy; optionally, the substituent is selected from halogen, C1-C3 alkyl, C1-C3 alkoxy, hydroxyl, amino;

[0114] Said a, a', p, p', b, b', q, q', c, c', r', and d' each independently represent the number of nucleotides, wherein: a' is selected from an integer of 3-8; p' is selected from an integer of 0-3; b' is selected from an integer of 4-13; q' is selected from an integer of 0-4; c' is selected from an integer of 3-9; r' is selected from an integer of 0-3; d' is selected from an integer of 0-9; a is selected from an integer of 4-7; p is selected from an integer of 0-1; b is selected from an integer of 4-8; q is selected from an integer of 0-4; c is selected from an integer of 6-10; and p', q', r', p, q are not all 0 at the same time, and 0≤q'+r'≤4.

[0115] In some embodiments, a' is selected from an integer of 3-8; p' is selected from 0 or 1; b' is selected from an integer of 4-13; q' is selected from 0 or 1; c' is selected from an integer of 3-9; r' is selected from 0 or 1; d' is selected from an integer of 1-8; a is selected from an integer of 4-7; p is selected from 1; b is selected from an integer of 4-8; q is selected from 0 or 1; c is selected from an integer of 6-10.

[0116] In some embodiments, the nucleotide N is selected from 2′-O-methyl modified nucleotides and 2′-fluoro-modified nucleotides, and the nucleotide X is selected from 2′-O-methoxyethyl modified nucleotides and 2′-O-methyl modified nucleotides, more preferably 2′-O-methoxyethyl modified nucleotides.

[0117] In some embodiments, the sense strand is 17-21 nucleotides in length and the antisense strand is 19-23 nucleotides in length.

[0118] In some embodiments, in the direction from the 5' end to the 3' end, at least three nucleotides among the nucleotides at positions 7-10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides or 2'-O-methoxyethyl-modified nucleotides; the nucleotides at positions 2, 6, 14 and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, any one of the nucleotides at positions 9, 10, 11 and 12 is selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides or 2'-O-methoxyethyl-modified nucleotides.

[0119] In some embodiments, in the direction from the 5' end to the 3' end, at least three nucleotides among the nucleotides at positions 7-10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, at most two nucleotides among the nucleotides at positions 5, 12, and 18 are selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, any one of the nucleotides at positions 9-12 is selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides.

[0120] In some embodiments, each nucleotide of the double-stranded oligonucleotide is independently selected from a modified nucleotide, wherein the modification of the sense strand and the antisense strand is selected from any one of the following (1)-(8):

[0121] (1) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 9, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides;

[0122] (2) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 12, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides;

[0123] (3) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 9, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides;

[0124] (4) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 12, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides;

[0125] (5) In the direction from the 5' end to the 3' end, the nucleotide at position 5, or position 12, or position 18 of the nucleotide sequence in the sense strand is selected from a 2'-O-methoxyethyl modified nucleotide, the nucleotides at positions 7 to 10 are selected from a 2'-fluoro modified nucleotide, and the nucleotides at the remaining positions are selected from a 2'-O-methyl modified nucleotide; the nucleotides at positions 2, 6, 9, 14, and 16 of the nucleotide sequence in the antisense strand are selected from a 2'-fluoro modified nucleotide, and the nucleotides at the remaining positions are selected from a 2'-O-methyl modified nucleotide;

[0126] (6) In the direction from the 5' end to the 3' end, the nucleotide at position 5, or position 12, or position 18 of the nucleotide sequence in the sense strand is selected from a 2'-O-methoxyethyl modified nucleotide, the nucleotides at positions 7 to 10 are selected from a 2'-fluoro modified nucleotide, and the nucleotides at the remaining positions are selected from a 2'-O-methyl modified nucleotide; the nucleotides at positions 2, 6, 12, 14, and 16 of the nucleotide sequence in the antisense strand are selected from a 2'-fluoro modified nucleotide, and the nucleotides at the remaining positions are selected from a 2'-O-methyl modified nucleotide;

[0127] (7) In the direction from the 5' end to the 3' end, the nucleotide at position 5, or position 12, or position 18 of the nucleotide sequence in the sense strand is selected from a 2'-O-methoxyethyl modified nucleotide, the nucleotides at positions 7 to 10 are selected from a 2'-fluoro modified nucleotide, and the nucleotides at the remaining positions are selected from a 2'-O-methyl modified nucleotide; the nucleotides at positions 2, 6, 9, 14, and 16 of the nucleotide sequence in the antisense strand are selected from a 2'-fluoro modified nucleotide, the nucleotide at position 15 is selected from a 2'-O-methoxyethyl modified nucleotide, and the nucleotides at the remaining positions are selected from a 2'-O-methyl modified nucleotide;

[0128] (8) In the direction from the 5' end to the 3' end, the nucleotide at position 5, or the 12th position, or the 18th position of the nucleotide sequence in the sense strand is selected from a 2'-O-methoxyethyl modified nucleotide, the nucleotides at positions 7 to 10 are selected from a 2'-fluoro modified nucleotide, and the nucleotides at the remaining positions are selected from a 2'-O-methyl modified nucleotide; the nucleotides at positions 2, 6, 12, 14, and 16 of the nucleotide sequence in the antisense strand are selected from a 2'-fluoro modified nucleotide, the nucleotide at position 15 is selected from a 2'-O-methoxyethyl modified nucleotide, and the nucleotides at the remaining positions are selected from a 2'-O-methyl modified nucleotide.

[0129] Optionally, in the direction from 5' end to 3' end, the terminal nucleotides at the 5' end of the sense strand contain 1 or 2 consecutive phosphorothioate bonds; and / or the 5' end and 3' end of the antisense strand each independently contain 1 or 2 consecutive phosphorothioate bonds.

[0130] In some embodiments, in the nucleic acid chimera described herein, the nucleotide unit I has a structure shown in formula (102):

[0131] In some embodiments, in the nucleic acid chimera described herein, the nucleotide unit I has a structure shown in formula (103):

[0132] In some embodiments, in the nucleic acid chimera described herein, each M1 is independently selected from a ligand capable of binding to an asialoglycoprotein receptor.

[0133] In some embodiments, in the nucleic acid chimera disclosed herein, each M1 is independently selected from the structure represented by formula (201a) or formula (201b) or an isomer thereof or a pharmaceutically acceptable salt thereof:

[0134] When M1 is located at the terminal position of the terminal nucleotide unit I, M1 is selected from the structure represented by formula (201a) or an isomer thereof or a pharmaceutically acceptable salt thereof;

[0135] When M1 is not located at the end of the terminal nucleotide unit I, M1 is selected from the structure represented by formula (201b) or an isomer thereof or a pharmaceutically acceptable salt thereof;

[0136] wherein A is selected from an optionally substituted 4-10 membered aliphatic ring;

[0137] X is selected from NH, O or S;

[0138] L1 is selected from wherein h is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0139] Each R is independently selected from H, optionally substituted C 1-6 Alkyl or optionally substituted C 1-6 alkoxy;

[0140] L2 is selected from optionally substituted C2-C 20 Alkylene or R La and R Lb Independently selected from optionally substituted C 1-10 Alkylene, i is selected from 1, 2, 3, 4 or 5;

[0141] Y is selected from O or S;

[0142] M 1a Selected from galactose or its derivatives Z is selected from hydroxyl or sulfhydryl;

[0143] p is selected from 1, 2 or 3;

[0144] q is selected from 1, 2 or 3.

[0145] In some embodiments, A is selected from

[0146] In some embodiments, A is selected from

[0147] In some embodiments, X is selected from NH.

[0148] In some embodiments, L1 is

[0149] In some embodiments, each R is independently selected from H.

[0150] In some embodiments, L2 is selected from C 1-10 Alkylene or Among them, R La and R La Independently selected from C 1-5 Alkylene, i is 1, 2 or 3.

[0151] In some embodiments, i is selected from 1.

[0152] In some embodiments, each L2 is independently selected from

[0153] In some embodiments, M 1a Selected from galactosamine derivatives.

[0154] In some embodiments, M 1a Selected from

[0155] In some embodiments, Z is selected from hydroxy.

[0156] In some embodiments, Z is selected from thiol.

[0157] In some embodiments, p is selected from 1.

[0158] In some embodiments, q is selected from 1.

[0159] In some embodiments, in the nucleic acid chimera disclosed herein, each M1 is independently selected from the structure represented by Formula (202a) or Formula (202b) or an isomer thereof or a pharmaceutically acceptable salt thereof:

[0160] Wherein, when M1 is located at the terminal position of the terminal nucleotide unit I, M1 is selected from the structure represented by formula (202a) or an isomer thereof or a pharmaceutically acceptable salt thereof;

[0161] When M1 is not located at the end of the terminal nucleotide unit I, M1 is selected from the structure represented by formula (202b) or its isomer or a pharmaceutically acceptable salt thereof.

[0162] In some embodiments, the nucleic acid chimeras described herein,

[0163] Each M1 is independently selected from the structure represented by formula (203a) or formula (203b) or an isomer thereof or a pharmaceutically acceptable salt thereof:

[0164] Wherein, when M1 is located at the terminal position of the terminal nucleotide unit I, M1 is selected from the structure represented by formula (203a) or its isomer or pharmaceutically acceptable salt thereof;

[0165] When M1 is not located at the end of the terminal nucleotide unit I, M1 is selected from the structure represented by formula (203b) or its isomer or a pharmaceutically acceptable salt thereof.

[0166] In some embodiments, in the nucleic acid chimera described herein, each M1 is independently selected from any of the following structures or isomers thereof or pharmaceutically acceptable salts thereof:

[0167] Wherein, when M1 is located at the terminal position of the nucleotide unit I, M1 is selected from or its isomer or pharmaceutically acceptable salt, or or an isomer thereof or a pharmaceutically acceptable salt thereof;

[0168] When M1 is not located at the end of the nucleotide unit I, M1 is selected from its isomers or pharmaceutically acceptable salts, or or an isomer thereof or a pharmaceutically acceptable salt thereof.

[0169] In some embodiments, in the nucleic acid chimera disclosed herein, the nucleotide unit I has a structure represented by formula (104) or an isomer thereof or a pharmaceutically acceptable salt thereof:

[0170] In some embodiments, the nucleic acid chimera described in the present disclosure has a structure represented by formula (105) or an isomer thereof or a pharmaceutically acceptable salt thereof:

[0171] Wherein, Z is OH;

[0172] AS represents the antisense strand of a double-stranded oligonucleotide molecule; SS represents the sense strand of a double-stranded oligonucleotide molecule;

[0173] In some embodiments, the nucleic acid chimera described in the present disclosure has a structure represented by formula (106) or an isomer thereof or a pharmaceutically acceptable salt thereof:

[0174] Wherein, AS represents the antisense strand of the double-stranded oligonucleotide molecule; SS represents the sense strand of the double-stranded oligonucleotide molecule;

[0175] Z is OH;

[0176] Optionally, j1 is selected from 0 or 1, j2 is selected from an integer of 1-3, and k is selected from 1.

[0177] In some embodiments, the nucleic acid chimera provided by the present disclosure includes a nucleotide unit III having a structure represented by formula (301), or an isomer thereof, or a pharmaceutically acceptable salt thereof:

[0178] Wherein, each M1 independently comprises a ligand capable of binding to a cell receptor; specifically, each M1 is independently selected from the structure shown in formula (201b) or an isomer thereof or a pharmaceutically acceptable salt thereof:

[0179] A, X, L1, R, L2, Y, M 1a , p, q are as defined above;

[0180] wherein Y3 is selected from the sense strand and Y4 is selected from the sense strand, or Y3 is selected from the antisense strand and Y4 is selected from the sense strand; Y3 and Y4 in the nucleotide unit III correspond to the same target gene or different target genes; the correspondence means that the sense strand is identical or substantially identical to a continuous stretch of nucleotides in the mRNA of the target gene, or the antisense strand is complementary or substantially complementary to a continuous stretch of nucleotides in the mRNA of the target gene; and when Y3 is selected from the antisense strand and Y4 is selected from the sense strand, the antisense strand of Y3 and the sense strand of Y4 in the nucleotide unit III do not form complementary base pairing;

[0181] j4, j5, j6, and j7 are each independently selected from integers of 0-3, and at least one of j6 and j7 is not 0; when Y3 is selected from the sense chain, at least one of j4 and j5 is not 0.

[0182] In some embodiments, when Y3 is selected from the sense strand and Y4 is selected from the sense strand, the 5' end of Y3 and the 5' end of Y4 are conjugated, or the 3' end of Y3 and the 3' end of Y4 are conjugated; and Dt has the structure shown in (401), or a stereoisomer thereof, or a tautomer thereof:

[0183] Wherein, A, X, L1, R, L2, p, q are as defined above;

[0184] Dt' is selected from

[0185] In some embodiments, when Y3 is selected from the antisense strand and Y4 is selected from the sense strand, the 5' end of Y3 and the 3' end of Y4 are conjugated, or the 3' end of Y3 and the 5' end of Y4 are conjugated; and Dt is selected from a group not having the structure shown in (501), or a stereoisomer thereof, or a tautomer thereof:

[0186] wherein t1 is selected from an integer of 0-5, t2 is independently selected from 0 or 1, and Base is selected from nucleoside base A, U, G, C or T; and when t1 is 0, j6 is not 0.

[0187] In some embodiments, when Y3 is selected from the sense strand and Y4 is selected from the sense strand, Dt has the structure shown in (402), or a stereoisomer thereof, or a tautomer thereof:

[0188] In some embodiments, Dt' is selected from

[0189] In some embodiments, when Y3 is selected from the sense strand and Y4 is selected from the sense strand, Dt has the structure shown in (403), or a stereoisomer thereof, or a tautomer thereof:

[0190] In some embodiments, when Y3 is selected from the sense strand and Y4 is selected from the sense strand, the nucleotide unit III has a structure represented by formula (301a), or an isomer thereof, or a pharmaceutically acceptable salt thereof:

[0191] wherein each Za is independently selected from a hydroxyl group or a thiol group.

[0192] In some embodiments, when Y3 is selected from the sense strand and Y4 is selected from the sense strand, the nucleic acid chimera further comprises two antisense strands, the nucleotide sequence of one antisense strand is at least partially reverse complementary to the nucleotide sequence of the Y3 sense strand in nucleotide unit III, and the nucleotide sequence of the other antisense strand is at least partially reverse complementary to the nucleotide sequence of the Y4 sense strand in nucleotide unit III.

[0193] In some embodiments, when Y3 is selected from the antisense strand and Y4 is selected from the sense strand, the nucleotide unit III has a structure represented by formula (301b), or an isomer thereof, or a pharmaceutically acceptable salt thereof:

[0194] wherein each Zb is independently selected from a hydroxyl group or a thiol group.

[0195] In some embodiments, when Y3 is selected from the antisense strand and Y4 is selected from the sense strand, the nucleotide unit III has a structure represented by formula (302b), or an isomer thereof, or a pharmaceutically acceptable salt thereof:

[0196] wherein each Zb is independently selected from a hydroxyl group or a thiol group.

[0197] In some embodiments, when Y3 is selected from the antisense strand and Y4 is selected from the sense strand, the nucleic acid chimera comprises two nucleotide units III, and the Y3 antisense strand and the Y4 sense strand in the same nucleotide unit III correspond to the same target gene or different target genes, and the Y3 antisense strand and the Y4 sense strand in the same nucleotide unit III do not form complementary base pairing;

[0198] The nucleotide sequence of the antisense strand of Y3 in the first nucleotide unit III and the nucleotide sequence of the sense strand of Y4 in the second nucleotide unit III are at least partially reverse complementary;

[0199] The nucleotide sequence of the sense strand of Y4 in the first nucleotide unit III and the nucleotide sequence of the antisense strand of Y3 in the second nucleotide unit III are at least partially reverse complementary.

[0200] In some embodiments, when Y3 is selected from the antisense strand and Y4 is selected from the sense strand, the nucleic acid chimera further comprises a sense strand and an antisense strand, wherein the nucleotide sequence of the sense strand and the nucleotide sequence of the antisense strand of Y3 in nucleotide unit III are at least partially reverse complementary; and the nucleotide sequence of the antisense strand and the nucleotide sequence of the sense strand of Y4 in nucleotide unit III are at least partially reverse complementary.

[0201] In some embodiments, the nucleic acid chimera consists of two nucleotide units III, wherein the Y3 position in the first nucleotide unit III is the antisense strand of the siRNA corresponding to target gene A and Y4 is the sense strand of the siRNA corresponding to target gene B, and the Y4 position in the second nucleotide unit III is the sense strand of the siRNA corresponding to target gene A and Y3 is the antisense strand of the siRNA corresponding to target gene B; and, the antisense strand Y3 of the siRNA corresponding to target gene A in the first nucleotide unit III and the sense strand Y4 of the siRNA corresponding to target gene A in the second nucleotide unit III are at least partially reverse complementary; and, the sense strand Y4 of the siRNA corresponding to target gene B in the first nucleotide unit III and the antisense strand Y3 of the siRNA corresponding to target gene B in the second nucleotide unit III are at least partially reverse complementary;

[0202] Among them, the target gene A and the target gene B can be the same or different; and the antisense chain Y3 of the siRNA corresponding to the target gene A in the first nucleotide unit III and the sense chain Y4 of the siRNA corresponding to the target gene B do not form a base complementary pairing, and the sense chain Y4 of the siRNA corresponding to the target gene A in the second nucleotide unit III and the antisense chain Y3 of the siRNA corresponding to the target gene B do not form a base complementary pairing.

[0203] In some embodiments, Y3 and Y4 in formula (301) independently represent the sense strand or the positive strand of siRNA; the siRNAs may correspond to the same or different target genes.

[0204] In some embodiments, the nucleic acid chimera disclosed herein may include two nucleotide units III, wherein the Y3 position in the first nucleotide unit III is the antisense strand of an siRNA corresponding to target gene A, and the Y4 position is the sense strand of an siRNA corresponding to target gene B, and target genes A and B are the same or different; and the antisense strand sequence of the siRNA corresponding to target gene A in the first nucleotide unit III is complementary or at least partially reverse complementary to the sense strand sequence of the siRNA corresponding to target gene A in the second nucleotide unit III. Optionally, target gene A and target gene B are different target genes.

[0205] Composition

[0206] In a second aspect, the present disclosure provides a composition comprising the nucleic acid chimera described in the present disclosure and a physiologically acceptable excipient.

[0207] In a third aspect, the present disclosure provides the nucleic acid chimera for use in treating a disease or condition caused by dysregulated expression of at least one gene.

[0208] Preferably, the present disclosure provides the nucleic acid chimera for use in simultaneously treating diseases or disorders caused by dysregulated expression of two genes.

[0209] use

[0210] In a fourth aspect, the present disclosure provides use of the nucleic acid chimera in the preparation of a drug for treating a disease or condition caused by dysregulated expression of at least one gene.

[0211] Preferably, the present disclosure provides use of the nucleic acid chimera in preparing a medicament for treating a disease or condition caused by dysregulated expression of two genes.

[0212] method

[0213] In a fifth aspect, the present disclosure provides a method for treating a disease or disorder, comprising administering the nucleic acid chimera of the present disclosure to an individual in need of treatment.

[0214] In some embodiments, the nucleic acid chimera of the present disclosure is administered to the individual subcutaneously or intravenously.

[0215] In some embodiments, after in vivo administration, the nucleic acid chimeras described herein disassemble to yield at least two independent double-stranded oligonucleotide molecules, each targeting a portion of an mRNA transcribed from one or more target genes, which may be the same or different.

[0216] Unless otherwise specified, the siRNA sequences used in this disclosure were synthesized by Suzhou Beixin Biotechnology Co., Ltd.; the PCR primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.; and the experimental animals, C57BL / 6J mice, were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.

[0217] Unless otherwise stated, the real-time PCR detection data for in vitro and in vivo siRNA activity experiments in each embodiment of the present disclosure all used the ΔΔCt method to perform relative quantitative calculation of the target gene mRNA in each test group. The calculation method is summarized as follows:

[0218] ΔCt(test group) = Ct(test group target gene) – Ct(test group reference gene)

[0219] ΔCt(control group) = Ct(control group target gene) – Ct(control group internal reference gene)

[0220] ΔΔCt(test group)=ΔCt(test group)-ΔCt(control group average)

[0221] ΔΔCt(control group) = ΔCt(control group) - ΔCt(control group average)

[0222] The mRNA expression level of the target gene in the test group was normalized with the control group as the benchmark, and the remaining expression level of the target gene mRNA in the control group was defined as 100%.

[0223] Relative residual expression level of target gene mRNA in the test group = 2 -ΔΔCt (Test group) × 100%

[0224] Test group target gene mRNA inhibition rate = 100% - test group target gene mRNA relative expression level

[0225] Unless otherwise stated, the reagent ratios in the examples of this disclosure are calculated by volume ratio (v / v); the activity experimental data are calculated by The experimental data were plotted and analyzed using GraphPad prism 8.0 software.

[0226] Unless otherwise stated, the reagents used in the examples of the present disclosure were purchased from Beijing Coupling Technology Co., Ltd., wherein the information of the main reagents is shown in Table 1.

[0227] Table 1 Reagent information

[0228] Among them, CPG stands for controlled pore glass (Controlled Pore Glass) carrier.

[0229] Preparation Example 1 Preparation of Compound CR01008

[0230] The synthetic route of compound CR01008 is as follows:

[0231] (1-1) Synthesis of Compound 2

[0232] Compound 1 (trans-4-(Boc-amino)cyclohexylcarboxaldehyde, 10.0 g, 1.0 eq) and formaldehyde solution (8.9 g, 37% by mass, 2.4 eq) were dissolved in 33 ml of methanol, and 13 ml of a 45.3% by mass KOH aqueous solution was added dropwise. After the addition was complete, the mixture was stirred at 25 ° C for 30 minutes, heated to 60 ° C and refluxed at 60 ° C for 2 hours. After the reaction was completed, the reaction solution was cooled to room temperature and evaporated to dryness under reduced pressure to obtain a crude white solid. A small amount of water was added to the crude product to slurry, and filtered to obtain compound 2 (9 g, yield 78.9%) as a white solid. MS-ESI (m / z) = 260 [M + H] + .

[0233] (1-2) Synthesis of Compound 3

[0234] Compound 2 (9 g, 1 eq) prepared in step (1.1) was dissolved in 70 ml of 1,4-dioxane. A 4 M solution of hydrogen chloride in 1,4-dioxane (45 ml) was added, and the mixture was stirred at 25°C for 1 hour. After completion of the reaction, the reaction solution was evaporated to dryness under reduced pressure to obtain compound 3 (6.8 g, 100% yield) as a white solid.

[0235] (1-3) Synthesis of Compound 5

[0236] Compound 3 (1.8 g, 2.0 eq) prepared in step (1.2), compound 4 (5-[[(2R,3R,4R,5R,6R)-3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)-2-tetrahydropyranyl]oxy]pentanoic acid, 2.1 g, 1.0 eq), and N,N-diisopropylethylamine (3.5 g, 6.0 eq, abbreviated as DIEA) were dissolved in N,N-dimethylformamide (15 ml, abbreviated as DMF, CAS No. 68-12-2). HBTU (1.9 g, 1.1 eq) was added, and the mixture was stirred at 25°C under a N2 atmosphere for 3 hours. After completion of the reaction, the reaction solution was evaporated to dryness under reduced pressure and reverse purification (22% acetonitrile in water) was performed to obtain compound 5 (1.78 g, yield 64.4%) as a white solid. MS-ESI (m / z) = 589 [M+H] + .

[0237] (1-4) Synthesis of Compound 6

[0238] Compound 5 (1.54 g, 1.0 eq) was dissolved in 15 ml of pyridine. The reaction system was cooled to 0°C using an ice-water bath and 4,4'-dimethoxytriphenylmethane (1.32 g, 1.5 eq, DMTrCl, CAS No. 40615-36-9) was added at 0°C. The reaction was allowed to react at 25°C for 3 hours. 15 ml of methanol was added to the reaction solution to quench the reaction. After completion of the reaction, the reaction solution was evaporated to dryness under reduced pressure and purified by reverse purification (60% acetonitrile in water by volume) to obtain compound 6 (1 g, 42.7% yield) as a yellow solid. MS-ESI (m / z) = 891 [M+H] + .

[0239] (1-5) Synthesis of Compound CR01008

[0240] Compound 6 (1.08 g, 1.0 eq) was dissolved in 20 ml of anhydrous dichloromethane, and DCI (115 mg, 0.8 eq) and compound 7 (bis(diisopropylamino)(2-cyanoethoxy)phosphine, 732 mg, 2.1 eq) were added, respectively. The atmosphere was purged with nitrogen three times, and the mixture was stirred at 25°C for 2 hours. After completion of the reaction, 20 ml of saturated aqueous sodium bicarbonate solution was added to the reaction solution, and the mixture was extracted three times with 20 ml of dichloromethane (3×20 ml). The organic phases were combined and evaporated to dryness under reduced pressure. After reverse purification (72 vol% acetonitrile in water), the mixture was dried under vacuum for 12 hours to obtain compound CR01008 (1 g, 76.0% yield) as a white powder. MS-ESI (m / z) = 1091 [M+Na] + .

[0241] 1H NMR(400MHz, DMSO-d6)δ1.05(d,J=6.7Hz,6H).1.14(d,J=6.7Hz,6H),1.37–1.17(m,5H),1.60–1.40(m,6H),1.68–1.62(m,1H),1.80(s,3H), 1.80(s,3H),1.92(s,3H),2.02(s,5H),2.13(s,3H),2.71(t,J=5.9Hz,2H),2.79(d,J=8.4Hz,1H),2.87(d,J=8.4Hz,1H),3.36(s,1H),3.58– 3.39(m,3H),3.69–3.60(m,2H),3.75(s,7H),3.90(dt,J=11.2,8.8Hz,1H),4.05(s,3H),4.51(d,J=8.4Hz,1H),4.99(dd,J=11.3,3.4Hz,1H) ,5.24(d,J=3.4Hz,1H),5.78(s,1H),6.93–6.87(m,4H),7.35–7.21(m,7H),7.44–7.37(m,2H),7.66(d,J=7.8Hz,1H),7.84(d,J=9.2Hz,1H).

[0242] Preparation Example 2 Preparation of Compound CR01008Z

[0243] The synthetic route of compound CR01008Z is as follows:

[0244] (2-1) Synthesis of Compound 9

[0245] Compound 6 (500 mg) prepared in step (1.4) was dissolved in 10 ml of dichloromethane, and compound 8 (succinic anhydride, 112 mg), DMAP (6.8 mg), and triethylamine (226.2 mg, abbreviated as TEA or Et3N) were added. The atmosphere was purged with nitrogen three times, and the mixture was stirred at 25°C for 16 hours. Flash purification was performed to obtain compound 9 (300 mg, yield 53.6%). MS-ESI (m / z) = 1013 [M+Na] + .

[0246] (2-2) Synthesis of Compound CR01008Z

[0247] Compound 9 (50 mg), aminoCPG (1.25 g, 80 μmol / g, 0.1 mmol), HBTU (27 mg), and DIEA (12 mg) prepared in step (1.6) were added to a 20 ml sample vial and shaken for 16 hours. After the reaction, the reaction solution was filtered to obtain a filter cake, which was washed once with 10 ml of acetonitrile (1×10 ml) and then dried in vacuo. The dried filter cake, DMAP (3 mg), Cap1 (10 ml, 200 V), and Cap2 (1 ml, 20 V) were added to a 20 ml sample vial and shaken for 6 hours. After the reaction, the reaction solution was filtered to obtain a filter cake, which was washed once with 10 ml of acetonitrile (1×10 ml) and then dried in vacuo to obtain compound CR01008Z (1.03 g, loading 20-30 μmol / g).

[0248] Cap1 and Cap2 are capping reagents, Cap1 is a 20% by volume N-methylimidazole mixed solution in pyridine / acetonitrile, with a volume ratio of pyridine to acetonitrile of 3:5; Cap2 is a 20% by volume acetic anhydride solution in acetonitrile.

[0249] Preparation Example 3 Preparation of Compound NM090

[0250] The synthetic route of compound NM090 is as follows:

[0251] (3-1) Synthesis of Compound 11

[0252] Compound 3 (3 g, 15.3 mmol, 1.0 eq), compound 10 (2.4 g, 15.2 mmol, 1.0 eq, 6-azidohexanoic acid, CAS No. 79598-53-1), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (4.38 g, 22.95 mmol, 1.5 eq, EDCI, CAS No. 25952-53-8), and 1-hydroxybenzotriazole (3.15 g, 22.95 mmol, 1.5 eq, HOBt, CAS No. 2592-95-2) were dissolved in DMF (150 ml). DIEA (6 g, 3.0 eq) was added under ice bath, and the mixture was stirred at room temperature for 2-3 hours. The reaction was completed. Water (100 ml) was added to the reaction solution, and the mixture was extracted twice with ethyl acetate (100 ml each time). The organic phases were separated and combined, dried, concentrated, and then purified by reverse phase purification (eluent: acetonitrile / water = 30 / 70, v / v) to obtain compound 11 (3 g, yield 75%) as a white solid. MS ESI (m / z) = 299 [M+H] + .

[0253] (3-2) Synthesis of Compound 12

[0254] Compound 11 (3 g, 11.19 mmol, 1.0 eq) was dissolved in pyridine (30 ml). DMTrCl (3.7 g, 11.19 mmol, 1.0 eq) was added in an ice bath. The atmosphere was replaced with nitrogen three times and stirred at room temperature for 3 hours. The reaction was quenched with methanol (50 ml). The reaction mixture was concentrated, water (50 ml) was added, and the mixture was extracted three times with ethyl acetate (50 ml each time). The organic phases were separated and combined, dried, concentrated, and then purified by reverse phase chromatography (eluent: acetonitrile / water = 60 / 40, v / v) to obtain compound 12 (1.9 g, 26% yield) as a light yellow solid. MS ESI (m / z) = 601 [M+H] + .

[0255] (3-3) Synthesis of Compound 13

[0256] Compound 12 (200 mg, 0.33 mmol, 1 eq) was dissolved in anhydrous dichloromethane (2 ml). Succinic anhydride (67 mg, 0.66 mmol, 2 eq) and TEA (100 mg, 1.0 mmol, 3 eq) were added, respectively. The atmosphere was purged with nitrogen three times and stirred at room temperature for 12 hours. The reaction was completed. Dichloromethane (10 ml) was added to the reaction solution for dilution, followed by extraction with water (10 ml). The organic phase was separated, dried, concentrated, and then purified by reverse phase chromatography (eluent: acetonitrile / water = 31 / 69, v / v) to obtain compound 13 (160 mg, yield 69.5%) as a white powder. MS ESI (m / z) = 701 [M+H] + .

[0257] (3-4) Synthesis of Compound NM090

[0258] Compound 13 (39 mg, 0.056 mmol, 1 eq) was dissolved in anhydrous acetonitrile (2 ml), and HBTU (32 mg, 1.5 eq), DIEA (15 mg, 2 eq) and aminoCPG (1.43 g, 2 eq) were added respectively. The mixture was replaced with nitrogen three times and shaken at room temperature for 12 hours. The mixture was filtered and the filter cake was rinsed with acetonitrile three times (10 ml each time). After drying, acetonitrile (10 ml) was added first, and then Cap1 (8 ml) and Cap2 (1 ml) were added. The mixture was shaken for 5 hours, filtered, and the filter cake was rinsed with acetonitrile three times (10 ml each time). The mixture was dried in vacuo for 12 hours to obtain compound NM090 (1 g, the measured loading value was 40-60 umol / g) as a white solid.

[0259] Preparation Example 4 Preparation of Compound NM091

[0260] The synthetic route of compound NM091 is as follows:

[0261] (4-1) Synthesis of Compound 15

[0262] Compound 3 (3 g, 15.3 mmol, 1.0 eq), compound 14 (1.92 g, 15.2 mmol, 1.0 eq, CAS No. 30964-00-2), EDCI (4.38 g, 22.95 mmol, 1.5 eq), and HOBt (3.15 g, 22.95 mmol, 1.5 eq) were dissolved in DMF (150 ml). DIEA (6 g, 3.0 eq) was added under ice-cooling and stirred at room temperature for 2-3 hours. The reaction was complete. Water (100 ml) was added to the reaction solution, which was then extracted twice with ethyl acetate (100 ml each time). The organic phase was dried and concentrated, and then purified by reverse phase purification (eluent: acetonitrile / water = 30 / 70, v / v) to obtain compound 15 (3 g, 75% yield) as a white solid. MS ESI (m / z) = 268 [M+H] + .

[0263] (4-2) Synthesis of Compound 16

[0264] Compound 15 (3 g, 11.19 mmol, 1.0 eq) was dissolved in pyridine (30 ml). DMTrCl (3.7 g, 11.19 mmol, 1.0 eq) was added in an ice bath. The atmosphere was replaced with nitrogen three times and the mixture was stirred at room temperature for 3 hours. The reaction was quenched with methanol (50 ml) and the reaction was complete. The reaction solution was concentrated, water (50 ml) was added, and the mixture was extracted three times with ethyl acetate (50 ml each time). The organic phases were separated and combined, dried, concentrated, and then purified by reverse phase chromatography (eluent: acetonitrile / water = 60 / 40, v / v) to obtain compound 16 (1.6 g, yield 26%) as a light yellow solid. MS ESI (m / z) = 569 [M+H] + .

[0265] (4-3) Synthesis of Compound 17

[0266] Compound 16 (1.6 g, 2.8 mmol, 1.5 eq) was dissolved in anhydrous dichloromethane (20 ml), and DCI (250 mg, 2.24 mmol, 0.8 eq) and compound 7 (875 mg, 3.1 mmol, 1.1 eq) were added, respectively. The atmosphere was purged with nitrogen three times and stirred at room temperature for 2 hours. The reaction was completed. Saturated aqueous sodium bicarbonate (20 ml) was added to the reaction solution, and the mixture was extracted three times with dichloromethane (20 ml each time). The organic phases were separated and combined, dried, concentrated, and then purified by reverse phase purification (eluent: acetonitrile / water = 91 / 9, v / v) to obtain compound NM091 (1 g, yield 46.3%) as a white powder. MS ESI (m / z) = 770 [M+H] + .

[0267] 1 H NMR(400MHz, DMSO-d6)δ7.67(d,J=7.8Hz,1H),7.43–7.36(m,2H),7.35–7.17(m,8H),6.88(dd ,J=15.5,8.5Hz,4H),3.75(s,6H),3.72(s,1H),3.70–3.61(m,2H),3.51(dq,J=16.9,6.8Hz,2H ),2.90–2.67(m,6H),2.16(td,J=7.1,2.7Hz,2H),2.05(t,J=7.3Hz,2H),1.58(tt,J=21.1,11 .4Hz,6H),1.46–1.37(m,2H),1.35–1.19(m,4H),1.13(d,J=6.7Hz,6H),1.05(d,J=6.7Hz,6H).

[0268] Preparation Example 5 Preparation of siRNA and siRNA Chimeras

[0269] (5-1) Synthesis of the positive chain (SS)

[0270] Using phosphoramidite nucleic acid solid phase synthesis, compound CR01008Z was used as the starting cycle, and nucleoside monomers were linked one by one in the 3'-5' direction according to the nucleotide sequence. During the synthesis process, compound CR01008 was considered as a nucleoside monomer.

[0271] Each linking of a nucleoside monomer involves four steps: deprotection, coupling, capping, and oxidation or sulfurization. The synthesis conditions are given below:

[0272] The nucleoside monomer was prepared into an acetonitrile solution with a concentration of 0.1 M.

[0273] The deprotection reaction conditions for each step were identical: 25°C, 70 seconds, a 3% vol. dichloroacetic acid solution in dichloromethane as the deprotection reagent, and a 5:1 molar ratio of dichloroacetic acid to the 4,4'-dimethoxytrityl protecting group on the solid support.

[0274] The conditions for each coupling reaction were the same. The coupling reaction conditions were: temperature 25°C, a molar ratio of the nucleic acid sequence attached to the solid support to the nucleoside monomer of 1:10, a molar ratio of the nucleic acid sequence attached to the solid support to the coupling reagent of 1:65, a reaction time of 600 seconds, a 0.5 M solution of 5-ethylthio-1H-tetrazole in acetonitrile as the coupling reagent, and a 0.2 M solution of hydrogenated xanthan gum in acetonitrile / pyridine (1:1 volume ratio of acetonitrile to pyridine) as the thiolation reagent.

[0275] The capping reaction conditions were identical for each step. The capping reaction conditions were: 25°C; 2 minutes; a 1:1 molar ratio of Cap1 and Cap2; Cap1: a 20% by volume N-methylimidazole solution in pyridine / acetonitrile (3:5 volume ratio of pyridine to acetonitrile); and Cap2: a 20% by volume solution of acetic anhydride in acetonitrile. The molar ratio of the N-methylimidazole in Cap1 to the acetic anhydride in Cap2 to the nucleic acid sequence attached to the solid support was 1:1:1.

[0276] The oxidation reaction conditions were identical for each step. The oxidation reaction conditions were: temperature, 25°C; reaction time, 3 seconds; oxidizing agent concentration, 0.05 M iodine solution; a molar ratio of iodine to the nucleic acid sequence attached to the solid support during the coupling reaction, 30:1; and the oxidation reaction was performed in a water / pyridine mixture (1:9 by volume). The sulfidation reaction conditions were: temperature, 25°C; reaction time, 360 seconds; thiolation agent concentration, 0.2 M hydrogenated xanthan gum in pyridine solution; a molar ratio of thiolation agent to the nucleic acid sequence attached to the solid support during the coupling reaction, 4:1; and the sulfidation reaction was performed in a water / pyridine mixture (1:9 by volume).

[0277] After the last nucleoside monomer is connected, the nucleic acid sequence connected to the solid phase support is cut, deprotected, purified, desalted, and then freeze-dried to obtain the positive chain, wherein:

[0278] Cleavage and deprotection conditions were as follows: the synthesized nucleotide sequence attached to a solid support was added to 0.5 ml / μmol of 25% ammonia water at 55°C for 16 hours, the solvent was removed, and the product was concentrated to dryness in vacuo. After the ammonia treatment, the product was dissolved in 0.4 ml / μmol of N-methylpyrrolidone relative to the amount of single-stranded nucleic acid, followed by the addition of 0.3 ml / μmol of triethylamine and 0.6 ml / μmol of triethylamine trihydrofluoride to remove the 2'-O-TBDMS protection from the ribose.

[0279] Purification and desalting conditions: Nucleic acid purification was achieved using a preparative ion chromatography column (Source 15Q) with a NaCl gradient elution. Specifically, eluent 1 consisted of 20 mM sodium phosphate (pH 8.1) in a water / acetonitrile mixture (9:1 volume ratio of water to acetonitrile); eluent 2 consisted of 1.5 M sodium chloride, 20 mM sodium phosphate (pH 8.1) in a water / acetonitrile mixture (9:1 volume ratio of water to acetonitrile); the eluent ratio was eluent 1:eluent 2 (100:0) to (50:50). The product eluates were collected and combined, and desalted using a reversed-phase chromatography column. Desalting conditions included using a Sephadex column with Sephadex G25 as the filler and eluting with deionized water.

[0280] Detection: Purity was determined using ion exchange chromatography (IEX-HPLC); molecular weight was determined using liquid chromatography-mass spectrometry (LC-MS, purchased from Waters, model: LCT Premier). The measured molecular weight was compared with the theoretical value. If the measured value was consistent with the theoretical value, a positive chain as shown in Table 2 was obtained.

[0281] Table 2 Sequence information and detection results of the positive chain

[0282] Table 3 Unmodified nucleotide sequence information corresponding to the positive chain

[0283] (5-2) Synthesis of antisense strand AS

[0284] The antisense strand is synthesized using the phosphoramidite nucleic acid solid-phase synthesis method. Starting with a solid-phase support (CPG or PS), the nucleoside monomers are linked one by one in a 3'-5' direction according to the nucleotide sequence. Each nucleoside monomer linking step involves four steps: deprotection, coupling, capping, and oxidation or sulfurization. The reaction conditions are the same as those for the sense strand synthesis in step (5-1).

[0285] Detection: Purity was determined using ion exchange chromatography (IEX-HPLC); molecular weight was determined using liquid chromatography-mass spectrometry (LC-MS). The measured molecular weight was compared with the theoretical value. If the measured value was consistent with the theoretical value, the antisense strand shown in Table 4 was obtained.

[0286] Table 4 Sequence information and detection results of antisense strand

[0287] Table 5 Unmodified nucleotide sequence information corresponding to the antisense strand

[0288] (5-3) Synthesis of head-to-tail single-stranded

[0289] A head-to-tail single-stranded sequence comprises two subsequences, designated as a first target and a second target, respectively. The first target and the second target are each independently selected from the sense strand and the antisense strand, and the 3' end of the first target and the 5' end of the second target are conjugated and linked. For example, the first target and the second target may both be selected from the sense strand, or the first target may be selected from the antisense strand and the second target may be selected from the sense strand, or the first target may be selected from the sense strand and the second target may be selected from the antisense strand.

[0290] Head-to-tail single-stranded nucleic acids are synthesized using the phosphoramidite solid-phase nucleic acid synthesis method. Starting with a solid support (CPG support, PS support, or compound CR01008Z), the nucleoside monomers are linked one by one in a 3'-5' direction according to the nucleotide sequence. During the synthesis, compound CR01008 is treated as a single nucleoside monomer.

[0291] Each connection of a nucleoside monomer includes four steps of reaction: deprotection, coupling, capping, oxidation or sulfurization; the reaction conditions are the same as those for the synthesis of the positive chain in step (5-1).

[0292] Detection: Purity was detected using ion exchange chromatography (IEX-HPLC); molecular weight was detected using liquid spectrometry (LC-MS), and the measured molecular weight was compared with the theoretical value. If the measured value was consistent with the theoretical value, it indicated that a head-to-tail single strand formed by conjugating the 3' end of the sense strand and the 5' end of the sense strand as shown in Table 6, and a head-to-tail single strand formed by conjugating the 3' end of the antisense strand and the 5' end of the sense strand as shown in Table 8 were obtained.

[0293] Table 6 Sequence information and detection results of head-to-tail single strands formed by conjugation of the 3' end of the sense strand and the 5' end of the sense strand

[0294] The structural formula of the head-to-tail single chain formed by conjugating the 3' end of the sense chain and the 5' end of the sense chain is:

[0295] in, Represents the sense chain (first target), Represents the sense strand (second target).

[0296] In RZ899 / 597-M1-SS, j1=0, j2=3, j3=0.

[0297] In RZ899 / 597-M2-SS, j1=0, j2=3, j3=3.

[0298] In RZ899 / 597-M3-SS, j1=1, j2=1, j3=1.

[0299] In RZ899 / 597-M4-SS, j1=2, j2=2, j3=2.

[0300] The naked sequence information corresponding to the head-to-tail sense strand formed by conjugating the 3' end of the sense strand and the 5' end of the sense strand in Table 6 is shown in Table 7.

[0301] Table 7 Unmodified nucleotide sequence information of the head-to-tail single strand formed by conjugating the 3' end of the sense strand and the 5' end of the sense strand

[0302] Table 8 Sequence information and detection results of the head-to-tail single strand formed by conjugation of the 3' end of the antisense strand and the 5' end of the sense strand

[0303] The structural formula of the head-to-tail single chain formed by the conjugation of the 3' end of the antisense chain and the 5' end of the sense chain is:

[0304] in, represents the antisense strand (first target), Represents the sense strand (second target).

[0305] In RZ597AS / 899SS-M16, t1=0, j6=3, j7=0.

[0306] In RZ597AS / 899SS-M16.2, t1=3, t2=0, Base represents the nucleobase T, j6=3, j7=0.

[0307] In RZ597AS / 899SS-M16.3, t1=3, t2=1, Base represents the nucleobase U, j6=3, and j7=0.

[0308] In RZ597AS / 899SS-M2, t1=3, t2=0, Base represents the nucleobase T, j6=0, and j7=3.

[0309] In RZ899AS / 597SS-M21, t1=3, t2=0, Base represents the nucleobase T, j6=0, and j7=3.

[0310] The naked sequence information corresponding to the head-to-tail sense strand formed by conjugating the 3' end of the antisense strand and the 5' end of the sense strand in Table 8 is shown in Table 9.

[0311] Table 9 Unmodified nucleotide sequence information of the head-to-tail single strand formed by conjugating the 3' end of the antisense strand and the 5' end of the sense strand

[0312] (5-4) Synthesis of head-to-head single-stranded

[0313] The head-to-head single-stranded protein contains two subsequences, which are respectively named subsequence (first target) and subsequence (second target). The subsequence (first target) and subsequence (second target) are independently selected from the sense chain and the antisense chain, and the 3' end of the subsequence (first target) and the 3' end of the second target are conjugated and connected through a click chemistry reaction.

[0314] Specifically, the subsequence (first target) and the subsequence (second target) are both selected from the sense strand, and the 3' end of the sense strand and the 3' end of the sense strand are conjugated and connected by a click chemistry reaction.

[0315] (5-4-1) Synthesis of subsequences

[0316] Subsequences were synthesized using phosphoramidite nucleic acid solid-phase synthesis. Using a solid-phase support (CPG support, PS support, or compound CR01008Z) as the starting cycle, nucleoside monomers were linked one by one in a 3'-5' direction according to the nucleotide sequence. During the synthesis process, compounds CR01008, NM090, and NM091 were each considered a nucleoside monomer.

[0317] Each connection of a nucleoside monomer includes four steps of reaction: deprotection, coupling, capping, oxidation or sulfurization; the reaction conditions are the same as those for the synthesis of the positive chain in step (5-1).

[0318] Detection: Purity was determined by ion exchange chromatography (IEX-HPLC); molecular weight was determined by liquid chromatography-mass spectrometry (LC-MS). The measured molecular weight was compared with the theoretical value. If the measured value was consistent with the theoretical value, the subsequence shown in Table 12 was obtained.

[0319] (5-4-2) Click Chemistry Reaction

[0320] The sense chain (first target) was dissolved in 40 μL H2O and 40 μL carbonate buffer (PH = 9.2, 0.2 M) to form a sense chain (first target) solution with a concentration of 0.08 μmol; the sense chain (second target) was dissolved in 80 μL H2O to form a sense chain (second target) solution with a concentration of 0.08 μmol; the sense chain (first target) solution and the sense chain (second target) solution were mixed and vortexed to obtain a first mixed solution.

[0321] Tris(3-hydroxypropyltriazolemethyl)amine (100 μL, abbreviated as THPTA, CAS No. 760952-88-3) and CuSO 4 ·5H 2 O (20 μL) were mixed and shaken at 40° C. for 5 min to obtain a second mixed solution.

[0322] 9.6 μL of the second mixed solution was added to the first mixed solution and vortexed; 3.2 μL of a 0.1 mol / L aqueous solution of sodium ascorbyl palmitate (CAS No. 134-03-2) was added and vortexed; the mixture was reacted at 40° C. for 0.5 hour to obtain a head-to-head single chain formed by the head-to-head conjugation connection of the 3' end of the sense chain and the 3' end of the sense chain as shown in Table 12.

[0323] Table 10 Sequence information of head-to-head single strands formed by conjugation of the 3' end of the sense strand and the 3' end of the sense strand

[0324] The structural formula of the head-to-head single chain formed by the conjugation of the 3' end of the sense chain and the 3' end of the sense chain is:

[0325] in, Represents the sense chain (first target), Represents the sense strand (second target).

[0326] In RZ899SS / 597SS-M26, j4=3, j5=0, j6=3, j7=0, Za is

[0327] In RZ899SS / 597SS-M26.2, j4=0, j5=2, j6=2, j7=0, Za is

[0328] In RZ899SS / 597SS-M26.3, j4=0, j5=3, j6=3, j7=0, Za is

[0329] In RZ899SS / 597SS-M28, j4=2, j5=0, j6=2, j7=2, Za is

[0330] In RZ899SS / 597SS-M28.2, j4=2, j5=2, j6=2, j7=2, Za is

[0331] Among them, the naked sequence corresponding to the head-to-head single chain in Table 10 is shown in Table 11.

[0332] Table 11 Unmodified nucleotide sequence information of the head-to-head single strand formed by conjugating the 3' end of the sense chain and the 3' end of the sense chain

[0333] (5-5) siRNA Synthesis

[0334] According to the sequence numbers used for siRNA in Table 12, each sequence was mixed in an equimolar ratio, dissolved in deionized water, heated to 95°C, slowly cooled to room temperature, and maintained at room temperature for 10 minutes to allow the sense and antisense strands to form a double-stranded structure through hydrogen bonding, thereby obtaining the siRNA shown in Table 12.

[0335] Table 12 siRNA sequence information

[0336] (5-6) Synthesis of siRNA Chimeras

[0337] According to the sequence numbering used for the siRNA chimeras in Table 13, each sequence was mixed in an equimolar ratio, dissolved in deionized water, and heated to 95°C. The mixture was slowly cooled to room temperature and maintained at room temperature for 10 minutes to allow the sense and antisense strands to form a double-stranded structure through hydrogen bonding, thereby obtaining the siRNA chimeras shown in Table 13.

[0338] Table 13 Sequence information of siRNA chimeras

[0339] The molecular weight of the synthesized compound was detected by liquid chromatography-mass spectrometry (LC-MS), and the actual molecular weight was consistent with the theoretical molecular weight, indicating that the theoretically desired nucleic acid chimera had been obtained.

[0340] Biological detection experiments

[0341] Unless otherwise stated, the reagents, consumables, and instruments used in the biological detection experiments of the present disclosure are all derived from commercially available products. The main reagents and consumables and their sources are shown in Table 14, and the main instruments and equipment and their sources are shown in Table 15.

[0342] Table 14 Main reagents and consumables

[0343] Table 15 Main instruments and equipment

[0344] Example 1

[0345] Evaluation of the inhibitory activity of nucleic acid chimeras on superoxide dismutase 1 (SOD1) and angiopoietin-like 3 (ANGPTL3) in primary mouse hepatocytes.

[0346] This example evaluates the inhibitory activity of four multi-target nucleic acid chimeras with different structures, RZ899 / 597-M1, RZ899 / 597-M2, RZ899 / 597-M3, and RZ899 / 597-M4, and a mixture of RZ899058 and RZ597104, on the target genes SOD1 and ANGPTL3 in primary C56BL / 6j mouse hepatocytes.

[0347] (1-1) Isolation of primary mouse hepatocytes

[0348] Primary mouse hepatocytes were extracted from fresh liver tissue of C56BL / 6j mice. The specific operation steps were as follows: the mice were anesthetized by intraperitoneal injection of 10% chloral hydrate solution, fixed and their abdomen and chest disinfected with 75% ethanol. The surgical instruments were sterilized, and the abdominal cavity was opened to expose the portal vein and inferior vena cava. The indwelling needle was fitted with a heparin cap and connected to a scalp needle connected to an infusion pump bottle (0.5mM EDTAHBSS perfusion solution). The needle was inserted into the inferior vena cava and perfused at a rate of 120 drops / min. The portal vein was cut open and the perfusion solution was allowed to flow out of the cut portal vein. The perfusion was continued for 4 minutes, followed by replacement with 0.8mg / mL type IV collagenase HBSS solution (Sigma, C5138) (containing 0.08% DN I enzyme (Sigma, DN25)) and continued perfusion for 8 minutes. The perfused liver was removed from the animal and washed with HBSS (containing Ca 2+ Mg 2+ The liver was cleaned and placed in a sterile culture dish. DMEM complete medium (DMEM medium + 10% serum) was added to mince the liver. The cell suspension was filtered through a cell sieve to remove undigested tissue and connective tissue. The cell suspension was centrifuged at 800 rpm for 3 min, and the supernatant was discarded. DMEM complete medium was added again, and the suspension was centrifuged to obtain primary mouse hepatocytes.

[0349] (1-2) Cell culture and transfection

[0350] Add DMEM complete medium to adjust the cell density to 2×10^ 5 cells / mL to obtain a primary mouse hepatocyte suspension. The cells were then seeded into a 12-well culture plate pre-coated with rat tail collagen type I (coating method was as described in the solarbio (C8062) instructions at 2 μg / cm 2The volume of cell suspension added was 1000 μL / well, i.e. the cell amount was 2×10^ 5 cells / well.

[0351] Each siRNA chimera was diluted with PBS to a 5 μM siRNA chimera working solution (based on siRNA chimera). RZ899058 and RZ597104 were diluted with PBS buffer to a 5 μM working solution (based on siRNA) for RZ899058 and a 5 μM working solution (based on siRNA) for RZ597104, respectively. 2 μL / well of each siRNA chimera working solution was added to the 12-well culture plate. For the RZ899058+RZ597104 group, 2 μL / well of both RZ899058 and RZ597104 working solutions were added to the same well. This resulted in a final transfection concentration of 10 nM for each siRNA in each well. Two wells were set up for each siRNA. Two additional wells were treated with 2 μL / well of PBS buffer as blank controls. Swirl the plate to mix evenly.

[0352] The culture plate was placed in a cell culture incubator at 37° C. and 5% CO 2 and cultured for 24 h.

[0353] (1-3) Detection

[0354] RNA extraction: The total RNA of each group of primary hepatocyte samples was extracted using the fully automatic nucleic acid extraction instrument and nucleic acid extraction kit produced by Zhejiang Hanwei Technology Co., Ltd. according to the method described in the instructions.

[0355] Reverse transcription reaction: 1000 ng of total RNA was extracted from primary hepatocyte samples and reverse transcription was performed using the Promega Reverse Transcription System (A3500) and Oligo(dT) 15 Prepare 20 μL of reverse transcription system according to the kit instructions for reverse transcription primers and complete the reverse transcription reaction. After the reaction is complete, add 80 μL of RNase-free water to the reverse transcription system to obtain the cDNA solution for real-time PCR detection.

[0356] Real-time PCR detection: using ABI SYBR TMSelect Master Mix (Catalog number: 4472908) reagent, and prepare 20 μL Real-time PCR reaction system per PCR detection well according to the method described in the kit instructions. Each detection system contains 5 μL of cDNA template obtained by the above reverse transcription reaction, 10 μL SYBR TM Prepare the Select Master Mix, 0.5 μL of a 10 μM upstream primer, 0.5 μL of a 10 μM downstream primer (primer sequences are shown in Table 16), and 4 μL of RNase-Free H2O. The prepared reaction system was placed on an ABI StepOnePlus PCR instrument and amplified using a three-step real-time PCR protocol: pre-denaturation at 95°C for 10 minutes, followed by denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds. Repeat the denaturation, annealing, and extension process for 40 cycles. Gene expression differences were calculated using the ΔΔCt method described above.

[0357] Table 16 Primer sequence table

[0358] (3-4) Analysis

[0359] The test results are shown in Table 17 and Figure 1.

[0360] Table 17 Inhibitory activity of target gene SOD1 / ANGPTL3 in primary mouse hepatocytes

[0361] The results showed that the nucleic acid chimeras RZ899 / 597-M1, RZ899 / 597-M2, RZ899 / 597-M3, and RZ899 / 597-M4 could all effectively reduce the mRNA expression of the target genes SOD1 and ANGPTL3. Among them, the inhibitory activity of the target genes of RZ899 / 597-M2 and RZ899 / 597-M4 was equivalent to or higher than that of the RZ899058+RZ597104 mixture group.

[0362] Example 2

[0363] IC of nucleic acid chimeras against target genes SOD1 and ANGPTL3 in primary mouse hepatocytes 50 Evaluation of inhibitory activity.

[0364] This example evaluated the IC values ​​of siRNA chimeras RZ899 / 597-M2 and RZ899 / 597-M4, and a mixture of RZ899058 and RZ597104 against target genes SOD1 and ANGPTL3 in primary hepatocytes of C56BL / 6j mice.50 Inhibitory activity.

[0365] (2-1) Isolation of primary mouse hepatocytes: Same as step (1-1) in Example 1.

[0366] (2-2) Cell culture and transfection

[0367] Primary mouse hepatocytes were obtained from fresh liver tissue of C56BL / 6j mice, with a cell count of 2×10^ 5 The cells / well were seeded in a 12-well culture plate, and RZ899 / 597-M2 and RZ899 / 597-M4 were gradiently diluted with PBS buffer to 50 μM, 10 μM, 2 μM, 0.4 μM, 0.08 μM, 0.016 μM, and 0.0032 μM siRNA chimera working solutions (calculated as siRNA chimera); RZ899058 was gradiently diluted with PBS buffer to 50 μM, 10 μM, 2 μM, 0.4 μM, 0.08 μM, 0.016 μM, and 0.0032 μM RZ899058 working solutions; RZ597104 was gradiently diluted with PBS buffer to 50 μM, 10 μM, 2 μM, 0.4 μM, 0.08 μM, 0.016 μM, and 0.0032 μM RZ597104 working solutions.

[0368] siRNA chimera group: 2 μL / well of siRNA chimera working solution of each concentration was added to the above 12-well culture plate, equivalent to the final transfection concentration of each siRNA chimera being 100 nM, 20 nM, 4 nM, 0.8 nM, 0.16 nM, 0.032 nM, and 0.0064 nM (calculated as siRNA); RZ899058 + RZ597104 mixture group: RZ899058 working solution and RZ597104 working solution of the same concentration were added to the same well of the 12-well culture plate, equivalent to the final transfection concentrations of the RZ899058 + RZ597104 mixture group being 00 nM, 20 nM, 4 nM, 0.8 nM, 0.16 nM, 0.032 nM, and 0.0064 nM (calculated as siRNA), respectively. For each experimental group, set up two culture wells for each final transfection concentration; add 2 μL / well of PBS buffer to the other two culture wells as blank controls. Shake the culture plate to mix thoroughly. Continue incubating at 37°C, 5% CO2 in a cell culture incubator for 24 hours.

[0369] (2-3) Detection: Same as step (1-3) of Example 1.

[0370] IC50 curve fitting was performed using the four-parameter log(inhibitor) vs. response-variable slope (four parameters) model in GraphPad Prism 8.0. The IC50 concentration was calculated using the following formula:

[0371] X=10^(LogIC50-Log((Top-Bottom) / (50-Bottom)-1) / HillSlope). The parameters in the formula can be obtained from the GraphPad prism 8.0 analysis result table.

[0372] The test results are shown in Tables 18 and 19, and Figures 2 and 3.

[0373] Table 18 Inhibitory activity of target gene SOD1 / ANGPTL3 in primary mouse hepatocytes

[0374] Table 19 IC of target gene SOD1 / ANGPTL3 in primary mouse hepatocytes 50 value

[0375] The results showed that the IC50 values ​​of the RZ899 / 597-M2 group for gene inhibition of SOD1 and ANGPTL3 were 0.3655nM and 0.5364nM, respectively, and the IC50 values ​​of the RZ899 / 597-M4 group for gene inhibition of SOD1 and ANGPTL3 were 0.4762nM and 1.2998nM, respectively, both of which were better than the 0.4976nM and 2.1649nM of the RZ899058+RZ597104 mixture group.

[0376] Example 3

[0377] Evaluation of the inhibitory activity of nucleic acid chimeras against target genes ANGPTL3 and SOD1 in mice.

[0378] This example uses the in vivo target gene inhibitory activity assessment method to evaluate the inhibitory activity of multi-targeted siRNA chimeras RZ899 / 597-M2, RZ899 / 597-M4, and the RZ899058+RZ597104 mixture on the target genes SOD1 and ANGPTL3 in mice.

[0379] (3-1) Animal grouping, drug administration, and tissue sample collection

[0380] 6-8 week old C57BL / 6j mice were randomly divided into 4 groups according to body weight, with 15 mice in each group. The mice in each group were administered the above-mentioned siRNA chimeras by subcutaneous administration in the abdomen, wherein each mouse in the PBS control group was administered a dose of 5 mL / kg (calculated on a mouse basis), each mouse in the RZ899 / 597-M2 and RZ899 / 597-M4 experimental groups was administered a dose of 6 mg (calculated as siRNA) / kg (calculated as mouse basis) (equivalent to a dose of 3 mg (calculated as siRNA) / kg (calculated as mouse basis) for each target siRNA), and the RZ899058+RZ597104 mixture group was administered a mixture of 6 mg (calculated as siRNA) / kg (calculated as mouse basis) (equivalent to a dose of 3 mg (calculated as siRNA) / kg (calculated as mouse basis) for each target siRNA. For example, the day of administration was recorded as the first day (D0), and 5 mice in each group were killed on the 7th day (D7), the 28th day (D28), and the 56th day (D56) after administration. The animals were grossly dissected, and the liver tissues were collected and cut into several 2 mm 3 The small pieces were preserved with RNAlater.

[0381] (3-2) Detection

[0382] RNA extraction: An appropriate amount of liver tissue samples were removed from RNAlater and disrupted in a Tissuelyser II fully automatic tissue homogenizer for 60 seconds. Total RNA was extracted from each liver tissue sample using a fully automatic nucleic acid extractor and nucleic acid extraction kit from Zhejiang Hanwei Technology Co., Ltd. according to the instructions.

[0383] Reverse transcription reaction and real-time PCR detection: the same as steps (1-3) in Example 1.

[0384] (3-3) Analysis

[0385] The test results are shown in Table 20, Figures 4 and 5.

[0386] Table 20 Inhibitory activity of target gene SOD1 / ANGPTL3 in mice

[0387] The above results show that in mice, the mRNA inhibitory effects of the multi-targeted conjugates RZ899 / 597-M2 and RZ899 / 597-M4 on SOD1 and ANGPTL3 are comparable to or slightly better than those of the RZ899058+RZ597104 mixture group.

[0388] Example 4

[0389] Evaluation of the inhibitory activity of multi-target siRNA chimeras against superoxide dismutase 1 (SOD1) and angiopoietin-like 3 (ANGPTL3) in primary mouse hepatocytes.

[0390] In this example, the inhibitory activity of the multi-target siRNA chimeras RZ899 / 597-M16, RZ899 / 597-M16.2, RZ899 / 597-M21, and RZ899 / 597-M21.2, as well as the control siRNA mixture RZ899058 + RZ598104, against the target genes SOD1 and ANGPTL3 was evaluated using a mouse primary liver cell activity assessment method. The dual-target ligation scheme for RZ899 / 597-M16, RZ899 / 597-M16.2, RZ899 / 597-M21, and RZ899 / 597-M21.2 all involved linking the sense strand (SS) of one target to the antisense strand (AS) of the other, with the 5' end of the SS strand connected to the 3' end of the AS strand. The difference lies in the different linker and vector conjugation methods.

[0391] The primer sequence information used in this experiment is the same as Table 16.

[0392] The results of Example 4 demonstrate that the multi-targeted conjugates described in this study were all able to effectively reduce the mRNA expression of the target genes SOD1 and ANGPTL3. Specifically, RZ899 / 597-M16.2, RZ899 / 597-M21, and RZ899 / 597-M21.2 demonstrated comparable inhibitory activity against the target genes as the RZ899058 + RZ597104 mixture ( Figure 6 , Table 21).

[0393] Table 21 Inhibitory activity of the target gene SOD1 / ANGPTL3 in mouse primary hepatocytes after administration of siRNA chimeras

[0394] Example 5

[0395] Evaluation of the inhibitory activity of multi-target siRNA chimeras against SOD1 and ANGPTL3 in primary mouse hepatocytes.

[0396] This example evaluated the inhibitory activity of dual-target siRNA chimeras RZ899 / 597-M26, RZ899 / 597-M26.2, RZ899 / 597-M26.3, RZ899 / 597-M28, and RZ899 / 597-M28.2, as well as a control siRNA mixture RZ899058 + RZ598104, against the target genes SOD1 and ANGPTL3 using a mouse primary liver cell activity assessment method. The sense strands of these dual-target siRNA conjugates all have a click-chemistry linking mechanism where the 3' end of the sense strand of one target is linked to the 3' end of the sense strand of the other target. The difference lies in the carrier conjugation method.

[0397] Mouse primary hepatocyte isolation, cell culture, and transfection procedures were as described previously. The density of mouse primary hepatocytes was adjusted to 2 × 10 ^5 cells / mL and inoculated into a 12-well culture plate pre-coated with rat tail collagen type I. The volume of cell suspension added was 1000 μL / well, that is, the cell amount was 2×10 ^5 cells / well. Each group of multi-targeted siRNA conjugates was diluted with PBS to a working solution of 5 μM (in terms of siRNA). 2 μL / well of the multi-targeted siRNA conjugate working solution was added to the above-mentioned 12-well culture plate, respectively, of which the RZ899058+RZ597104 group was prepared by adding 2 μL / well of RZ899058 and 2 μL / well of RZ597104 to the same culture well. This is equivalent to the final transfection concentration of each multi-targeted siRNA conjugate in each well being 10 nM (in terms of siRNA), and 2 culture wells were set up for each multi-targeted siRNA conjugate. 2 μL / well of PBS was added to the other two culture wells as blank control wells. Shake the culture plate to mix evenly. Place the culture plate in a cell culture incubator at 37°C and 5% CO2 and continue to culture for 24 hours. RNA extraction and detection were as described above, and the primers were as shown in Example 4.

[0398] The results of Example 5 showed that the multi-target siRNA chimeras RZ899 / 597-M26, RZ899 / 597-M26.2, RZ899 / 597-M26.3, RZ899 / 597-M28, and RZ899 / 597-M28.2 were all able to effectively reduce the mRNA expression levels of the target genes SOD1 and ANGPTL3. Among them, RZ899 / 597-M26.3, RZ899 / 597-M28, and RZ899 / 597-M28.2 had higher inhibitory activity against dual-target genes compared to the control ( Figure 7 , Table 22).

[0399] Table 22 Inhibitory activity of target gene SOD1 / ANGPTL3 in primary mouse hepatocytes after administration of siRNA chimeras

[0400] Example 6

[0401] Evaluation of the inhibitory activity of multi-target siRNA chimeras against SOD1 and ANGPTL3 in mice.

[0402] This example evaluated the inhibitory activity of the siRNA chimera RZ899 / 597-M16.3, which simultaneously targets the SOD1 and ANGPTL3 genes, and a control siRNA mixture RZ899058 + RZ598104, against the target genes SOD1 and ANGPTL3 in C57BL / 6j mice. The dual-target linkage pattern of RZ899 / 597-M16.3 consisted of one target sense strand (SS) linked to the other target antisense strand (AS), with the 5' end of the SS strand connected to the 3' end of the AS strand.

[0403] 6-8 week old C57BL / 6j mice were randomly divided into 4 groups according to body weight, with 15 mice in each group. Each group of mice was given the above siRNA conjugate by subcutaneous administration in the abdomen. The dose of each mouse in the PBS control group was 5 mL / kg, and the dose of each mouse in the siRNA conjugate experimental group was 6 mg / kg (calculated as siRNA) in a volume of 5 mL / kg. The day of administration was recorded as day 0 (D0). After administration, 5 mice in each group were killed on the 7th day (D7), the 14th day (D14), and the 28th day (D28). The animals were grossly dissected, and the liver tissue was collected and cut into several 2 mm 3 Small pieces were preserved in RNAlater. For the experiment, appropriate liver tissue samples were removed and homogenized for 60 seconds using a Tissuelyser II automated tissue homogenizer. RNA was extracted and detected as described above, and gene expression differences were calculated using the ΔΔCt method. Primers were as described in Example 4.

[0404] The results of Example 6 showed that in mice, the multi-target conjugate RZ899 / 597-M16.3 had comparable inhibitory effects on the SOD1 gene at D7, D14, and D28 compared to the RZ899058+RZ597104 mixture, and had a higher inhibitory effect on the ANGPTL3 gene ( Figures 8-9 , Table 23).

[0405] Table 23 Inhibitory activity of target gene SOD1 / ANGPTL3 in mice after administration of siRNA chimeras

[0406] Example 7

[0407] Evaluation of the inhibitory activity of multi-target siRNA chimeras against SOD1 and ANGPTL3 in mice.

[0408] In this example, an in vivo mouse evaluation method was used to evaluate the inhibitory activity of siRNA chimeras RZ899 / 597-M26.3 and RZ899 / 597-M28.2, which simultaneously target SOD1 and ANGPTL3 genes, on the target genes SOD1 and ANGPTL3 in C57BL / 6j mice.

[0409] 6-8 week old C57BL / 6j mice were randomly divided into 4 groups according to body weight, with 15 mice in each group. Each group of mice was given the above-mentioned siRNA chimera by subcutaneous administration in the abdomen. The dose of each mouse in the PBS control group was 5 mL / kg, and the dose of each mouse in the siRNA chimera experimental group was 6 mg / kg (calculated as siRNA) in a volume of 5 mL / kg. The day of administration was recorded as day 0 (D0). After administration, 5 mice in each group were killed on the 7th day (D7), the 14th day (D14), and the 28th day (D28). The animals were dissected, and the liver tissue was collected and cut into several 2 mm 3 Small pieces were preserved in RNAlater. For the experiment, appropriate liver tissue samples were removed and homogenized for 60 seconds using a Tissuelyser II automated tissue homogenizer. RNA was extracted and detected as described above, and gene expression differences were calculated using the ΔΔCt method. Primers were as described in Example 4.

[0410] The results of Example 7 showed that in mice, RZ899 / 597-M26.3 had comparable inhibitory activity against the target genes SOD1 and ANGPTL3 compared to the RZ899058+RZ597104 mixture at D7, D14, and D28. RZ899 / 597-M28.2 exhibited greater inhibitory activity against both target genes SOD1 and ANGPTL3 compared to the RZ899058+RZ597104 mixture ( Figures 10-11 , Table 24).

[0411] Table 24 Inhibitory activity of target gene SOD1 / ANGPTL3 in mice after administration of siRNA chimeras

[0412] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A nucleic acid chimera, characterized in that: The nucleic acid chimera comprises a nucleotide unit I, and the nucleotide unit I has a structure shown in formula (101): Wherein, each X1, X2, X3 is independently selected from Each M1 independently contains a ligand capable of binding to a cell receptor; Each M2 is independently selected from a modified or unmodified nucleotide; each m is independently selected from 1, 2 or 3, and each n is independently selected from an integer of 0-3; Wherein, X1, X2, and X3 may be the same or different; Wherein, each Y1 and Y2 independently represents a sense strand of a double-stranded oligonucleotide, and each of the sense strands may correspond to the same target gene or different target genes; the correspondence means that the sense strand is identical or substantially identical to a continuous nucleotide segment in the mRNA of the target gene; j1, j2, j3 are independently selected from integers of 0-3, and j2≠0; k is selected from integers of 1-5.

2. The nucleic acid chimera according to claim 1, characterized in that The nucleic acid chimera further comprises a nucleotide unit II, wherein the nucleotide unit II comprises an antisense strand that is complementary or substantially complementary to the sense strand of the double-stranded oligonucleotide, the number of the antisense strands is equal to the number of the sense strands, and each of the antisense strands is at least partially complementary to the sense strand corresponding thereto to form a duplex region; wherein the antisense strand is complementary or at least partially complementary to the mRNA of at least one target gene; Optionally, the antisense strands are respectively and independently complementary or at least partially complementary to mRNAs of two or at least two target genes; Optionally, the sense strand and the antisense strand of the double-stranded oligonucleotide each comprise at least partially modified nucleotides; Optionally, the sense strand and / or the antisense strand comprises at least one phosphorothioate bond; Optionally, the sense strand and the antisense strand comprise at least 15 consecutive nucleotides, wherein the antisense strand is complementary to the mRNA of the target gene; Optionally, after in vivo administration, the chimera is optionally cleaved at X2 or at the position connected to X2, thereby decomposing the independent double-stranded oligonucleotide parts.

3. The nucleic acid chimera according to claim 1, characterized in that k is selected from 1; Optionally, j1 is selected from 0, j2 is selected from 3, and j3 is selected from 0; Optionally, j1 is selected from 0, j2 is selected from 3, and j3 is selected from 3; Optionally, j1 is selected from 1, j2 is selected from 1, and j3 is selected from 1; Optionally, j1 is selected from 2, j2 is selected from 2, and j3 is selected from 2; Optionally, j1 is selected from 3, j2 is selected from 3, and j3 is selected from 0; Optionally, j1 is selected from 3, j2 is selected from 0, and j3 is selected from 3; Optionally, m is selected from 1 or 2 or 3, and each n is independently selected from 0 or 1; Preferably, m is selected from 1 and n is selected from 0; or, m is selected from 1 and n is selected from 1.

4. The nucleic acid chimera according to any one of claims 1 to 3, characterized in that The modified nucleotides are independently selected from abasic nucleotides, 2'-halogenated modifications, 2'-deoxy modified nucleotides or 2'-O-(CH2) n2 -R1 modified nucleotide, or quasi-nucleotide; the quasi-nucleotide is selected from one or more of PNA, MNA, BNA, LNA, GNA, TNA and UNA; wherein n2 is selected from 0, 1 or 2; R1 is selected from optionally substituted C 1-6 Alkyl, optionally substituted C 1-6 Alkoxy or -Si(R 1a )3, each R 1a are independently selected from optionally substituted C 1-6 Alkyl or optionally substituted C 1-6 Alkoxy, the substitution means that one or more hydrogen atoms on the alkyl or alkoxy are replaced by a substituent; optionally, the substituent is independently selected from C1-C3 alkyl, C1-C3 alkoxy or halogen; Optionally, 2'-O-(CH2) n2 -R1 is selected from 2'-O-CH3, 2'-O-CH2-CH3, 2'-O-TBDMS, 2'-O-TIPS, 2'-O-TOM, 2'-O-CH2-O-CH2-CH3, 2'-O-CH2-O-CH2-CF3 or 2'-O-CH2-CH2-O-CH3; Optionally, the sense strand and the antisense strand of the double-stranded oligonucleotide are complementary or substantially complementary to form a duplex, as shown in the following formula: SS:5'-(N)a'-(X)p'-(N)b'-(X)q'-(N)c'-(X)r'-(N)d'-3' AS:3'-(N)a-(X)p-(N)b-(X)q-(N)c-5', Wherein, SS represents the sense strand, and AS represents the antisense strand; all nucleotides of the sense strand and the antisense strand are modified nucleotides; The Ns each independently represent the following modified nucleotides: 2'-O-methyl modified nucleotides, 2'-fluorine modified nucleotides, 2'-deoxy-modified nucleotides; The Xs are each independently a 2′-O-methoxyethyl modified nucleotide, a 2′-O-methyl modified nucleotide or a 2′-O(CH2) n3 OR3 substituted modified nucleotides; wherein n3 is 1 or 2, R3 is selected from substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy; optionally, the substituent is selected from halogen, C1-C3 alkyl, C1-C3 alkoxy, hydroxyl, amino; Said a, a', p, p', b, b', q, q', c, c', r', d' each independently represents the number of nucleotides, wherein: a' is selected from an integer of 3-8; p' is selected from an integer of 0-3; b' is selected from an integer of 4-13; q' is selected from an integer of 0-4; c' is selected from an integer of 3-9; r' is selected from an integer of 0-3; d' is selected from an integer of 0-9; a is selected from an integer of 4-7; p is selected from an integer of 0-1; b is selected from an integer of 4-8; q is selected from an integer of 0-4; c is selected from an integer of 6-10; and p', q', r', p, q are not 0 at the same time, and 0≤q'+r'≤4; Preferably, a' is selected from an integer of 3-8; p' is selected from 0 or 1; b' is selected from an integer of 4-13; q' is selected from 0 or 1; c' is selected from an integer of 3-9; r' is selected from 0 or 1; d' is selected from an integer of 1-8; a is selected from an integer of 4-7; p is selected from 1; b is selected from an integer of 4-8; q is selected from 0 or 1; c is selected from an integer of 6-10; Preferably, the nucleotide N is selected from 2′-O-methyl modified nucleotides and 2′-fluoro-modified nucleotides, and the nucleotide X is selected from 2′-O-methoxyethyl modified nucleotides and 2′-O-methyl modified nucleotides, and more preferably 2′-O-methoxyethyl modified nucleotides; Optionally, the sense strand is 17-21 nucleotides long, and the antisense strand is 19-23 nucleotides long; Optionally, in the duplex, in the direction from the 5' end to the 3' end, at least three nucleotides of the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides or 2'-O-methoxyethyl-modified nucleotides; the nucleotides at positions 2, 6, 14 and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, any one of the nucleotides at positions 9, 10, 11 and 12 is selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides or 2'-O-methoxyethyl-modified nucleotides; Optionally, in the duplex, in the direction from the 5' end to the 3' end, at least three nucleotides of the nucleotides at positions 7-10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, at most two nucleotides of the nucleotides at positions 5, 12, and 18 are selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, any one of the nucleotides at positions 9-12 is selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; Optionally, each nucleotide of the double-stranded oligonucleotide is independently selected from modified nucleotides, wherein in the duplex, the modifications of the sense strand and the antisense strand are selected from any one of the following (1)-(8): (1) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 9, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; (2) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 12, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; (3) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 9, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; (4) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 12, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; (5) In the direction from the 5' end to the 3' end, the nucleotide at position 5, or the 12th position, or the 18th position of the nucleotide sequence in the sense strand is selected from 2'-O-methoxyethyl modified nucleotides, the nucleotides at positions 7 to 10 are selected from 2'-fluoro modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl modified nucleotides; the nucleotides at positions 2, 6, 9, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl modified nucleotides; (6) In the direction from the 5' end to the 3' end, the nucleotide at position 5, or the 12th position, or the 18th position of the nucleotide sequence in the sense strand is selected from 2'-O-methoxyethyl modified nucleotides, the nucleotides at positions 7 to 10 are selected from 2'-fluoro modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl modified nucleotides; the nucleotides at positions 2, 6, 12, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl modified nucleotides; (7) In the direction from the 5' end to the 3' end, the nucleotide at position 5, or the 12th position, or the 18th position of the nucleotide sequence in the sense strand is selected from 2'-O-methoxyethyl modified nucleotides, the nucleotides at positions 7 to 10 are selected from 2'-fluoro modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl modified nucleotides; the nucleotides at positions 2, 6, 9, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl modified nucleotides; (8) In the direction from the 5' end to the 3' end, the nucleotide at position 5, or the 12th position, or the 18th position of the nucleotide sequence in the sense strand is selected from 2'-O-methoxyethyl modified nucleotides, the nucleotides at positions 7 to 10 are selected from 2'-fluoro modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl modified nucleotides; the nucleotides at positions 2, 6, 12, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl modified nucleotides; Optionally, in the duplex, in the direction from the 5' end to the 3' end, the terminal nucleotides at the 5' end of the sense strand contain 1 or 2 consecutive phosphorothioate bonds; and / or, the 5' end and the 3' end of the antisense strand each independently contain 1 or 2 consecutive phosphorothioate bonds.

5. The nucleic acid chimera according to any one of claims 1 to 3, characterized in that: The nucleotide unit I has a structure shown in formula (102): The definition of each substituent is as defined in any one of claims 1 to 3.

6. The nucleic acid chimera according to any one of claims 1 to 3, characterized in that: The nucleotide unit I has a structure shown in formula (103): The definition of each substituent is as defined in any one of claims 1 to 3.

7. The nucleic acid chimera according to claim 1, characterized in that Each M1 is independently selected from a ligand capable of binding to an asialoglycoprotein receptor.

8. The nucleic acid chimera according to any one of claims 1 to 7, characterized in that: Each M1 is independently selected from the structure represented by formula (201a) or formula (201b) or an isomer thereof or a pharmaceutically acceptable salt thereof: Wherein, when M1 is located at the terminal position of the nucleotide unit I, M1 is selected from the structure represented by formula (201a) or its isomer or a pharmaceutically acceptable salt thereof; When M1 is not located at the end of the nucleotide unit I, M1 is selected from the structure represented by formula (201b) or its isomer or a pharmaceutically acceptable salt thereof; wherein A is selected from an optionally substituted 4-10 membered aliphatic ring; X is selected from NH, O or S; L1 is selected from wherein h is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; Each R is independently selected from H, optionally substituted C 1-6 Alkyl or optionally substituted C 1-6 Alkoxy; L2 is selected from optionally substituted C2-C 20 Alkylene or R La and R Lb are independently selected from optionally substituted C 1-10 Alkylene, i is selected from 1, 2, 3, 4 or 5; Y is selected from O or S; M 1a Selected from galactose or its derivatives; Z is selected from hydroxyl or thiol; p is selected from 1, 2 or 3; q is selected from 1, 2 or 3; Optionally, A is selected from Optionally, A is selected from Optionally, X is selected from NH; Optionally, L1 is selected from Optionally, each R is independently selected from H; Optionally, L2 is selected from C 1-10 Alkylene or Among them, R La and R La Independently selected from C 1-5 Alkylene, i is 1, 2 or 3; Optionally, i is selected from 1; Optionally, each L2 is independently selected from Optionally, Y is selected from O; Optionally, M 1a Selected from galactosamine derivatives; Optionally, M 1a Selected from Optionally, Z is selected from hydroxyl; Optionally, p is selected from 1; Optionally, q is selected from 1.

9. The nucleic acid chimera according to claim 8, characterized in that Each M1 is independently selected from the structure represented by formula (202a) or formula (202b) or an isomer thereof or a pharmaceutically acceptable salt thereof: Wherein, when M1 is located at the terminal position of the nucleotide unit I, M1 is selected from the structure represented by formula (202a) or its isomer or a pharmaceutically acceptable salt thereof; When M1 is not located at the end of the nucleotide unit I, M1 is selected from the structure represented by formula (202b) or its isomer or a pharmaceutically acceptable salt thereof.

10. The nucleic acid chimera according to claim 8, characterized in that Each M1 is independently selected from the structure represented by formula (203a) or formula (203b) or an isomer thereof or a pharmaceutically acceptable salt thereof: Wherein, when M1 is located at the terminal position of the nucleotide unit I, M1 is selected from the structure represented by formula (203a) or its isomer or a pharmaceutically acceptable salt thereof; When M1 is not located at the end of the nucleotide unit I, M1 is selected from the structure represented by formula (203b) or its isomer or a pharmaceutically acceptable salt thereof.

11. The nucleic acid chimera according to claim 8, characterized in that Each M1 is independently selected from any of the following structures or isomers thereof or pharmaceutically acceptable salts thereof: Wherein, when M1 is located at the terminal position of the nucleotide unit I, M1 is selected from or its isomer or pharmaceutically acceptable salt, or or an isomer thereof or a pharmaceutically acceptable salt thereof; When M1 is not located at the end of the nucleotide unit I, M1 is selected from or its variant or a pharmaceutically acceptable salt thereof, or or an isomer thereof or a pharmaceutically acceptable salt thereof.

12. The nucleic acid chimera according to claim 1, characterized in that The nucleotide unit I has a structure represented by formula (104) or an isomer thereof or a pharmaceutically acceptable salt thereof: The definitions of the substituents are the same as those in claims 8-11.

13. The nucleic acid chimera according to claims 1 to 12, characterized in that: The nucleic acid chimera has a structure represented by formula (105) or an isomer thereof or a pharmaceutically acceptable salt thereof: Preferably, Z is OH; Among them, AS represents the antisense strand of the double-stranded oligonucleotide molecule; SS represents the sense strand of the double-stranded oligonucleotide molecule.

14. The nucleic acid chimera according to claims 1-13, characterized in that: The nucleic acid chimera has a structure represented by formula (106) or an isomer thereof or a pharmaceutically acceptable salt thereof: Among them, AS represents the antisense strand of the double-stranded oligonucleotide molecule; SS represents the sense strand of the double-stranded oligonucleotide molecule; Z is OH; Optionally, j1 is selected from 0 or 1, j2 is selected from an integer of 1-3, and k is selected from 1.

15. A nucleic acid chimera, characterized in that The nucleic acid chimera comprises a nucleotide unit III, wherein the nucleotide unit III has a structure represented by formula (301), or an isomer thereof, or a pharmaceutically acceptable salt thereof: Wherein, each M1 is independently selected from the structure shown in formula (201b) or its isomer or a pharmaceutically acceptable salt thereof: A, X, L1, R, L2, Y, M 1a , p, q as defined in any one of claims 1 to 14; wherein Y3 is selected from the sense strand and Y4 is selected from the sense strand, or, Y3 is selected from the antisense strand and Y4 is selected from the sense strand; Y3 and Y4 in the nucleotide unit III correspond to the same target gene or different target genes; the correspondence means that the sense strand is identical or substantially identical to a continuous nucleotide stretch in the mRNA of the target gene, or the antisense strand is complementary or substantially complementary to a continuous nucleotide stretch in the mRNA of the target gene; j4, j5, j6, and j7 are each independently selected from integers of 0-3, and at least one of j6 and j7 is not 0; when Y3 is selected from the sense strand, at least one of j4 and j5 is not 0; Wherein, when Y3 is selected from the sense strand and Y4 is selected from the sense strand, the 5' end of Y3 and the 5' end of Y4 are conjugated, or the 3' end of Y3 and the 3' end of Y4 are conjugated; and Dt has the structure shown in (401), or a stereoisomer thereof, or a tautomer thereof: Wherein, A, X, L1, R, L2, p, q are as defined in any one of claims 1-14; Dt' is selected from When Y3 is selected from the antisense strand and Y4 is selected from the sense strand, the Y3 antisense strand and the Y4 sense strand in the nucleotide unit III do not form a base complementary pairing; the 5' end of Y3 and the 3' end of Y4 are conjugated to each other, or the 3' end of Y3 and the 5' end of Y4 are conjugated to each other; and Dt is selected from a structure not having the structure shown in (501), or a stereoisomer thereof, or a tautomer thereof: wherein t1 is selected from an integer of 0-5, t2 is independently selected from 0 or 1, Base is selected from a nucleoside base A, U, G, C or T; and when t1 is When 0, j6 is not 0.

16. The nucleic acid chimera according to claim 15, characterized in that When Y3 is selected from the sense strand and Y4 is selected from the sense strand, Dt has the structure shown in (402), or a stereoisomer thereof, or a tautomer thereof: Optionally, Dt' is selected from Optionally, when Y3 is selected from the sense strand and Y4 is selected from the sense strand, Dt has the structure shown in (403), or a stereoisomer thereof, or a tautomer thereof: Optionally, when Y3 is selected from the sense strand and Y4 is selected from the sense strand, the nucleotide unit III has a structure represented by formula (301a), or an isomer thereof, or a pharmaceutically acceptable salt thereof: wherein each Za is independently selected from a hydroxyl group or a thiol group; Optionally, when Y3 is selected from the sense strand and Y4 is selected from the sense strand, the nucleic acid chimera further comprises two antisense strands, the nucleotide sequence of one antisense strand being at least partially reverse complementary to the nucleotide sequence of the sense strand of Y3 in nucleotide unit III, and the nucleotide sequence of the other antisense strand being at least partially reverse complementary to the nucleotide sequence of the sense strand of Y4 in nucleotide unit III.

17. The nucleic acid chimera according to claim 15, characterized in that When Y3 is selected from the antisense strand and Y4 is selected from the sense strand, the nucleotide unit III has a structure represented by formula (301b), or an isomer thereof, or a pharmaceutically acceptable salt thereof: wherein each Zb is independently selected from a hydroxyl group or a thiol group; The Y3 antisense strand and the Y4 sense strand in the nucleotide unit III correspond to the same target gene or different target genes, and the Y3 antisense strand and the Y4 sense strand in the nucleotide unit III do not form complementary base pairing; Optionally, when Y3 is selected from the antisense strand and Y4 is selected from the sense strand, the nucleotide unit III has a structure represented by formula (302b), or an isomer thereof, or a pharmaceutically acceptable salt thereof: wherein each Zb is independently selected from a hydroxyl group or a thiol group; The Y3 antisense strand and the Y4 sense strand in the nucleotide unit III correspond to the same target gene or different target genes, and the Y3 antisense strand and the Y4 sense strand in the nucleotide unit III do not form complementary base pairing; Optionally, when Y3 is selected from the antisense strand and Y4 is selected from the sense strand, the nucleic acid chimera comprises two nucleotide units III, and the Y3 antisense strand and the Y4 sense strand in the same nucleotide unit III correspond to the same target gene or different target genes, and the Y3 antisense strand and the Y4 sense strand in the same nucleotide unit III do not form a complementary base pairing; The nucleotide sequence of the antisense strand of Y3 in the first nucleotide unit III and the nucleotide sequence of the sense strand of Y4 in the second nucleotide unit III are at least partially reverse complementary; The nucleotide sequence of the sense strand of Y4 in the first nucleotide unit III and the nucleotide sequence of the antisense strand of Y3 in the second nucleotide unit III are at least partially reverse complementary; Optionally, when Y3 is selected from the antisense strand and Y4 is selected from the sense strand, the nucleic acid chimera further comprises a sense strand and an antisense strand, wherein the nucleotide sequence of the sense strand and the nucleotide sequence of the antisense strand of Y3 in nucleotide unit III are at least partially reverse complementary; The nucleotide sequence of the sense strand is at least partially reverse complementary to the nucleotide sequence of the sense strand of Y4 in nucleotide unit III.

18. A composition comprising the nucleic acid chimera according to any one of claims 1 to 17 and a physiologically acceptable excipient.

19. The nucleic acid chimera according to any one of claims 1 to 17, for use in treating at least one disease or condition caused by dysregulated gene expression.

20. Use of the nucleic acid chimera according to any one of claims 1 to 17 in the preparation of a medicament for treating a disease or condition caused by dysregulated expression of at least one gene.

21. A method of treating a disease or disorder comprising administering to an individual in need of treatment the nucleic acid chimera of any one of claims 1-17.

22. The method of claim 21, wherein the nucleic acid chimera is administered to the individual subcutaneously or intravenously.

23. The method according to claim 21 or 22, wherein after in vivo administration, the nucleic acid chimera dissociates to obtain at least two independent double-stranded oligonucleotide molecules, each targeting a portion of mRNA transcribed from one or more target genes, which may be the same or different.

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