Oligonucleotide conjugate and use thereof

Abstract

Provided are an oligonucleotide conjugate and use thereof. The oligonucleotide conjugate has a structure represented by formula II: C-(X) n-(B) k-A (formula II), wherein C is a group with relatively good lipid solubility; X represents an oligonucleotide for regulating gene expression levels, and n≥1; B represents a linking arm, and k≥1; A represents a group capable of reacting with a free active group of albumin to form a covalent bond. The oligonucleotide conjugate can have significantly higher in vivo oligonucleotide delivery efficiency and good in vivo stability and enables targeted delivery of the oligonucleotide to multiple extrahepatic tissues, such as hepatic tissue, renal tissue, and proximal convoluted tubule tissue, in vivo.

Description

Oligonucleotide conjugates and uses thereof

[0001] Cross-reference to related applications

[0002] This patent application claims the priority benefit of Chinese patent application No. CN2024105301278, filed on April 29, 2024, the entire contents of which are incorporated herein for all purposes. TECHNICAL FIELD

[0003] The present application belongs to the technical field of biopharmaceuticals, and specifically relates to an oligonucleotide conjugate and a delivery method or system for oligonucleotides, and more specifically, to an oligonucleotide conjugate capable of being coupled with albumin, and an oligonucleotide delivery system that achieves delivery of oligonucleotides by coupling with albumin. BACKGROUND

[0004] Nucleic acids are the genetic material of living organisms, and can directly guide protein synthesis through transcription or reverse transcription, translation, or can play an important regulatory role in protein synthesis. Oligonucleotides are a general term for short-chain nucleotide compounds. Oligonucleotide drugs are another type of new drug designed to target pathogenic RNA genes, following small molecule drugs and protein drugs, including antisense oligonucleotides (ASO), small interfering RNA (siRNA), micro-RNA (miRNA), and aptamers, etc. Compared with traditional drugs, oligonucleotide drugs have the advantages of high efficiency, specificity, long-acting, targeting non-druggable targets, not easy to develop drug resistance, short development cycle, etc. In addition, oligonucleotide drugs have a wide range of indications, including tumors, viral infectious diseases, kidney diseases, cardiovascular diseases, immune diseases, metabolic diseases, etc.

[0005] Due to the selective permeability of the cell membrane, highly negatively charged oligonucleotide drugs are difficult to pass through the membrane to reach the intracellular, and oligonucleotide drugs are easily degraded in the blood and body, with low stability, which seriously hinders the development and application of oligonucleotide drugs. Therefore, the delivery carrier problem and the stability problem are great challenges faced by the development of oligonucleotide drugs.

[0006] The first siRNA drug Patisiran of Alnylam Pharmaceuticals was approved for marketing in 2018, and LNP became the first carrier for successful delivery of siRNA; in 2019, Alnylam successfully developed another siRNA delivery carrier trivalent N-acetylgalactosamine (GalNAc). Compared with LNP, GalNAc targets the delivery of siRNA by binding to the receptor ASGPR, has higher specificity, longer drug efficacy and no hepatotoxicity. However, both of the two carriers deliver siRNA into liver tissue parenchymal cells; the lack of extrahepatic targeting delivery system limits the siRNA treatment of non-liver tissue diseases.

[0007] In the past 50 years, the related research on the in vivo targeted delivery of polypeptide small molecule drugs using serum albumin as a carrier has been gradually reported in large quantities. For example, the polypeptide drug Exenatide can bind to serum albumin in vivo to achieve targeted delivery of Exenatide and improve the in vivo stability of Exenatide. However, the albumin delivery carrier has great limitations, such as low delivery efficiency and limited delivery tissue types.

[0008] In summary, improving the in vivo delivery efficiency of oligonucleotide drugs, optimizing the multi-tissue delivery ability of albumin carriers for oligonucleotide drugs, and improving the in vivo stability of oligonucleotide drugs are the problems to be solved for expanding the indications of oligonucleotide drugs and benefiting non-liver tissue disease patients with oligonucleotide drugs. SUMMARY

[0009] The purpose of the present application is to provide an oligonucleotide drug delivery method or delivery system, and relates to an oligonucleotide conjugate and its application. The present application solves the technical problems of low in vivo delivery efficiency and narrow tissue delivery range of oligonucleotide drug albumin conjugates by designing and optimizing the conjugate structure, enhances the in vivo stability of oligonucleotide drugs, and realizes the in vivo efficient delivery of oligonucleotide drugs and the delivery of liver tissues and extrahepatic tissues.

[0010] In a first aspect, the present application provides an oligonucleotide conjugate having the structure shown in Formula I: n -(B) k -A (Formula I)

[0011] wherein:

[0012] X represents an oligonucleotide for regulating the expression level of a gene, n≥1;

[0013] B represents a connecting arm, k≥1;

[0014] A represents a group capable of reacting with the free active group of albumin to form a covalent bond.

[0015] In the present invention, the free active group is selected from one or more of the following groups: thiol, primary amine, and hydroxyl.

[0016] Further, the A can be selected from one or more of the following groups: maleimide and its derivative groups, divinyl sulfone and its derivative groups, acrylamide and its derivative groups, a-cyanoacrylamide and its derivative groups, 2-butynoamide and its derivative groups, carboxylic acid and its derivative groups.

[0017] Preferably, the A is a maleimide derivative group; the structure of the maleimide derivative group is as follows: or,

[0018] Preferably, the A is a divinyl sulfone derivative group; further preferably, the divinyl sulfone derivative group is selected from one or more of the following compounds:

[0019] or,

[0020] Preferably, the A is a carboxylic acid and its derivative group; the carboxylic acid and its derivative group includes carboxylic acid activated ester and its derivative group.

[0021] Further preferably, the A can be selected from one or more of the following groups:

[0022] In formula I, B is a connecting arm, selected from a non-cleavable linker or a cleavable linker.

[0023] In the present invention, the B can be -(B1) j -(B2) q -(B3) p -(B4) m -(B5) h -;

[0024] wherein: the B1 is selected from a C18-containing group; the B2 is selected from a S-S bond-containing group; the B3 is selected from an NH2amino-containing group; the B4 is selected from a polypeptide-containing group; the B5 is selected from a multiple C atom or O atom-containing group; the B5 end is connected to A; J, q, p, m, h are each independently selected from an integer from 0 to 20, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and the like.

[0025] Preferably, J = an integer from 0 to 20; q = 0 or 1; p = 0 or 1; m = 0 or 1; h = 0 or 1.

[0026] Preferably, B1, B2, B3 are each independently selected from the group consisting of optionally substituted linear or branched saturated or unsaturated alkyl, optionally substituted linear or branched saturated or unsaturated heteroalkyl.

[0027] In some embodiments, the number of atoms of the saturated or unsaturated alkyl or heteroalkyl is 1-100 (such as 1-50, such as 1-30, such as 1-20, such as 1-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30), the heteroatom(s) in the heteroalkyl is selected from one, two, three or four of O, S, N, P.

[0028] In some embodiments, the substituent(s) in the optionally substituted corresponding group(s) is / are selected from Ra.

[0029] In particular, Ra can be selected from carbonyl, hydroxyl, halogen (such as F, Cl, Br, I), amino, alkylamino (such as C 1-3 alkyl-NH-, (C 1-3 alkyl)2-N-), alkoxy (such as C 1-3 alkoxy, such as methoxy, ethoxy), hydroxyalkyl (such as hydroxyC 1-3 alkyl-, such as -CH2OH). Preferably, Ra is -CH2OH.

[0030] In some embodiments, B1, B2, B3 are each independently selected from the group consisting of: -(R1) j1 -(R2) j2 -(R3) j3 - wherein each R1, R2, R3 can be the same or different, R1, R2, R3 are each independently selected from -CH2CH2O-, -CH2-, -C(O)-, -O-, -NH-, -P(O)(OH)O-, -S-S-; j1, j2, j3 are each independently selected from an integer from 0-100 (such as 0-50, 0-30, 0-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20); R1, R2, R3 are optionally substituted with Ra.

[0031] In some embodiments, B1 is selected from -(R1) j1 - each R1 is each independently selected from -CH2CH2O-, -CH2-.

[0032] In some embodiments, B1 is selected from -(R1) j1 -P(O)(OH)O-(R3) j3each R1, R3 is independently selected from -CH2CH2O-, -CH2-.

[0033] In some embodiments, B2 is selected from -(R1) j1 -S-S-(R3) j3 -O-, -(R1) j1 -P(O)(OH)O-, -P(O)(OH)O-, each R1and R3is independently selected from -CH2-.

[0034] In some embodiments, B3 is selected from -(R1) j1 -NH-, each R1is independently selected from -CH2-.

[0035] Preferably, said B1 group is a bond, or

[0036] Preferably, said B2 group is a bond,

[0037] Preferably, said B3 group is a bond,

[0038] Preferably, said B4 is selected from a bond, or

[0039] Preferably, said B5 group is selected from -(R4)m1-(R5)m2-(R6)m3-, wherein each R4, R5, R6may be the same or different, R4, R5, R6is independently selected from -C(O)-, -CH2-, -C(O)CH2-, -NH-, -CH2CH2O-, C3-C8cycloalkylene (such as cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane), C6-C14arylene (such as phenylene, naphthylene), 3-8 membered heterocyclylene (such as azetidinylene, tetrahydrofuranylene, tetrahydropyrrolylene, morpholylene, piperidinylene, piperazinylene, etc.), 5-8 membered heteroarylene (such as furanylene, thienylene, pyrrolylene, pyrazolylene, pyridinylene, pyrimidinylene, etc.); m1, m2, m3 is independently selected from an integer of 0-20 (such as an integer of 0-10, such as an integer of 0-5, such as an integer of 0-3, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10).

[0040] In some embodiments, R4, R6are each independently selected from -C(O)-, -CH2-, -C(O)CH2-, -NH-, -CH2CH2O-, R5is selected from C3-C8 cycloalkylene (such as cyclopropanediyl, cyclobutanediyl, cyclopentanediyl, cyclohexanediyl, cycloheptanediyl).

[0041] In some embodiments, B5is selected from a bond, -C(O)-C 3-8 cycloalkyl-C 1-6 alkyl, preferably -C(O)-C 5-6 cycloalkyl-C 1-3 alkyl.

[0042] For example, said B5may be a bond, or wherein m4= 0-12.

[0043] In particular, said B can be selected from one or more of the following compounds:

[0044] In formula I, the connecting arm B can be covalently linked to the oligonucleotide X through a phosphorothioate or a phosphorothioate .

[0045] In the present application, said X is an oligonucleotide for regulating the expression level of a gene, which is used to inhibit the expression of a target protein by mediating the degradation of the mRNA of the target gene, or to achieve the regulation of the selective splicing of pre-mRNA by steric hindrance, so as to ultimately achieve the purpose of disease treatment.

[0046] Said oligonucleotide for regulating the expression level of a gene is siRNA, ASO or miRNA.

[0047] The sequence of said oligonucleotide is at least partially complementary to the mRNA of a target gene, and U in the sequence of said oligonucleotide can be replaced by T.

[0048] Preferably, when n≥2, said (X) n is formed by at least 2 oligonucleotides (in series or in parallel).

[0049] In some embodiments, said X is siRNA, said siRNA comprising a sense strand and an antisense strand, said sense strand and antisense strand being partially or completely complementary.

[0050] Preferably, said sense strand and antisense strand have 15-25 nucleotides of complementarity (such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 nucleotides of complementarity).

[0051] In some embodiments, each of the sense strand and the antisense strand independently comprises 13-40 nucleotides (e.g., 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 nucleotides).

[0052] When the oligonucleotide X is an siRNA, the coupling site of the C on the siRNA is: coupling at the 5' end or the 3' end of the sense strand of the siRNA; or coupling at a certain nucleotide of the sense strand of the siRNA, including but not limited to coupling at the sugar ring or the base thereof.

[0053] In some embodiments, the B is linked to the 5' end or the 3' end of the sense strand of the siRNA.

[0054] Alternatively, when the X is an ASO or an miRNA, the coupling site of the B on the ASO or the miRNA is: coupling at the 5' end and the 3' end of the single strand of the ASO or the miRNA, or coupling at a certain nucleotide of the single strand of the ASO or the miRNA, including but not limited to coupling at the sugar ring or the base thereof.

[0055] Further, the oligonucleotide conjugate provided by the present application further comprises a modified group C linked to the X; the oligonucleotide conjugate has a structure shown in Formula II: C-(X) n -(B) k -A (Formula II)

[0056] wherein the definitions of X, B, A, n, and k are the same as defined above for Formula I.

[0057] The C is a group with better liposolubility, and is selected from one or more of saturated alkanes, unsaturated alkanes, saturated fatty acids, unsaturated fatty acids, and cholesterols with different chain lengths.

[0058] In the present application, when the oligonucleotide X is an siRNA, the coupling site of the C on the siRNA is: coupling at the 5' end or the 3' end of the sense strand of the siRNA; or coupling at a certain nucleotide of the sense strand of the siRNA, including but not limited to coupling at the sugar ring or the base thereof.

[0059] Alternatively, when the oligonucleotide X is an ASO or an miRNA, the coupling site of the C on the ASO or the miRNA is: coupling at the 5' end or the 3' end of the single strand of the ASO or the miRNA; or coupling at a certain nucleotide of the single strand of the ASO or the miRNA, including but not limited to coupling at the sugar ring or the base thereof.

[0060] Preferably, the C can have (M) e-(N) w -P- represents a structure wherein:

[0061] said M has a structure of Q-Linker 1 or said Q represents a saturated alkane having at least 8 carbon atoms, a C10-C30 unsaturated alkane comprising 1-5 unsaturated double bonds, a C10-C30 fatty acid having a double bond content of < 5, or a combination thereof; or, said Q is

[0062] In some embodiments, Q is selected from optionally substituted R or R-C(O)-, wherein R is selected from a C7-C30 straight chain or branched chain saturated or unsaturated alkane, a C7-C30 straight chain or branched chain saturated or unsaturated fatty acid, preferably C7-C24, more preferably C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24.

[0063] In some embodiments, said straight chain or branched chain unsaturated alkane or straight chain or branched chain unsaturated fatty acid comprises 1-10 -C=C- and / or -C≡C-, preferably comprises 1-6, more preferably comprises 1, 2, 3, 4, 5, or 6.

[0064] In some embodiments, R is selected from a straight chain saturated alkane, a straight chain unsaturated alkane, a straight chain saturated fatty acid, a straight chain unsaturated fatty acid.

[0065] said Linker 1 can further have a structure of -L2-L3-, wherein, -L2- is selected from optionally substituted 1-50 atom straight chain or branched chain saturated or unsaturated alkylene, optionally substituted 1-50 atom straight chain or branched chain saturated or unsaturated heteroalkylene, the heteroatoms in said heteroalkylene are selected from one or more of O, N, S, P, the optional substituents are selected from Ra; -L3- is selected from a bond, a 3-8 membered heterocyclyl, a 5-6 membered heteroaryl.

[0066] In some embodiments, -L2- is selected from -NH-(CH2CH2O)m4-C 1-3 alkyl-, -N(C 1-3 alkyl)-(CH2CH2O)m4-C 1-3 alkyl-, -NH-(CH2CH2O)m4-CH2CH2-, -N(C 1-3 alkyl)-(CH2CH2O)m4-CH2CH2-, -O-C 1-12 alkyl-, -NH-C 1-12 alkyl-, -N(C 1-3 alkyl)-C 1-12alkyl-, -(CH2CH20)m5-, optionally substituted -C

[0067] In some embodiments, the Linker 1 has the formula:

[0068] In some embodiments, the N has the formula: -N1-N2-N3-N4-N5-, wherein,

[0069] -N1- is selected from the group consisting of a bond, optionally substituted -C 1-20 alkyl-, optionally substituted heteroalkylene of 1-20 atoms;

[0070] -N2- is selected from the group consisting of a bond, optionally substituted -NH-C 1-8 alkyl-NH-, optionally substituted -NH-C 1-8 alkyl-NH-C 1-6 alkyl-, optionally substituted -NH-C 1-8 alkyl-NH-1-6 membered heteroalkyl-;

[0071] -N3- is selected from the group consisting of a bond, optionally substituted -C 1-6 alkyl-,

[0072] -N4- is selected from the group consisting of a bond, optionally substituted -C 1-6 alkyl-, optionally substituted heteroalkylene of 1-6 atoms;

[0073] -N5- is selected from the group consisting of a bond, optionally substituted divalent branching group, optionally substituted trivalent branching group;

[0074] wherein the heteroatoms in the heteroalkylene are selected from one, two, three, or four of O, N, P, S, and the optional substituents are selected from Ra.

[0075] In some embodiments, -N1- is selected from the group consisting of optionally substituted -C 1-6 alkyl-, -(CH2CH20)m5-, optionally substituted -C 1-6 alkyl-(CH2CH20)m5-, optionally substituted -C 1-6 alkyl-(CH2CH20)m5-C 1-6 alkyl-, -(CH2CH20)m5-C 1-6 alkyl-; -N2- is selected from the group consisting of a bond, optionally substituted -NH-C(O)-C 1-6 alkyl-C(O)-NH-, optionally substituted -NH-C(O)-C 1-6alkyl-C(O)-NH-C 1-6 alkyl-; -N3- is selected from the group consisting of a bond, hydroxymethyl- substituted C 1-3 alkyl-, hydroxymethyl-substituted-C 1-6 alkyl-OC 1-6 alkyl-, -N4- is selected from the group consisting of a bond, or -O-C 1-6 alkyl-; -N5- is selected from the group consisting of: a bond,

[0076] In some embodiments, -N1- is selected from the group consisting of optionally substituted methylene, ethylene, propylene, butylene, pentylene, hexylene, -C 1-3 alkyl-OCH2CH2-, -C 1-3 alkyl-(OCH2CH2)2-, -C 1-3 alkyl-(OCH2CH2)3-, -C 1-3 alkyl-(OCH2CH2) 1-3 -C 1-3 alkyl-; -N2- is selected from the group consisting of a bond, optionally substituted -NH-C(O)-methylene-C(O)-NH-, -NH-C(O)-ethylene-C(O)-NH-, -NH-C(O)-propylene-C(O)-NH-, -NH-C(O)-butylene-C(O)-NH-, -NH-C(O)-pentylene-C(O)-NH-, optionally substituted -NH-C(O)-C 1-3 alkyl-C(O)-NH-C 1-3 alkyl-; -N3- is selected from the group consisting of a bond, hydroxymethyl- substituted C 1-3 alkyl-OC 1-2 alkyl-, -N4- is selected from the group consisting of a bond, or -O-C 1-3 alkyl-; -N5- is selected from the group consisting of: a bond,

[0077] In some embodiments, the N represents

[0078] (M) e -(N) w In -P-, the P represents

[0079] e = 1, 2, or 3; w = 0, 1, 2, or 3.

[0080] In some embodiments, C is selected from the group consisting of Formula C-1, Formula C-2, or Formula C-3:

[0081] wherein Rb, Rc, Rd are each independently selected from the group consisting of an optionally substituted C6-C50 linear or branched saturated alkyl, an optionally substituted C6-C50 linear or branched unsaturated alkyl, an optionally substituted C6-C50 linear or branched saturated fatty acid, an optionally substituted C6-C50 linear or branched unsaturated fatty acid, ti, t2, t3, t4, t5, t6, t7, t8 are each independently selected from an integer from 0 to 10, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0082] In some embodiments, ti, t2, t3, t4, t5, t6, t7, t8 can each independently be 1.

[0083] In some embodiments, Rb, Rc, Rd are each independently selected from the group consisting of an optionally substituted C7-C36 linear or branched saturated alkyl, an optionally substituted C7-C36 linear or branched unsaturated alkyl, an optionally substituted C7-C36 linear or branched saturated fatty acid, an optionally substituted C7-C36 linear or branched unsaturated fatty acid.

[0084] In some embodiments, Rb, Rc, Rd are each independently selected from the group consisting of an optionally substituted C7-C24 linear or branched saturated alkyl, an optionally substituted C7-C24 linear or branched unsaturated alkyl, an optionally substituted C7-C24 linear or branched saturated fatty acid, an optionally substituted C7-C24 linear or branched unsaturated fatty acid.

[0085] In some embodiments, Rb, Rc, Rd are each independently selected from the group consisting of an optionally substituted C12-C36 linear or branched saturated alkyl, an optionally substituted C12-C36 linear or branched unsaturated alkyl, an optionally substituted C12-C36 linear or branched saturated fatty acid, an optionally substituted C12-C36 linear or branched unsaturated fatty acid.

[0086] In some embodiments, Rb, Rc, Rd are each independently selected from the group consisting of an optionally substituted C12-C24 linear or branched saturated alkyl, an optionally substituted C12-C24 linear or branched unsaturated alkyl, an optionally substituted C12-C24 linear or branched saturated fatty acid, an optionally substituted C12-C24 linear or branched unsaturated fatty acid.

[0087] In some embodiments, Rb, Rc, and Rd are each independently selected from optionally substituted straight-chain or branched saturated alkyl groups of C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, and C24, or optionally substituted C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, and C24. Straight-chain or branched unsaturated alkyl groups, optionally substituted C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24 straight-chain or branched unsaturated fatty acids; optionally substituted C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24 straight-chain or branched unsaturated fatty acids.

[0088] In some embodiments, with respect to Rb, Rc, and Rd, the unsaturated alkyl or unsaturated fatty acid contains 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) -C=C- or -C≡C-.

[0089] In some embodiments, the unsaturated alkyl or unsaturated fatty acid contains 1, 2, 3, 4, 5 or 6 -C=C-.

[0090] In some implementations, Rb, Rc, and Rd are each independently selected from: Re-(CH2)m6-(CH2)6-, Re-(CH2)m7-CH=CH-(CH2)m8-, Re-(CH2)m9-(C 1-3 Alkyl-CH=CH-C 1-3 alkyl)m 10 -(CH2)m 11 -、Re-(CH2)m9-(CH=CH-CH2)m 10 -(CH2)m 11 -, Re-(CH2)m9-(CH=CH-CH2CH2)m 10 -(CH2)m 11 -, Re-(CH2)m9-(CH2CH=CH-CH2)m 10 -(CH2)m 11 - where Re is selected from hydrogen or -COOH, m6, m7, m8, m9, m 11each independently selected from an integer from 1 to 24, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, m 10 selected from an integer from 1 to 6, such as 1, 2, 3, 4, 5, 6, wherein the sum of m7and m8or the sum of m9, m 10 and m 11 is greater than 7, preferably greater than 12, further preferably from 12 to 24.

[0091] In some embodiments, each of Rb, Rc, Rd is independently selected from: Re-(CH2)m6-(CH2)6-, Re is selected from hydrogen, m6 is selected from 10, 12, 14, 16, 18, 20, 22, 24.

[0092] In some embodiments, each of Rb, Rc, Rd is independently selected from: Re-(CH2)m9-(CH=CH-CH2)m 10 -(CH2)m 11 -, Re is hydrogen, m9=2, m 10 =5, m 11 =2; or Re is hydrogen, m9=8, m 10 =1, m 11 =6.

[0093] In some embodiments, each of Rb, Rc, Rd is independently selected from:

[0094] In some embodiments, the oligonucleotide conjugate of Formula II has a structure of Formula II-a: C-(X) n -Z (Formula II-a)

[0095] wherein C, X, n are defined as above for Formula II, and Z is selected from Z2, Z3, Z6, Z7, Z8, Z9, Z10, Z11:

[0096] In some embodiments, C is selected from Formula C-1, Formula C-2, Formula C-3.

[0097] In some embodiments, n is 1.

[0098] In some embodiments, X is an siRNA.

[0099] In some embodiments, n is 1, and C-X is selected from the group consisting of S1A1, S1A2, S1A3, S1A4, S1A5, S1A6, S1A7, S1A8, S1A9, S2A1, S2A2, S2A3, S2A4, S2A5, S2A6, S2A7, S2A8, S2A9, S3A1, S3A2, S3A3, S3A4, S3A5, S3A6, S3A7, S3A8, S3A9, S1E1, S1E2, S1E3, S1E4, S1E5, S1E6, S1E7, S1E8, S1E9, S1E10, S1E11, S1E12, S1E13, S2E1, S2E2, S2E3, S2E4, S2E5, S2E6, S2E7, S2E8, S2E9, S2E10, S2E11, S2E12, S2E13, S3E1, S3E2, S3E3, S3E4, S3E5, S3E6, S3E7, S3E8, S3E9, S3E10, S3E11, S3E12, S3E13, S1C1, S1C2, S1C3, S1C4, S2C1, S2C2, S2C3, S2C4, S3C1, S3C2, S3C3, S3C4, as shown in Table 1 below.

[0100] In some embodiments, C and Z are each independently conjugated to the sense strand of the siRNA.

[0101] In some embodiments, C and Z are each independently conjugated to the 5' end or 3' end of the sense strand of the siRNA.

[0102] The present application also provides an oligonucleotide conjugate having the structure shown in Formula III: C-(X)n n (Formula III);

[0103] wherein C, X, n are defined as above.

[0104] In some embodiments, C-X has the structure shown in Table 1.

[0105] Table 1: C-X structure

[0106] wherein, represents an siRNA, represents a sense strand, represents an antisense strand, and C is conjugated to the 5' end or 3' end of the siRNA.

[0107] In a second aspect, the present application further provides a method for preparing the oligonucleotide conjugate, comprising:

[0108] When the oligonucleotide is an siRNA, the siRNA is prepared according to the following steps:

[0109] S1, (X) n -(B) k Preparation of sense strand; S2, (X) n -(B) k Preparation of double strand; S3, preparation of modification group C; S4, C-(X) n -(B) k Preparation of sense strand; S5, C-(X) n -(B) k Preparation of double strand; S6, (X) n (C)-(B) k Preparation of sense strand; S6, (X) n (C)-B k Preparation of double strand.

[0110] When the oligonucleotide is an ASO or a miRNA, it is prepared according to the following steps:

[0111] S1, (X) n -(B) k Preparation of single strand; S2, preparation of modification group C; S3, C-(X) n -(B) k Preparation of single strand or X n (C)-(B) k Preparation of single strand.

[0112] In a third aspect, the present application further provides a protein conjugate comprising an oligonucleotide conjugate of Formula I or Formula II and albumin; the thiol and / or primary amine group of the albumin is conjugated to the oligonucleotide conjugate through its A.

[0113] In some embodiments, the protein conjugate has a structure of Formula IV or Formula V: C-(X) n -(B) k -A'-albumin (Formula IV) (X) n -(B) k -A'-albumin (Formula V)

[0114] wherein C, X, B, n, k are defined as above for Formula II, and A' is selected from groups that can conjugate to albumin.

[0115] In some embodiments, A' is selected from or -S-.

[0116] In some embodiments, the protein conjugate of Formula IV has a structure of Formula IV-1: C-(X) n-Z-albumin (Formula IV-1)

[0117] Z is selected from Z4, Z5, Z12.

[0118] In some embodiments, the protein conjugate of Formula V has a structure of Formula V-1 (X) n -Z-albumin (Formula V-1)

[0119] Z is selected from Z4, Z5, Z12.

[0120] The albumin includes, but is not limited to, natural albumin, synthetic albumin, modified albumin, etc.; wherein the natural albumin can be isolated from different mammalian species, including but not limited to, bovids, equids, felids, canids, leporids, suids, camellids, rodents, and primates, preferably bovine, equine, caprine, ovine, feline, leporine, porcine, camelid, alpaca, rat, mouse, guinea pig, non-human primate (such as simian, monkey, baboon, chimpanzee) and human, more preferably bovine, equine, canine, caprine, ovine, porcine, camelid, rat, mouse, monkey and human; and preferably, laboratory animals, such as mouse, rat, rabbit, guinea pig, hamster, monkey, dog, cat, pig, sheep, horse, etc. The synthetic albumin can be obtained by chemical synthesis, biological synthesis, recombinant production, transgenic animal production, etc. The albumin is preferably human albumin, more preferably human blood albumin isolated from human plasma.

[0121] The albumin is an albumin form present in plasma or serum in a mammal, preferably selected from one or more of the following: an albumin fraction and / or a prealbumin fraction isolated by electrophoresis, an albumin fraction capable of being salt-analytically precipitated by ammonium sulfate, an albumin fraction capable of being isolated by blue gel affinity chromatography, gel filtration chromatography or ion exchange chromatography, an albumin fraction capable of being isolated by low-temperature ethanol method, or any combination thereof.

[0122] In a fourth aspect, the present application further provides a pharmaceutical composition comprising the oligonucleotide conjugate or the protein conjugate and a pharmaceutically acceptable carrier.

[0123] In a fifth aspect, the present application further provides use of the oligonucleotide conjugate, the protein conjugate, or the pharmaceutical composition in the preparation of a targeted drug delivery system or a targeted drug having tissue specificity.

[0124] The target site is selected from one or more of the following tissues and / or organs or cells in the tissues and / or organs: liver, kidney, heart, spleen, stomach, muscle, brain, blood, tumor or nervous system.

[0125] The cells in the tissue and / or organ are normal cells or abnormal cells. In one embodiment, the abnormal cells are tumor cells.

[0126] In a sixth aspect, the present application further provides use of the oligonucleotide conjugate or the protein conjugate or the pharmaceutical composition in the preparation of a drug delivery system for conjugating albumin in vivo.

[0127] In a seventh aspect, the present application further provides a method for modulating the expression of a target gene and / or the activity, expression level or circulating level of a protein encoded by the target gene in a target site of a subject, comprising the step of: administering to a subject in need a prophylactically or therapeutically effective amount of the oligonucleotide conjugate or the protein conjugate or the pharmaceutical composition.

[0128] The subject is a mammal, including but not limited to, bovine, equine, feline, canine, leporine, porcine, camelid, rodent and primate, preferably bovine, equine, caprine, ovine, feline, leporine, porcine, camelid, llama, rat, mouse, guinea pig, non-human primate (such as simian, monkey, baboon, chimpanzee) and human, more preferably bovine, equine, canine, caprine, ovine, porcine, camelid, rat, mouse, monkey and human; still preferably, laboratory animals, such as mouse, rat, rabbit, guinea pig, hamster, monkey, dog, cat, pig, sheep, horse.

[0129] The target site is selected from one or more of the following tissues and / or organs or cells in the tissues and / or organs: liver, kidney, heart, spleen, stomach, muscle, brain, blood, tumor or nervous system.

[0130] The cells in the tissue and / or organ are normal cells or abnormal cells.

[0131] The abnormal cells can be tumor cells.

[0132] In the method, the administration route includes oral, parenteral, intravenous, intramuscular, transdermal, sublingual, inhalation, hepatic artery perfusion, renal artery perfusion, etc.; the administration timing includes prophylactic, therapeutic, etc.; the administration frequency is once a day, once a week, once every 4 weeks, once a month, once every three months, once every six months, once every nine months, once a year, etc.; the administration regimen includes single dose, multiple doses, combination, etc.

[0133] In an eighth aspect, the present application further provides a drug delivery method, comprising the step of: administering to a subject in need a prophylactically or therapeutically effective amount of the oligonucleotide conjugate, thereby delivering the oligonucleotide to a target site in the subject.

[0134] The subject is a mammal, including but not limited to, bovine, equine, feline, canine, leporine, porcine, camelid, rodent and primate, preferably bovine, equine, caprine, ovine, feline, leporine, porcine, camelid, llama, rat, mouse, guinea pig, non-human primate (such as simian, monkey, baboon, chimpanzee) and human, more preferably bovine, equine, canine, caprine, ovine, porcine, camelid, rat, mouse, monkey and human; and still preferably, laboratory animals, such as mouse, rat, rabbit, guinea pig, hamster, monkey, dog, cat, pig, sheep, horse.

[0135] The target site is selected from one or more of the following tissues and / or organs or cells in the tissues and / or organs: liver, kidney, heart, spleen, stomach, muscle, brain, blood, tumor or nervous system.

[0136] The cells in the tissues and / or organs are normal cells or abnormal cells. The abnormal cells can be tumor cells.

[0137] Compared with the prior art, the present application has the following beneficial effects:

[0138] 1. The present application connects the oligonucleotide with the oligonucleotide conjugate through the group capable of reacting with the free active group of the protein to form a covalent bond and the connecting arm, so that the oligonucleotide has the targeting delivery performance in specific tissues in vivo.

[0139] 2. The present application further increases the modification group C on the oligonucleotide conjugate, which can improve the distribution ability of the conjugate in specific tissues in vivo and improve the knockdown ability of the target gene in these specific tissues, and at the same time, improve the stability of the oligonucleotide conjugate.

[0140] In summary, the oligonucleotide conjugate provided by the present application can improve the in vivo delivery efficiency of the oligonucleotide and improve its in vivo stability, and can target the delivery of the oligonucleotide to multiple extrahepatic tissues such as liver tissue, kidney tissue and proximal tubule tissue. BRIEF DESCRIPTION OF DRAWINGS

[0141] Figure 1 shows the distribution of siRNA in different tissues after siRNA coupled with maleimide group is administered to mice in effect implementation example 1.

[0142] Figure 2 shows the distribution and metabolism of siRNA in different tissues after siRNA coupled with maleimide group is administered to mice in effect implementation example 2.

[0143] Figure 3 shows the knockdown of target gene C5 mRNA in the liver after different doses of siRNA conjugate are administered to mice in effect implementation example 3.

[0144] Figure 4 shows the change of tumor volume of mice in effect example 4 after administration of siRNA conjugate targeting VEGFA.

[0145] Figure 5 shows the knockdown of target gene C5 mRNA in liver of mice in effect example 5 after administration of siRNA conjugated with different linkers.

[0146] Figure 6 shows the knockdown of target gene C5 mRNA in liver of mice in effect example 6 after administration of siRNA modified with monovalent aliphatic hydrocarbon and the distribution of siRNA in liver and kidney.

[0147] Figure 7 shows the knockdown of target gene C5 mRNA in liver of mice in effect example 7 after administration of siRNA modified with divalent aliphatic hydrocarbon and the distribution of siRNA in liver and kidney.

[0148] Figure 8 shows the knockdown of target gene C5 mRNA in liver of mice in effect example 8 after administration of siRNA modified with trivalent aliphatic hydrocarbon and the distribution of siRNA in liver and kidney.

[0149] Figure 9 shows the knockdown of target gene C5 mRNA in liver of mice in effect example 9 after administration of siRNA modified with monovalent unsaturated aliphatic hydrocarbon.

[0150] Figure 10 shows the knockdown of target gene C5 mRNA in liver of mice in effect example 10 after administration of siRNA modified with divalent unsaturated aliphatic hydrocarbon.

[0151] Figure 11 shows the knockdown of C5 mRNA in liver of mice in effect example 11 after administration of siRNA modified with trivalent unsaturated aliphatic hydrocarbon.

[0152] Figure 12 shows the knockdown of target gene C5 mRNA in liver of mice in effect example 12 after administration of siRNA modified with fatty acid.

[0153] Figure 13 shows the knockdown of target gene C5 mRNA in liver of mice in effect example 13 after administration of siRNA with maleimide modification at 3' and 5' end of sense strand.

[0154] Figure 14 shows the knockdown of target gene SOD1 mRNA and the distribution of siRNA in different tissues of mice in effect example 14 after administration of siRNA with different modification groups.

[0155] Figure 15 shows the knockdown of target gene KRAS mRNA and the distribution of siRNA in different tissues of mice in effect example 15 after administration of siRNA with different modification groups.

[0156] Figure 16 shows the knockdown of target gene ANGPTL3, TTR mRNA and the distribution of siRNA in different tissues of mice after administration of siRNA containing different modification groups in Example 16.

[0157] Definitions of terms

[0158] The term "nucleotide", without further specification, can be used herein to refer to an unmodified nucleotide, a modified nucleotide or a surrogate moiety, as well as a nucleoside analogue or derivative. Those skilled in the art will recognize that guanine, cytosine, adenine and uracil can be replaced by other moieties without significantly altering the base pairing properties of an oligonucleotide comprising nucleotides with such replacement moieties. The partial nucleotide abbreviations and structures are as follows in Table 2:

[0159] Table 2. Nucleotide abbreviations and their structures

[0160] In the present application, in all nucleotide sequences, the capital letters C, G, U, A represent the base composition of the nucleotide; the lower case letter m represents that the 2'-hydroxyl group of the ribose group of the nucleotide to the left of the letter m is replaced by methoxyl; the lower case letter f represents that the 2'-hydroxyl group of the ribose group of the nucleotide to the left of the letter f is replaced by fluorine; the capital letter and number C16 represent that the 2'-hydroxyl group of the ribose group of the nucleotide to the left of the number letter C16 is replaced by 2'-O-hexadecyl modification; the lower case letter s represents that the phosphodiester bond between two nucleotides is replaced by a phosphorothioate diester bond.

[0161] The term "oligonucleotide", without further specification, generally refers to a short chain nucleic acid molecule composed of a small number of nucleotides connected by phosphodiester bonds or phosphorothioate diester bonds. The number of nucleotides is between 10 and 100, such as between 15 and 50. Oligonucleotides include single-stranded oligonucleotides such as antisense oligonucleic acids (ASOs) or double-stranded oligonucleotides such as small interfering RNAs (siRNAs).

[0162] [Corrected according to Rule 91 18.09.2025] The term "small interfering RNA (siRNA)" refers to a double-stranded RNA molecule composed of two completely complementary or partially complementary nucleotide chains (sense strand and antisense strand), usually 17-27 base pairs in length, such as 19, 20, 21, 22, 23, 24, 25 base pairs. In the present text denotes siRNA, "SS", denotes sense strand, "AS", antisense strand, when the above schematic structure appears in a specific structure, represents the nucleotide sequence in the corresponding number, such as the molecular structure numbered as "CH2301002Z2S1A1" is, wherein represents the nucleotide sequence numbered as CH2301002.

[0163] Albumin, HAS in this article refers to albumin.

[0164] In the present application, the nucleotide sequence is 5'-3' unless otherwise specified.

[0165] Depending on the context, the terms "modification", "conjugation" and "coupling" can be used interchangeably.

[0166] Depending on the context, the terms "expression amount", "content" and "level" can be used interchangeably when referring to the target gene mRNA. DETAILED DESCRIPTION

[0167] Some of the compounds in the following examples and their commercial routes are as follows:

[0168] Compound 1: its structural formula is Compound 2: its structural formula is Compound 3: its structural formula is Compound 3: its structural formula is

[0169] L96: its structural formula is:

[0170] Preparation Example

[0171] Preparation Example 1

[0172] The synthesis of oligonucleotide sequences is obtained by solid-phase phosphoramidite method. The specific synthesis process is divided into three parts of sense chain synthesis, antisense chain synthesis and annealing. The synthesis methods of sense and antisense chains are the same, that is, the nucleoside monomers are connected in the order of sequence nucleotide arrangement 3'-5' direction, and each step of nucleoside monomer connection contains four steps of deprotection, coupling, capping, oxidation or sulfuration. After all the nucleoside monomers are connected, the solid-phase carrier connected with nucleic acid is cut, deprotected, purified, desalted and freeze-dried, and the desired sense and antisense chains are obtained. If necessary, the sense and antisense chains are dissolved into 40mg / mL aqueous solution, mixed in equimolar ratio, heated at 50℃ for 15min, cooled at room temperature, annealed, and freeze-dried, to obtain the final siRNA compound.

[0173] According to the above conventional method, the synthesized siRNA is shown in Table 3, and the synthesized sense chain conjugate is shown in Table 4.

[0174] Table 3. siRNA

[0175] Table 4. Sense strand conjugate (5'-3')

[0176] Note: The nucleotide monomers containing Z1, Z6', Z7', Z8', Z9', Z10', Y1, Y2, Y3 in the table are commercially available.

[0177] Preparation of Example 2 Preparation of CH2301001Z2 siRNA (oligonucleotide conjugate)

[0178] The synthetic route is as follows

[0179] The specific synthesis steps are as follows:

[0180] 1) Take the desired siRNA-NH2 (CH2301001Z1) sense strand (CH2301001Z1 (SS)) (1.0 eq) and dissolve it in PBS, and dissolve compound 1 (15.0 eq) in DMF. Add the two solutions to a 1.5 mL centrifuge tube, and add an appropriate amount of PBS and DMF to make the volume ratio of the two solvents PBS / DMF 1:1. Then control the temperature of the centrifuge tube on the shaker at 25.0-30.0°C and the speed at 220 times / minute, and react for 2.0-3.0 hours.

[0181] After the end, dilute the reaction solution with DEPC water to the appropriate volume, desalt with a G25 (Spedex) desalting column, collect the effective components, and lyophilize at low temperature to remove the solvent to obtain the crude siRNA-L1 (CH2301001Z2) sense strand (CH2301001Z2 (SS)) (HPLC purity about 70%), and then further purify the crude product on a preparative HPLC. Dissolve the sample in an appropriate amount of DEPC water, and select an appropriate separation method to purify the sample. After collecting the target components, desalt the pure product with a G25 (Spedex) desalting column, and then lyophilize at low temperature to remove the solvent. Take the sample to HPLC, MALDI and gel electrophoresis to confirm the purity of the product;

[0182] 2) siRNA-L1 (CH2301001Z2) sense strand CH2301001Z2 (SS) and antisense strand were dissolved in DEPC water respectively, and quantified (concentration controlled at about 100 OD / mL) respectively, then the DEPC water solution of siRNA-L1 (CH2301001Z2) sense strand and the DEPC water solution of antisense strand were added into 1.5 mL centrifuge tube in proportion, hybridized at 25.0-30.0 °C for 30 minutes, quantified, and freeze-dried at low temperature to remove the solvent to obtain CH2301001Z2 siRNA pure product.

[0183] Preparation of siRNA-L2 (CH2301001Z3) siRNA

[0184] The synthetic route is as follows:

[0185] Specific synthesis steps are as follows:

[0186] 1) The required siRNA-NH2 (CH2301001Z1) sense strand (CH2301001Z1 (SS)) (1.0 eq) was dissolved in 0.1M NaOAc, and compound 2 (15.0 eq) was dissolved in DMF. The two solutions were added into 1.5 mL centrifuge tube, and appropriate amount of 0.1M NaOAc and DMF was added to make the volume ratio of the two solvents 0.1M NaOAc / DMF 1:9. Then the centrifuge tube was controlled at 25.0-30.0 °C and 220 times / min on a shaker, and reacted for 2.0-3.0 hours. After the reaction was completed, the reaction solution was diluted with DEPC water to an appropriate volume, desalted with G25 (Spedex) desalting column, and the effective components were collected. The solvent was removed by freeze-drying at low temperature to obtain the crude siRNA-L2 (CH2301001Z3) sense strand (CH2301001Z3 (SS)). Then the crude product was further purified on a preparative HPLC. The sample was dissolved in an appropriate amount of DEPC water, and the sample was purified by selecting an appropriate separation method. After the target components were collected, the pure product was desalted with G25 (Spedex) desalting column, and then freeze-dried at low temperature to remove the solvent. The sample was sent to HPLC, MALDI and gel electrophoresis to confirm the purity of the product;

[0187] 2) siRNA-L2 (CH2301001Z3) sense strand and antisense strand were dissolved in DEPC water respectively, and quantified (concentration controlled at about 100 OD / mL) respectively, then the DEPC water solution of siRNA-L2 (CH2301001Z3) sense strand and the DEPC water solution of antisense strand were added into 1.5 mL centrifuge tube in proportion, hybridized at 25.0-30.0 °C for 30 minutes, quantified, and freeze-dried at low temperature to remove the solvent to obtain CH2301001Z3 siRNA pure product.

[0188] Preparation of siRNA-L3 (CH2301001Z11) (oligonucleotide conjugate)

[0189] The synthetic route is as follows:

[0190] Specific synthesis steps:

[0191] 1) Dissolve the desired siRNA-NH2 (CH2301001Z1) sense strand (1.0 eq) in PBS and dissolve compound 3 (15.0 eq) in DMF. Add the two solutions to a 1.5 mL centrifuge tube, and add appropriate amounts of PBS and DMF to make the volume ratio of the two solvents PBS / DMF 9:1. Then, incubate the centrifuge tube on a shaker at 25.0-30.0°C and at a speed of 220 times per minute for 2.0-3.0 hours.

[0192] After the reaction is completed, dilute the reaction solution with DEPC water to the appropriate volume, desalt the solution with a G25 (Spedex) desalting column, collect the effective components, and lyophilize the solution at low temperature to remove the solvent to obtain the crude siRNA-L3 (CH2301001Z11). Then, further purify the crude product on a preparative HPLC. Dissolve the sample in an appropriate amount of DEPC water, and purify the sample by selecting an appropriate separation method. After collecting the target components, desalt the pure product with a G25 (Spedex) desalting column, and then lyophilize the solution at low temperature to remove the solvent. Take the sample to HPLC, DADI, and gel electrophoresis to confirm the purity of the product.

[0193] 2) Dissolve the siRNA-L3 (CH2301001Z11) sense strand (CH2301001Z11 (SS)) and its antisense strand in DEPC water, respectively, and quantitate them (control the concentration to be about 100 OD / mL). Then, add the DEPC water solution of the siRNA-L3 (CH2301001Z11) sense strand and the DEPC water solution of the antisense strand to a 1.5 mL centrifuge tube in the appropriate proportions, hybridize at 25.0-30.0°C for 30 minutes, quantitate, and lyophilize the solution at low temperature to remove the solvent to obtain the pure siRNA-L3 (CH2301001Z11).

[0194] Using the same method as described above, react the sense strand of siRNA-NH2 with different linkers to prepare sense strands with different linkers, and then hybridize with the antisense strand to prepare siRNA conjugated with Z2-Z10. The prepared siRNA conjugates are as follows in Table 5:

[0195] Table 5. siRNA conjugates

[0196] Preparation of Example 5 CH2301001Z4 (protein conjugate)

[0197] The synthetic route is as follows:

[0198] Specific synthesis steps:

[0199] Take the desired siRNA CH2301001Z2 (1.0 eq) and Albumin (2.5 eq) (Sigma) and dissolve them in PBS. Add the two solutions to a 1.5 mL centrifuge tube, then control the temperature at 25.0-30.0°C and the speed at 220 times / min on a shaker for 3.0 hours. After the reaction is completed, dilute the reaction solution to the appropriate volume with DEPC water, desalt with a G25 (Spedex) desalting column, collect the effective components, and freeze-dry to remove the solvent to obtain the crude product. Then further purify the crude product by using an ultrafiltration purification method to obtain the dsRNA-albumin conjugate. In the later stage, the purity of the protein conjugate CH2301001Z4 can be further improved by preparative HPLC.

[0200] Using the same method as in Example 4, siRNA conjugated with Z3 (CH2301001Z3) is coupled with albumin to obtain siRNA conjugated with albumin (CH2301001Z5), and siRNA conjugated with Z4-Z11 is coupled with albumin to obtain siRNA conjugated with albumin.

[0201] Preparation of Example 6 Preparation of fatty alkane or fatty acid siRNA conjugate

[0202] General preparation method of siRNA conjugate conjugated with fatty alkane or fatty acid:

[0203] wherein Y is selected from Y1, Y2, Y3, Z' is selected from Z1, Z6', Z7', Z8', Z9', Z10'; Z is selected from Z2, Z3, Z6, Z7, Z8, Z9, Z10, Z11, R is selected from Rb, Rc, Rd, Y' is selected from Y1': Y2': Y3':

[0204] Step one: react the sense strand conjugated with Y and Z' obtained from the solid phase synthesizer with azide compounds with different R to obtain sense strands conjugated with different fatty alkane or fatty acid.

[0205] Step two: further react the sense strands conjugated with different fatty alkane or fatty acid with linkers such as compounds 1, 2 or 3 to obtain sense strands conjugated with groups that can react with albumin, such as maleimide.

[0206] Step three: conjugate the sense strand conjugate with the antisense strand to obtain the target siRNA conjugate.

[0207] [Rule 91 correction 18.09.2025] The synthetic route of an exemplary monovalent aliphatic alkane or fatty acid conjugate is as follows:

[0208] Take 10.0 OD of compound 4 (CH2301002Z1Y1(SS)) into a 1.5 mL centrifuge tube, add 2.0 M TEAA (11.0 uL) and DECP water (14.0 uL) to dissolve, continue to add DMSO (36.0 uL), add 100.0 mM compound 5 DMSO solution (20.0 eq, 9.0 uL), 5.0 mM ascorbic acid solution (20.0 uL, 2.2 eq) and 10.0 mM Cu-TBTA (10.0 uL, 2.2 eq), protect under nitrogen for 3 hours. Desalt the reaction solution, freeze-dry, and detect the reaction conversion rate by HPLC. Finally, compound 6 (CH2301002Z1A1S1(SS)) is obtained.

[0209] Take 5.0 OD of compound 6 dissolved in 50.0 ul PBS Buffer, compound 1 dissolved in DMF, mix the two, shake bed for 3-4 h, desalt the reaction solution, freeze-dry, and detect the reaction conversion rate by HPLC. Then purify the product by preparative HPLC to obtain compound 7 (CH2301002Z2A1S1(SS)).

[0210] Dissolve the freeze-dried compound 7 in 50.0 uL DEPC water, dissolve the antisense strand (CH2301002(AS)) in 50.0 uL DEPC, mix the two, hybridize at room temperature for 30.0 min, and quantitatively freeze-dry the reaction solution to obtain the final product compound 8 (CH2301002Z2A1S1). The final product is characterized using HPLC, PAGE gel and MALDI.

[0211] [Rule 91 correction 18.09.2025] The synthetic route of an exemplary divalent aliphatic alkane or fatty acid conjugate is as follows:

[0212] [Rule 91 correction 18.09.2025] The synthetic route of an exemplary trivalent aliphatic alkane or fatty acid conjugate is as follows:

[0213] The following monovalent, divalent, and trivalent conjugates in Table 6 are prepared by the above similar method:

[0214] [Corrected according to Rule 91 on 18.09.2025] Table 6. siRNA conjugates

[0215] wherein, represents siRNA, the sequence of which is the corresponding sequence in the compound number, such as in CH2301002Z2S1A1, represents the corresponding sequence of CH2301002, represents the sense strand, represents the antisense strand, the aliphatic alkane or aliphatic acid is conjugated to the 5' end of siRNA.

[0216] Effect examples

[0217] Experimental cells and animals:

[0218] Cell lines: 293T, HeLa, A549 were purchased from the Chinese Academy of Sciences Cell Bank.

[0219] Unless otherwise specified, the in vivo efficacy experiments of the present application all use the following steps to administer drugs to animals, obtain blood samples or tissue samples for testing:

[0220] The experimental animals mice were randomly grouped, and administered by tail vein injection according to the experimental dose. After administration, at the sampling time point, 200-300 μL of blood was taken from each mouse; then euthanized, and the target tissue was taken. The blood sample was taken from the eye orbit, 200-300 μL per mouse, and the whole blood was placed at 4°C for 30 minutes, centrifuged at 1500g, 4°C, for 15 minutes, the supernatant was taken with a pipette, and stored at -80°C until testing. The tissue sample was obtained by euthanizing the mouse, dissecting, and taking liver, kidney, thigh muscle, heart, spleen, lung, cerebral cortex, hippocampus, intestinal epithelium, brainstem, cerebellum, thymus, midbrain, pancreas, duodenum, small intestine, adrenal gland, stomach, tongue, spinal cord, lymph node, tumor, etc. The tissues were cut and then placed in RNA protection solution, and then placed in a -4°C refrigerator overnight. The next day, the tissues were placed in a -20°C freezer.

[0221] Unless otherwise specified, the present application uses stem-loop method to detect the content of siRNA in mouse tissues or blood samples, and the steps are as follows:

[0222] Tissue sample 10 mg, add 980 μL PBS, homogenate; plasma sample 2 uL. After 196 μL homogenate was taken into each tube, 4 μL 12.5% Triton was added, heated at 95°C for 10 min. The lysis product was placed on ice for 10 min. Centrifuged at 12000 rpm, 4°C for 20 min, and the supernatant was taken for subsequent reverse transcription experiment. Reverse transcription was completed according to the standard operation of the HiScript II kit (Vazyme, R201-01). The reverse transcription primers were developed by Suzhou Gemma Gene Co., Ltd. The reverse transcription cDNA sample was diluted 5 times for QPCR detection. The dye method was used to detect the expression change of the target gene. The reaction system was configured according to the standard method of the 2×SYBR Green qPCR Master Mix (Adamas life, G8272-500T) kit instructions. The fluorescence quantitative PCR instrument (ABI, Steponeplus) was used for fluorescence quantitative PCR reaction. The experimental results were calculated according to the Ct value combined with the standard curve.

[0223] Unless otherwise specified, the present application uses RT-qPCR method to detect the mRNA content in mouse tissue or blood sample. The steps are as follows:

[0224] The mRNA extraction uses VAMNE Magnetic Cell / Tissue Total RNA Kit (Vazyme, RMA101-C2). The extraction product is used for subsequent reverse transcription experiment. The HiScript II Q RT SuperMix for qPCR (+gDNA wiper) (Vazyme, R223-01) is used to complete the reverse transcription according to the standard operation of the kit. The reverse transcription cDNA sample is diluted 5 times for QPCR detection. The dye method is used to detect the expression change of the target gene. The primers are developed by Suzhou Gemma Gene Co., Ltd. The reaction system is configured according to the standard method of the 2×SYBR Green qPCR Master Mix (Adamas life, G8272-500T) kit instructions. The fluorescence quantitative PCR instrument (ABI, Steponeplus) is used for fluorescence quantitative PCR reaction. The experimental results are calculated according to the Ct value using 2–ΔΔCt analysis to show the relative expression change of the target gene.

[0225] Effect example 1 in vivo efficacy experiment of siRNA conjugate

[0226] In this example, 6-8 week old, 18-30 gram, SPF female C57BL / 6J mice were used as experimental animals. They were grouped and administered once (day 0) according to the following table and sampled. The stem loop method was used to detect the amount of siRNA in each tissue of the mouse.

[0227] Table 7. Experimental grouping

[0228] The experimental results are shown in Figure 1. After CH2301001 enters the blood, it mainly enters the kidney and liver and is metabolized by the kidney and liver, and has poor ability to enter other tissues and organs. In the liver tissue, the distribution amount of CH2301001Z3 is equivalent to that of CH2301001G. The extrahepatic distribution of CH2301001Z3 is significantly higher than that of CH2301001G, and is about 2-7 times that of CH2301001G. In terms of sending siRNA to the spleen, kidney, lung, myocardium, skeletal muscle, tumor tissue, tongue, stomach, intestine, pancreas, vaginal tissue and other extrahepatic tissues, the siRNA-albumin delivery system is significantly stronger than GalNAc. Relative to CH2301001G and CH2301001, CH2301001Z2 and CH2301001Z3 remain in the blood for a longer time, indicating that albumin coupling enhances the plasma stability of siRNA. The siRNA mouse in vivo distribution data on the first day (1A in Figure 1), the seventh day (1B in Figure 1) and the fourteenth day (1C in Figure 1) after administration are consistent.

[0229] Effect Example 2 In vivo efficacy experiment of siRNA conjugate on PDX mice

[0230] In this experiment, the experimental animals are: nude mice, SPF, 36 female mice, weighing 18-30 grams, 6-8 weeks old, purchased from Jixiu. The mice were grouped according to the following table, administered once (on day 0) and sampled, and the amount of siRNA in each tissue of the mice was detected by stem-loop method.

[0231] Table 8. Experimental grouping

[0232] The experimental results are shown in Figure 2. On the seventh day after administration, the amount of CH2301001Z2 and CH2301001Z3 in each tissue and organ is significantly higher than that of CH2301001. In the liver tissue, the distribution amount of CH2301001Z3 is equivalent to that of CH2301001G. In the extrahepatic tissues (such as spleen, kidney, lung, myocardium, tumor tissue), the distribution amount of CH2301001Z3 is about 2-7 times that of CH2301001G. The experimental results show that the conjugate of the application can deliver siRNA to extrahepatic tissues, including spleen, kidney, lung, myocardium, skeletal muscle, tumor tissue, tongue, stomach, intestine, pancreas, vaginal tissue (A, B, C, D, E in Figure 2). Relative to CH2301001G and CH2301001, the plasma stability of CH2301001Z2 and CH2301001Z3 is significantly higher (F in Figure 2).

[0233] Example 3 siRNA conjugate single-dose gene silencing experiment

[0234] In this example, 6-8 week old, 18-30 gram, SPF female C57BL / 6J mice were used as experimental animals, which were grouped according to the following table and administered once (day 0) and sampled. The mRNA content of the target gene in each tissue of the mouse was detected by rt-qPCR.

[0235] Table 9. Experimental grouping

[0236] The experimental results are shown in Figure 3. CH2301002Z2 and CH2301002Z3 down-regulated the mRNA level of the target gene in liver tissue in a dose-dependent manner on day 7 after tail vein administration. CH2301002Z3 had a knockdown effect on the mRNA level of the target gene equivalent to that of CH2301002G 10 mg / kg when administered at 30 mg / kg, both of which down-regulated the mRNA level of the target gene by about 80%.

[0237] Example 4 siRNA conjugate inhibits tumor growth in vivo

[0238] In this example, 6-8 week old, 18-30 gram, SPF female PDX nude mice were used as experimental animals, which were purchased from Jisui Yakang. The tumors were inoculated subcutaneously in the mice, and the growth state of the tumor mass was observed after one week. When the tumor volume reached 100-200 mm 3 around, the mice were randomly grouped, and the tumor-bearing mice were administered from the tail vein according to the following table. The day of administration was designated as day 0. During the experiment, the tumor growth and the effect of treatment on the animals were monitored twice a week, including the activity, food intake and water intake of the experimental animals, tumor volume, body weight, eyes, fur, etc.

[0239] Tumor volume calculation method: for irregularly shaped tumors, L is calculated according to the longest diameter, and S is calculated according to the middle value of the short diameter perpendicular to the longest diameter. Tumor volume calculation: tumor volume TV (mm 3 ) = long diameter (L) x short diameter (S)2 / 2.

[0240] Table 10. Experimental grouping

[0241] The experimental results are shown in Figure 4. CH2301002Z3 and CH2301002Z6 significantly inhibited tumor growth (p<0.05) during the 21 days from day 0 to day 21 of administration. It can be seen that the conjugate of the application can deliver siRNA to the tumor, knock down the expression of the target gene vascular endothelial growth factor (VEGFA), and significantly inhibit tumor growth.

[0242] In vivo activity experiment of siRNA coupled with different linkers of Effect Example 5

[0243] In this example, 6-8 weeks old, 18-30 grams of SPF female nude mice were used as experimental animals, which were purchased from Jisui Yaoke. The animals were grouped and administered once (day 0) according to the table below and sampled. The mRNA content of the target gene in each tissue of the mouse was detected by rt-qPCR.

[0244] Table 11. Experimental grouping one

[0245] Table 12. Experimental grouping two

[0246] The experimental results are shown in Figure 5A (experimental grouping one) and Figure 5B (experimental grouping two). On the 7th day after administration, each siRNA coupled with different linkers showed significant knockdown effect of the target gene in liver tissue.

[0247] In vivo efficacy experiment of monovalent aliphatic alkane modified siRNA of Effect Example 6

[0248] In this example, 6-8 weeks old, 18-30 grams of SPF female C57BL / 6J mice were used as experimental animals, which were administered once (day 0) according to the table below and sampled. The mRNA content of the target gene in each tissue of the mouse was detected by rt-qPCR; the content of siRNA in each tissue of the mouse was detected by stem-loop method.

[0249] Table 13. Experimental grouping

[0250] The experimental results are shown in Figure 6. The results show that on the 7th day after administration, compared with siRNA without coupled delivery molecules, monovalent aliphatic alkane modified siRNA significantly silenced the expression of target gene mRNA in mouse liver tissue (Figure 6A). After introducing aliphatic alkane, the distribution ratio of siRNA in kidney and liver was 1:1-1:3; compared with CH2301002, the aliphatic alkane containing delivery system can significantly improve the enrichment of siRNA in kidney (Figure 6B).

[0251] In vivo efficacy experiment of bivalent aliphatic alkane modified siRNA of Effect Example 7

[0252] In this example, 6-8 weeks old, 18-30 grams of SPF female C57BL / 6J mice were used as experimental animals. The animals were grouped and administered once (day 0) according to the table below and sampled. The mRNA content of the target gene in each tissue of the mouse was detected by rt-qPCR; the content of siRNA in each tissue of the mouse was detected by stem-loop method.

[0253] Table 14. Experimental grouping

[0254] The experimental results are shown in Figure 7. The results show that, on the 7th day after administration, the siRNAs coupled with the delivery molecules significantly silenced the expression of the target gene mRNA in the liver tissues of mice, as compared with the siRNAs without the delivery molecules; the siRNAs with the introduction of the bivalent aliphatic alkyl at the 5' end of the sense strand increased the silencing efficiency of the target gene mRNA in the liver tissues of mice to different extents, as compared with the siRNAs without the introduction of the bivalent aliphatic alkyl (A in Figure 7). After the introduction of the aliphatic alkyl, the distribution ratio of the siRNAs in the kidney and the liver was 1:1-2:1; the siRNAs with the aliphatic alkyl delivery system significantly increased the enrichment of the siRNAs in the kidney, as compared with CH2301002 (B in Figure 7).

[0255] Effect Example 8 In vivo pharmacodynamic experiment of siRNA modified with trivalent aliphatic alkyl

[0256] In this example, 6-8-week-old, 18-30-gram, SPF female C57BL / 6J mice were used as experimental animals. The mice were administered once (on day 0) and sampled according to the following table. The content of the mRNA of the target gene in each tissue of the mice was detected by rt-qPCR; the content of the siRNA in each tissue of the mice was detected by stem-loop.

[0257] Table 15. Experimental grouping

[0258] The experimental results are shown in Figure 8. The results show that, on the 7th day after administration, the siRNAs coupled with the delivery molecules significantly silenced the expression of the target gene mRNA in the liver tissues of mice, as compared with the siRNAs without the delivery molecules; the siRNAs with the introduction of the trivalent aliphatic alkyl at the 5' end of the sense strand increased the silencing efficiency of the target gene mRNA in the liver tissues of mice to different extents, as compared with the siRNAs without the introduction of the trivalent aliphatic alkyl (A in Figure 8). After the introduction of the aliphatic alkyl, the distribution ratio of the siRNAs in the kidney and the liver was about 1 / 1; the siRNAs with the aliphatic alkyl delivery system significantly increased the enrichment of the siRNAs in the kidney, as compared with CH2301002 (B in Figure 8).

[0259] Effect Example 9 In vivo pharmacodynamic experiment of siRNA modified with monovalent unsaturated aliphatic alkyl

[0260] In this example, 6-8-week-old, 18-30-gram, SPF female C57BL / 6J mice were used as experimental animals. The mice were administered once (on day 0) and sampled according to the following table. The content of the mRNA of the target gene in each tissue of the mice was detected by rt-qPCR; the content of the siRNA in each tissue of the mice was detected by stem-loop.

[0261] Table 16. Experimental grouping

[0262] The experimental results are shown in Figure 9. On the 7th day after administration, compared with siRNA without conjugated delivery molecules, siRNA conjugated with delivery molecules were significantly silenced in the expression of target gene mRNA in mouse liver tissue delivered by albumin. Compared with siRNA delivered by albumin alone, the introduction of a monovalent unsaturated aliphatic alkane at the 5' end of the siRNA sense strand can increase the inhibition effect of siRNA on target genes in the liver. In other words, the introduction of a monovalent unsaturated aliphatic alkane can further improve the silencing efficiency of siRNA on mRNA in the liver.

[0263] Effect Example 10 In vivo pharmacodynamic experiment of siRNA modified by divalent unsaturated aliphatic alkane

[0264] In this example, 6-8 week old, 18-30 gram, SPF female C57BL / 6J mice were used as experimental animals. According to the following table, the animals were administered once (on day 0) and sampled. The content of mRNA of the target gene in each tissue of the mouse was detected by rt-qPCR; the content of siRNA in each tissue of the mouse was detected by stem loop method.

[0265] Table 17. Experimental grouping

[0266] The experimental results are shown in Figure 10. On the 7th day after administration, compared with siRNA without conjugated delivery molecules, siRNA conjugated with delivery molecules were significantly silenced in the expression of target gene mRNA in mouse liver tissue. Compared with siRNA conjugated with maleimide alone, the introduction of a divalent unsaturated aliphatic alkane at the 5' end of the siRNA sense strand increased the inhibition effect of siRNA on target genes in the liver to varying degrees.

[0267] Effect Example 11 In vivo pharmacodynamic experiment of siRNA modified by trivalent unsaturated aliphatic alkane

[0268] In this example, 6-8 week old, 18-30 gram, SPF female C57BL / 6J mice were used as experimental animals. According to the following table, the animals were administered once (on day 0) and sampled. The content of mRNA of the target gene in each tissue of the mouse was detected by rt-qPCR; the content of siRNA in each tissue of the mouse was detected by stem loop method.

[0269] Table 18. Experimental grouping

[0270] The experimental results are shown in Figure 11. On the 7th day after administration, the siRNA coupled with the delivery molecule significantly silenced the expression of the target gene mRNA in the liver tissue of the mice, as compared with the siRNA without the coupled delivery molecule. The introduction of the trivalent unsaturated aliphatic hydrocarbon at the 5' end of the sense strand of the siRNA increased the inhibition effect of the siRNA on the target gene in the liver to different extents.

[0271] Effect Example 12 In vivo efficacy experiment of siRNA modified with fatty acid

[0272] In this example, 6-8 week old, 18-30 gram, SPF female C57BL / 6J mice were used as experimental animals. The mice were administered once (on day 0) and sampled according to the following table. The content of mRNA of the target gene in each tissue of the mice was detected using rt-qPCR; the content of siRNA in each tissue of the mice was detected using the stem loop method.

[0273] Table 19. Experimental grouping

[0274] The experimental results are shown in Figure 12. On the 7th day after administration, the siRNA coupled with the fatty acid significantly silenced the expression of the target gene mRNA in the liver tissue of the mice.

[0275] Effect Example 13 In vivo efficacy experiment of siRNA modified at the 3' and 5' ends of the sense strand

[0276] In this example, 6-8 week old, 18-30 gram, SPF female C57BL / 6J mice were used as experimental animals. The mice were administered once (on day 0) and sampled according to the following table. The content of mRNA of the target gene in each tissue of the mice was detected using rt-qPCR.

[0277] Table 20. Experimental grouping

[0278] The information of the tested substances is shown in Table 21.

[0279] Table 21. Tested substances

[0280] Note: C16 indicates that the 2'-hydroxyl group of the ribose group of one nucleotide on the left side of C16 is modified and replaced with a 2'-O-hexadecyl group

[0281] The experimental results are shown in Figure 13. On the 7th day after administration, when the maleimide modification group was on the 3' and 5' ends of the sense strand of the siRNA, respectively, the delivery efficiency mediated by albumin was not different, and the similar delivery ability was exhibited.

[0282] Effect Example 14 In vivo efficacy experiment of siRNA targeting SOD1 (CH2301005)

[0283] The present example uses 6-8 week old, 18-30 gram weight, SPF grade female C57BL / 6J mice as experimental animals. The mice are grouped according to the table below, administered once (day 0) and sampled. The content of mRNA of the target gene in each tissue of the mouse is detected using rt-qPCR; the content of siRNA in each tissue of the mouse is detected using the stem loop method.

[0284] Table 22. Experimental grouping

[0285] The experimental results are shown in Figure 14. On day 7 after administration, the siRNA conjugate can well inhibit the expression of the target gene in the heart, liver, lung, and muscle after the introduction of aliphatic hydrocarbons (14A in Figure 14); and the siRNA conjugate can be better distributed in each tissue (14B in Figure 14).

[0286] Effect Example 15 In vivo efficacy experiment of aliphatic hydrocarbon modified siRNA targeting KRAS

[0287] The present example uses 4-6 week old, 18-30 gram weight, SPF grade female C57BL / 6J mice as experimental animals. The mice are grouped according to the table below, administered once (day 0) and sampled. The content of mRNA of the target gene in each tissue of the mouse is detected using rt-qPCR; the content of siRNA in each tissue of the mouse is detected using the stem loop method.

[0288] Table 23. Experimental grouping

[0289] The experimental results are shown in Figure 15. On day 7 after administration, the siRNA of the present application significantly inhibits the expression of the target gene after the introduction of aliphatic hydrocarbons; compared with siRNA not conjugated with maleimide, the inhibitory effect of siRNA conjugated with maleimide is significantly improved, and the inhibitory effect of siRNA further conjugated with aliphatic hydrocarbons is further enhanced (15A in Figure 15). Moreover, the introduction of aliphatic hydrocarbons significantly improves the distribution of siRNA in each tissue, and the distribution concentration of siRNA conjugated with maleimide and further conjugated with aliphatic acid in the heart, liver, spleen, stomach, kidney, muscle, brain, and blood is significantly improved compared with siRNA not conjugated with maleimide (15B in Figure 15), indicating that the conjugate of the present application can deliver siRNA to the above tissues.

[0290] Effect Example 16 In vivo efficacy experiment of aliphatic hydrocarbon modified siRNA targeting PCSK9 / ANGPTL3 / TTR (CH2301001 / CH2301004 / CH2301008)

[0291] This example used 4-6 week old, 18-30 gram in weight, SPF grade female C57BL / 6J mice as experimental animals. The mice were grouped and administered once (on day 0) and sampled according to the table below. The content of mRNA of the target gene in each tissue of the mice was detected using rt-qPCR; the content of siRNA in each tissue of the mice was detected using stem-loop method.

[0292] Table 24. Experimental grouping

[0293] The experimental results are shown in Figure 16. On day 7 after administration, the siRNA of the application significantly inhibited the expression of the target gene after the introduction of aliphatic hydrocarbons (16A in Figure 16, expression of ANGPTL3 mRNA in liver tissue; 16B in Figure 16, expression of TTR mRNA in different tissues); and the introduction of aliphatic hydrocarbons significantly improved the distribution of siRNA in each tissue, and the results showed that the distribution concentration of siRNA of the application in heart, liver, spleen, lung, kidney, muscle, brain and blood was significantly improved (16C in Figure 16), indicating that the conjugate of the application can deliver siRNA to the above tissues.

[0294] Effect Example 17 In vivo efficacy experiment of siRNA with 3' and 5' end modified siRNA

[0295] This example used 4-6 week old, 18-30 gram in weight, SPF grade female C57BL / 6J mice as experimental animals. The mice were grouped and administered once (on day 0) and sampled according to the table below. The content of mRNA of the target gene in each tissue of the mice was detected using rt-qPCR; the content of siRNA in each tissue of the mice was detected using stem-loop method.

[0296] Table 25. Experimental grouping

[0297] The information of the test substances is shown in Table 26.

[0298] [Corrected according to Rule 91 18.09.2025] Table 26. Test substances

[0299] The mRNA level detection result shows that the siRNA with the fatty alkyl introduced at the 5' end and the maleimide coupled at the 3' end and the siRNA with the fatty alkyl introduced at the 3' end and the maleimide coupled at the 5' end both significantly inhibit the expression of the target gene, and there is no significant difference in the inhibition level of the target gene expression. The siRNA level detection result shows that the siRNA with the fatty alkyl introduced at the 5' end and the maleimide coupled at the 3' end and the siRNA with the fatty alkyl introduced at the 3' end and the maleimide coupled at the 5' end both significantly improve the distribution of the siRNA in each tissue, and the distribution concentration of the siRNA of the application in the heart, liver, spleen, lung, kidney, muscle, brain and blood is significantly improved, indicating that the conjugate of the application can deliver the siRNA to the above-mentioned tissues.

[0300] Although the present application has been described in detail with general description and specific embodiments above, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the present application are within the scope of the present application claimed.

Claims

1. An oligonucleotide conjugate having the structure of Formula II: C-(X) n -(B) k -A (Formula II) wherein, C is a better lipid-soluble group selected from one or more of saturated alkanes of different chain lengths, unsaturated alkanes, saturated fatty acids, unsaturated fatty acids and cholesterol; X represents an oligonucleotide for regulating the expression level of a gene, n≥1; B represents a connecting arm, k≥1; A represents a group capable of reacting with the free active group of albumin to form a covalent bond.

2. The oligonucleotide conjugate of claim 1, wherein, C has (M) e - (N) w - P- indicated structure; wherein the structure of M is Q-Linker 1 or Q represents a saturated alkane having at least 8 carbon atoms, a C10-C30 unsaturated alkane containing 1-5 unsaturated double bonds, a C10-C30 fatty acid having a double bond content of < 5, or a combination thereof; or, Q is The N has the structure shown in the following formula: -N1-N2-N3-N4-N5-, wherein, -N1- is selected from a bond, optionally substituted -C1-20 alkyl-, optionally substituted heteroalkylene of 1-20 atoms; -N2- is selected from a bond, optionally substituted -NH-C1-8 alkyl-NH-, optionally substituted -NH-C1-8 alkyl-NH-C1-6 alkyl-, optionally substituted -NH-C1-8 alkyl-NH-1-6 membered heteroalkyl-; - N3- is selected from a bond, optionally substituted -C 1-6 alkyl-, - N4- is selected from a bond, Optional substitution of -C 1-6 Alkyl-, optionally substituted heteroalkylene groups of 1-6 atoms; -N5- is selected from a bond, optionally substituted divalent branched group, optionally substituted trivalent branched group; The P represents e = 1, 2 or 3; w = 0, 1, 2 or 3.

3. The oligonucleotide conjugate of claim 2, wherein, Q is selected from optionally substituted R or R-C(O)-, wherein R is selected from C7-C30 straight chain or branched chain saturated or unsaturated alkane, C7-C30 straight chain or branched chain saturated or unsaturated fatty acid, preferably C7-C24, more preferably C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24; and / or Linker 1 has the structure -L2-L3-, wherein -L2- is selected from the group consisting of an optionally substituted straight or branched chain saturated or unsaturated alkylene of 1-50 atoms, an optionally substituted straight or branched chain saturated or unsaturated heteroalkylene of 1-50 atoms, the heteroatoms in said heteroalkylene being selected from one or more of O, N, S, P, said optionally substituted substituents being selected from Ra; -L3- is selected from the group consisting of a bond, a 3-8 membered heterocyclyl, a 5-6 membered heteroaryl; preferably -L2- is selected from -NH-(CH2CH2O)m4-C 1-3 alkyl-, -N(C 1-3 alkyl)-(CH2CH2O)m4-C 1-3 alkyl-, -NH-(CH2CH2O)m4-CH2CH2-, 1-3 alkyl)-(CH2CH2O)m4-CH2CH2-, 1-12 alkyl-, -NH-C 1-12 alkyl-, -N(C 1-3 alkyl)-C 1-12 alkyl-, wherein m4 is selected from an integer from 1-12, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12; -L3- is selected from the group consisting of a bond, 4. The oligonucleotide conjugate of claim 2 or 3, wherein, Linker 1 is:

5. The oligonucleotide conjugate of any one of claims 2-4, wherein, -N1- is selected from the group consisting of -C 1-6 alkyl-, -(CH2CH20)m5-, optionally substituted -C 1-6 alkyl-(CH2CH20)m5-, optionally substituted -C 1-6 alkyl-(CH2CH20)m5-C 1-6 alkyl-, -(CH2CH20)m5-C 1-6 alkyl-; -N2- is selected from the group consisting of a bond, optionally substituted -NH-C(O)-C 1-6 alkyl-C(O)-NH-, optionally substituted -NH-C(O)-C 1-6 alkyl-C(O)-NH-C 1-6 alkyl-; - N3- is selected from the group consisting of a bond, hydroxymethyl- substituted C 1-3 alkyl-, hydroxymethyl-substituted-C 1-6 alkyl-OC 1-6 alkyl-, - N4- is selected from a bond, or -O-C 1-6 alkyl-; - N5- is selected from: a bond, Preferably, -N1- is selected from the group consisting of optionally substituted methylene, ethylene, propylene, butylene, pentylene, hexylene, -C 1-3 alkyl-OCH2CH2-, -C 1-3 alkyl-(OCH2CH2)2-, -C 1-3 alkyl-(OCH2CH2)3-, -C 1-3 alkyl-(OCH2CH2) 1-3 -C 1-3 alkyl-; -N2- is selected from the group consisting of a bond, optionally substituted -NH-C(O)- methylene-C(O)-NH-, -NH-C(O)-ethylene-C(O)-NH-, -NH-C(O)-propylene-C(O)-NH-, -NH-C(O)-butylene-C(O)-NH-, -NH-C(O)-pentylene-C(O)-NH-, optionally substituted -NH-C(O)-C 1-3 alkyl-C(O)-NH-C 1-3 alkyl-; -N3- is selected from the group consisting of a bond, hydroxymethyl- substituted ethylene, hydroxymethyl-substituted -C 1-3 alkyl-OC 1-2 alkyl-, - N4- is selected from a bond, or -O-C 1-3 alkyl-; - N5- is selected from: a bond, 6. The oligonucleotide conjugate of any one of claims 2-5, wherein, N is selected from:

7. The oligonucleotide conjugate of any one of claims 1-6, wherein, C is selected from the group consisting of the structure of Formula C-1, Formula C-2, or Formula C-3: wherein, Rb, Rc, Rd are each independently selected from optionally substituted C6-C50 straight chain or branched chain saturated alkyl, optionally substituted C6-C50 straight chain or branched chain unsaturated alkyl, optionally substituted C6-C50 straight chain or branched chain saturated fatty acid, optionally substituted C6-C50 straight chain or branched chain unsaturated fatty acid; t1, t2, t3, t4, t5, t6, t7, t8 are each independently selected from an integer from 0 to 10, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; Preferably, Rb, Rc, Rd are each independently selected from the group consisting of Re-(CH2)m6-(CH2)6-, Re-(CH2)m7-CH=CH-(CH2)m8-, Re-(CH2)m9-(C 1-3 alkyl-CH=CH-C 1-3 alkyl)m 10 -(CH2)m 11 - Re-(CH2)m9-(CH=CH-CH2)m 10 -(CH2)m 11 - Re-(CH2)m9-(CH=CH-CH2CH2)m 10 -(CH2)m 11 - Re-(CH2)m9-(CH2CH=CH-CH2)m 10 -(CH2)m 11 - wherein Re is selected from hydrogen or -COOH, m6, m7, m8, m9, m 11 are each independently selected from an integer from 1 to 24, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, m 10 are selected from an integer from 1 to 6, such as 1, 2, 3, 4, 5, 6, wherein the sum of m7 and m8 or the sum of m9, m 10 and m 11 is greater than 7, preferably greater than 12, further preferably from 12 to 24.

8. The oligonucleotide conjugate of claim 7, wherein, Rb, Rc, Rdare each independently selected from 9. The oligonucleotide conjugate of any one of claims 1-8, wherein, A is selected from one or more of the following groups: maleimide and derivative groups thereof, divinyl sulfone and derivative groups thereof, acrylamide and derivative groups thereof, a-cyanoacrylamide and derivative groups thereof, 2-butynoamide and derivative groups thereof, carboxylic acid and derivative groups thereof; Preferably, said A is selected from 10. The oligonucleotide conjugate of any one of claims 1-9, wherein, B is selected from a non-cleavable linker or a cleavable linker; Preferably, the B is -(B1)j-(B2)q-(B3)p-(B4)m-(B5)h-; wherein B1is selected from a C18-containing group; B2is selected from a S-S bond-containing group; B3is selected from a NH2amino-containing group; B4is selected from a polypeptide-containing group; B5is selected from a multiple C atom or O atom-containing group, and B5terminally connects with A; J, q, p, m, h are each independently selected from an integer from 0 to 20, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and the like; Preferably, B1, B2, B3are each independently selected from an optionally substituted straight chain or branched chain saturated or unsaturated alkyl, an optionally substituted straight chain or branched chain saturated or unsaturated heteroalkyl, Further preferably, B1, B2, B3are each independently selected from: -(R1)j1-(R2)j2-(R3)j3-, wherein each R1, R2, R3may be the same or different, and R1, R2, R3are each independently selected from -CH2CH2O-, -CH2-, -C(O)-, -O-, -NH-, -P(O)(OH)O-, -S-S-; j1, j2, j3are each independently selected from an integer from 0 to 100 (such as an integer from 0 to 50, an integer from 0 to 30, an integer from 0 to 20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20); R1, R2, R3are optionally substituted with Ra; Still more preferably, B1is: a bond, or and / or, B2 is: a bond, and / or B3 is a bond, and / or B4 is a bond, 11. The oligonucleotide conjugate of claim 10, wherein, B5is selected from -(R4)m1-(R5)m2-(R6)m3-, wherein each R4, R5, R6may be the same or different, and R4, R5, R6are each independently selected from -C(O)-, -CH2-, -C(O)CH2-, -NH-, -CH2CH2O-, C3-C8cycloalkylene (such as cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane), C6-C14arylene (such as phenylene, naphthylene), 3-8 membered heterocyclylene (such as azetidinylene, tetrahydrofuranylene, tetrahydropyrrolylene, morpholinylylene, piperidinylene, piperazinylene, and the like), 5-8 membered heteroarylene (such as furanylene, thienylylene, pyrrolylene, pyrazolylene, pyridinylene, pyrimidinylene, and the like); m1, m2, m3are each independently selected from an integer from 0 to 20 (such as an integer from 0 to 10, such as an integer from 0 to 5, such as an integer from 0 to 3, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); Preferably, B5is selected from a bond, -C(O)-C 3-8 cycloalkyl-C 1-6 alkyl; Further preferably, B5 is a bond, or 12. The oligonucleotide conjugate of any one of claims 1-11, wherein, The substituents in the optionally substituted corresponding groups are selected from the group consisting of Ra, Ra being selected from the group consisting of hydrogen, carbonyl, hydroxyl, halogen (e.g. F, Cl, Br, I), amino, alkylamino (e.g. C 1-3 alkyl-NH-, (C 1-3 alkyl)2-N-, alkoxyl (e.g. C 1-3 alkoxyl, e.g. methoxyl, ethoxyl), hydroxyalkyl (e.g. hydroxyC 1-3 alkyl-, e.g. -CH2OH); Ra is preferably -CH2OH; and / or B by phosphonate or phosphorothioate covalently linked to an oligonucleotide X.

13. The oligonucleotide conjugate of any one of claims 1-12, said B is selected from one or more of the following compounds: and / or A is 14. The oligonucleotide conjugate of any one of claims 1-13, wherein, X is a siRNA, an ASO, or a miRNA.

15. An oligonucleotide conjugate or protein conjugate having the structure shown below: (X)n-(B)k-A (Formula I), C-(X)n (Formula III), C-(X)n-(B)k-A’-albumin (Formula IV), or (X)n-(B)k-A’-albumin (Formula V), wherein C, X, B, n, k are defined as in any one of claims 1 to 14, A' is selected from groups which can be conjugated to albumin, preferably A' is selected from or -S-.

16. A pharmaceutical composition comprising the oligonucleotide conjugate or protein conjugate according to any one of claims 1-15 and a pharmaceutically acceptable carrier.

17. Use of the oligonucleotide conjugate or protein conjugate of any one of claims 1-15 or the pharmaceutical composition of claim 16 for the preparation of a targeted drug delivery system or a targeted drug having tissue specificity.

18. Use of the oligonucleotide conjugate or protein conjugate of any one of claims 1-15 or the pharmaceutical composition of claim 16 for the preparation of a drug delivery system for conjugation to albumin in vivo.

19. A method of modulating the expression of a gene of interest and / or the activity, expression level or circulating level of a protein encoded by the gene of interest in a target site in a subject, comprising the step of administering to a subject in need thereof a prophylactically or therapeutically effective amount of the oligonucleotide conjugate or protein conjugate of any one of claims 1-15 or the pharmaceutical composition of claim 16.

20. A method of drug delivery, comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of the oligonucleotide conjugate or protein conjugate of any one of claims 1-15 or the pharmaceutical composition of claim 16, thereby delivering the oligonucleotide to a target site in the subject.

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